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An eye for an eye

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<contrib contrib-type="author"><name><surname>Honavar</surname><given-names>Santosh G.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Raju</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib>

Oman Journal of Ophthalmology

<p>Anophthalmia can be congenital or acquired. Malignancy, infection, and trauma may lead to the removal of the eye by enucleation or evisceration. In addition, there are clinical situations where the eye may be blind, shrunken and deformed (phthisis bulbi). Irrespective of the etiology, the loss of an eye can have a profound physical, social, and psychological impact on an individual. The challenge in such situations lies in providing a life-like ocular prosthesis to restore the esthetics, and consequently help provide social rehabilitation.</p><p>The ocular prosthesis can be ready-made (stock eye) or custom-made. Stock eyes are supplied in three basic shapes: Oval, standard and three-cornered. Each shape has three sizes: Small, medium and large, and laterality-right and left. The iris is produced in three basic colors: Brown, hazel and blue with some overlap, and the scleral colors are also varied. One basic size of the iris (11.5 mm) is used, and it has a pupil opening of 3.5 mm.[<xref rid="ref1" ref-type="bibr">1</xref>] Cost and convenience are the major considerations in providing a stock eye, and a skilled ocularist is not necessary.</p><p>Custom-made prosthesis has the advantages of the better fit over the socket surface, stability, reduced incidence of discharge and socket infection, comfort, color match, and optimal upper and lower eyelid position and contour.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] In addition, it needs to be sparingly removed, only once every 3-4 weeks, to clean. It is known that the incidence of socket contraction is significantly low with a custom prosthesis as compared to a stock eye.</p><p>Numerous techniques for processing a custom ocular prosthesis exist. Mathews <italic>et al</italic>.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] have classified ocular impression and fitting techniques as follows:</p><sec id="sec1-1"><title>Direct Impression</title><p>An impression material is injected directly into the enucleated socket. Using the impression, a wax trial ocular prosthesis is made. It is fit, contour, and comfort are assessed. Then it is processed as done routinely.</p></sec><sec id="sec1-2"><title>Impression with Stock Ocular Tray</title><p>A stock ocular tray is placed in the socket. This tray has a hollow stem fastened in the middle through which an impression material is injected. Perforations in the tray aid flow and retention of the impression material. Subsequently, the impression is removed, and the wax pattern is fabricated. This wax trial prosthesis is placed in the socket, fitted, and modified as needed. It is then processed.</p></sec><sec id="sec1-3"><title>Impression with Modified Stock Ocular Tray</title><p>Exactly, the same as in type 2, but using a modified ocular tray.</p></sec><sec id="sec1-4"><title>Impression with Custom Ocular Tray</title><p>This technique involves attaching a suction rod to the patient's existing artificial eye or the custom conformer and investing it in an alginate mould. After the alginate sets, the prosthesis or conformer is removed and replaced with clear acrylic resin, which is then used as the &#x0201c;custom ocular tray.&#x0201d;</p></sec><sec id="sec1-5"><title>Impression using Stock Ocular Prosthesis</title><p>This method uses a stock ocular prosthesis as a tray to carry the impression material. An aesthetic stock eye is selected, and its peripheral and posterior aspects are ground to size. The stock eye is then lined with an impression material and inserted for the definitive impression. Alternately, the impression material can be injected directly into the socket and then reinforced by placement of the stock eye. The resulting impression is processed, providing a customized stock prosthesis.</p></sec><sec id="sec1-6"><title>Stock Prosthesis Modification</title><p>This technique involves fitting of a stock prosthesis by trimming and polishing.</p></sec><sec id="sec1-7"><title>Wax Scleral Blank Technique</title><p>An empirically manufactured wax scleral blank is tried in the given socket and suitably modified. After the addition of an iris button, it is further processed.</p><p>The major considerations in using a specific technique include patient's exacting requirements, time at disposal, cost involved, and the training level of the ocularist/technician. Obtaining impression with a stock or a modified impression tray, and incorporating a matching custom-painted iris button provides the most acceptable cosmesis, and a durable and a comfortable prosthesis [Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>]. A trained ocularist can fabricate 3-4 such prosthesis in a day, making it time- and cost-effective. We have been using this technique routinely in our clinical practice.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Anophthamic left socket in a 4-year-old child following enucleation (for retinoblastoma) by the myoconjunctival technique with a primary orbital implant (a), appears cosmetically optimal following a custom ocular prosthesis (b)</p></caption><graphic xlink:href="OJO-7-109-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Anophthamic left socket in a 60-year-old lady following enucleation (for malignant melanoma) by the myoconjunctival technique with a primary orbital implant (a), a custom ocular prosthesis (b) that exactly matches with her right eye</p></caption><graphic xlink:href="OJO-7-109-g002"/></fig><p>The steps in fabricating a custom ocular prosthesis comprise of the following:[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p> <list list-type="bullet"><list-item><p>Detailed evaluation of the socket by the oculoplasty surgeon to determine if the healing is complete and the socket is ready for prosthesis. In patients with contracted socket, the surgeon may want to deepen the fornices, increase the surface or build in the volume by simple surgical procedures prior to prosthesis fitting to optimize the ultimate cosmesis. Specific requirements if any (ptosis shelf, light-weight prosthesis, self-lubricating prosthesis) are discussed by the surgeon with the ocularist</p></list-item><list-item><p>Examination by the ocularist to determine the type of prosthesis and the nature of customization</p></list-item><list-item><p>Obtaining a socket impression using an appropriate sized ocular tray, generally matching with the size of the existing conformer that the surgeon had provided at the conclusion of the surgery</p></list-item><list-item><p>Preparation of the wax model for the socket</p></list-item><list-item><p>Careful centration of the prosthesis using interpupillary distance, Hirschberg's test, and an iris corneal button and fabrication of iris and pupil to match the fellow eye</p></list-item><list-item><p>This is followed by moulding in acrylic, tinting to match the scleral shade, incorporation of blood vessels, packing with the clear acrylic and final polishing</p></list-item><list-item><p>The final fit is assessed based on the following criteria-comfort, stability, three-planar position-vertical, horizontal, and frontal, eyelid position and contour, lagophthalmos, degree of prosthesis motility, iris color, iris position, horizontal visible iris diameter, pupil size, and scleral color and vascular pattern</p></list-item><list-item><p>Instructions on socket hygiene, prosthesis care, technique of prosthesis removal and insertion, and use of protective glasses.</p></list-item></list> </p><p>In this issue of the Journal, Sonune <italic>et al</italic>. reported excellent outcome in a patient where they used a simplified technique of integration of the socket impression to the back surface of a stock eye that had been selected by the ophthalmologist to match with the contralateral eye.[<xref rid="ref6" ref-type="bibr">6</xref>] The technique used by the authors is convenient and cost-effective, but the main disadvantage is the inability to exactly match the iris color and limited variations in iris size, which may render several patients unsuitable for this technique.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Fabrication of a custom ocular prosthesis using socket impression and customized iris button remains the gold standard in optimally rehabilitating an anophthalmic socket in a clinical facility where an oculoplasty surgeon works as a cohesive team with a dedicated ocularist. However, improvisations such as that used by Sonune <italic>et al</italic>. may indeed benefit a certain cross-section of patients where the major consideration may be cost and convenience, and the service is provided by someone who makes an ocular prosthesis only occasionally.</p></sec>

Vascular endothelial growth factor trap-eye and trap technology: Aflibercept from bench to bedside

<p>Anti-vascular endothelial growth factor (VEGF) currently used to treat eye diseases have included monoclonal antibodies, antibody fragments, and an aptamer. A different method of achieving VEGF blockade in retinal diseases includes the concept of a cytokine trap. Cytokine traps technology are being evaluated for the treatment of various diseases that are driven by excessive cytokine levels. Traps consist of two extracellular cytokine receptor domains fused together to form a human immunoglobulin G (IgG). Aflibercept/VEGF trap-eye (VTE) is a soluble fusion protein, which combines ligand-binding elements taken from the extracellular components of VEGF receptors 1 and 2 fused to the Fc portion of IgG. This protein contains all human amino acid sequences, which minimizes the potential for immunogenicity in human patients. This review presents the latest data on VTE in regard to the pharmacokinetics, dosage and safety, preclinical and clinical experiences. Method of the literature search: A systematic search of the literature was conducted on PubMed, Scopus, and Google Scholar with no limitation on language or year of publication databases. It was oriented to articles published for VTE in preclinical and clinical studies and was focused on the pharmacokinetics, dosage and safety of VTE.</p>

<contrib contrib-type="author"><name><surname>Al-Halafi</surname><given-names>Ali M.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Angiogenesis is important for several physiological processes, including embryonic and postnatal development, reproductive functions, and wound healing.[<xref rid="ref1" ref-type="bibr">1</xref>] A different endogenous inhibitors of angiogenesis have been identified, including endostatin, tumstatin, and vasostatin.[<xref rid="ref2" ref-type="bibr">2</xref>] However, despite such complexity, vascular endothelial growth factor (VEGF)-A appears to be necessary for growth of blood vessels in a variety of normal and pathological circumstances.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] The VEGF family consists of five related glycoproteins, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF).[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] VEGF has two main receptors in normal biological systems: VEGF receptors (VEGFR) 1 and VEGFR2. VEGFR2 mediates most of the endothelial cell proliferating activity of VEGF,[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] whereas VEGFR1 has other activities of VEGF, such as its chemo-attractant properties.[<xref rid="ref10" ref-type="bibr">10</xref>] Both receptors are important for the angiogenic-promoting properties of VEGF.</p><p>VEGF-A is responsible for vascular permeability, which is critical for normal physiological processes such as wound healing, but can potentially increase leakage in pathological diseases, such as neovascular age-related macular degeneration (AMD).[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] VEGF-A promotes the survival of hematopoietic stem cells; the migration of these cells appears to be promoted via interactions with VEGFR1.[<xref rid="ref10" ref-type="bibr">10</xref>] It has been suggested VEGF-A is a significant factor for the recruitment of monocytes and macrophages in an inflammatory neovascularization.[<xref rid="ref11" ref-type="bibr">11</xref>] Despite the fact that the role of VEGF-B in angiogenesis is not entirely understood, there is proof that it may be involved in inflammatory angiogenesis in various disease states, and may also modulate endothelial proliferation and vessel growth.[<xref rid="ref12" ref-type="bibr">12</xref>] VEGF-C and VEGF-D have a role in the development of the lymphatic system.[<xref rid="ref10" ref-type="bibr">10</xref>] In human tumors, over expression of VEGF has been frequently observed.[<xref rid="ref13" ref-type="bibr">13</xref>] Definitely, increased VEGF expression was established to correlate frequently with tumor progression, recurrence, and survival.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] VEGF trap-eye (VTE) made by fusing DNA sequences encoding the second immunoglobulin (Ig) domain of human VEGFR1 to the third Ig domain of human VEGFR2, is fused to the constant region of human IgG, which serves to create a homodimer of the fusion protein.[<xref rid="ref16" ref-type="bibr">16</xref>] The VTE forms a stable and inert complex with VEGF.[<xref rid="ref17" ref-type="bibr">17</xref>] It also binds its receptors as homo- or heterodimers.[<xref rid="ref9" ref-type="bibr">9</xref>] The trap binds VEGF very tightly and prevents it from activating its cell surface receptor. In contrast, anti-VEGF antibodies bind in a manner that allows the VEGF dimer to interact further with other molecules. VTE forms inert complexes with VEGF that minimizes the potential for VEGF-trap to interact with more than one VEGF-trap molecule.[<xref rid="ref17" ref-type="bibr">17</xref>]</p><p>This review presents the latest data on VTE in regard to the pharmacokinetics, dosage and safety, preclinical and clinical experiences.</p><sec id="sec2-1"><title>Method of the literature search</title><p>A systematic search of the literature was conducted on PubMed, Scopus, and Google Scholar with no limitation on language or year of publication databases. It was oriented to articles published for VTE in preclinical and clinical studies and was focused on the pharmacokinetics, dosage and safety of VTE.</p></sec></sec><sec id="sec1-2"><title>Pharmacokinetics</title><p>The pharmacokinetics and posterior segment distribution of intravitreal VTE were studied in rabbits.[<xref rid="ref18" ref-type="bibr">18</xref>] Maximum vitreous concentrations of free VTE were about 500 &#x003bc;g/ml 0.25-6 h following the 500-&#x003bc;g injection, and vitreous elimination half-life was approximately 4.5 days. In This model, VTE was found in both retina and choroid, from which it was eliminated with a similar half-life. Ten days after the injection, maximal plasma total VTE levels were 1.6 &#x003bc;g/ml. At week 4, vitreous free VTE was 10-fold greater than levels of excess bound VTE. Therefore, ocular VEGF production would likely be totally suppressed for &#x0003e;6 weeks following an intravitreal injection based on these results.[<xref rid="ref18" ref-type="bibr">18</xref>] It also shows an extended half-life, allowing for long-term blockade.[<xref rid="ref19" ref-type="bibr">19</xref>] Experimental studies with intravitreal injections display that VTE should penetrate all layers of the retina (molecular weight&#x0223c;110,000) with the least possible systemic exposure.[<xref rid="ref20" ref-type="bibr">20</xref>] In diabetic rats, an intravitreal injection of VTE was distributed to all retinal layers, with minimal systemic exposure.[<xref rid="ref21" ref-type="bibr">21</xref>] A recent study showed that the binding activity for the monoclonal antibody ranibizumab would be fairly close to that of the anti-VEGF monoclonal antibody bevacizumab, implying that VTE should have more extended binding activity than anti-VEGF monoclonal antibody.[<xref rid="ref22" ref-type="bibr">22</xref>] This study hypothesizes that VTE should have more durable effects than currently available VEGF blockers, which would likely result in longer intervals between doses. VTE theoretically binds VEGF more tightly than native receptors or monoclonal antibodies, which suggests that a much lower dose of VTE may be used versus anti-VEGF monoclonal antibodies.[<xref rid="ref16" ref-type="bibr">16</xref>] VTE blocks all VEGF-A isoforms and PlGF.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref19" ref-type="bibr">19</xref>] It also blocks all isoforms of VEGF-B.[<xref rid="ref23" ref-type="bibr">23</xref>] Consistent with this comprehensive receptor-blocking effect, VTE was shown <italic>in vitro</italic> to block several biological effects of VEGF, including potent blockade of the activation of VEGFR by VEGF and also complete blockade of VEGFR2-induced phosphorylation in cultured human umbilical vein endothelial cells.[<xref rid="ref16" ref-type="bibr">16</xref>]</p></sec><sec id="sec1-3"><title>Dosage and Safety</title><p>VTE/Aflibercept (EYLEA^&#x000ae;-Regeneron Pharmaceuticals, Inc., Tarrytown, New York, NY, USA and Bayer Healthcare Pharmaceuticals, Berlin, Germany) is a novel 115-kDa anti-VEGF agent. It is available in a single-use vial which contains 0.05 mL of VTE (40 mg/mL in 10 mmol/L sodium phosphate, 40 mmol/L sodium chloride, 0.03% polysorbate 20, and 5% sucrose, pH 6.2).[<xref rid="ref24" ref-type="bibr">24</xref>] Up to date, VTE is only available for intravitreal injection. No systemic effects have been reported in any phase I, phase II, or phase III trials with intravitreal administration of doses of up to 4 mg (&#x0003c;0.06 mg/kg; 0.057 mg/kg).[<xref rid="ref25" ref-type="bibr">25</xref><xref rid="ref26" ref-type="bibr">26</xref>]</p><p>A number of articles have been reporting a significant increase in systemic adverse effects in patients treated with intravitreal VEGF blocker agents.[<xref rid="ref27" ref-type="bibr">27</xref>] The Committee for Medicinal Products for Human Use showed an increase in cerebrovascular events with VTE.[<xref rid="ref28" ref-type="bibr">28</xref>] In contrast, the VTE: Investigation of Efficacy and Safety in Wet (VIEW) 1 and VIEW 2 studies stated &#x0201c;there was a similar overall incidence of systemic (nonocular) adverse events, serious systemic adverse events.&#x0201d;[<xref rid="ref29" ref-type="bibr">29</xref>]</p></sec><sec id="sec1-4"><title>Drug Actions</title><sec id="sec2-2"><title>Preclinical</title><p>Preclinical animal studies have determined the efficacy of VTE in several models of neovascularization in the eye, including the suppression of choroidal neovascular membrane (CNV) in mice and suppression of VEGF-induced breakdown of the blood&#x02013;retinal barrier. Subcutaneous injections of a single intravitreal injection of VTE markedly inhibited CNV in mice with laser-induced rupture of Bruch's membrane.[<xref rid="ref30" ref-type="bibr">30</xref>] Subcutaneous injection of VTE also significantly suppressed subretinal neovascularization in transgenic mice that express VEGF in photoreceptors.[<xref rid="ref30" ref-type="bibr">30</xref>] In a mouse model of suture-induced inflammatory corneal neovascularization, VTE have been shown to block angiogenesis.[<xref rid="ref11" ref-type="bibr">11</xref>] It also prevents the development of grade 4 CNV lesions in primates and strongly reduced proliferative activities of the retina to laser injury in adult cynomolgus monkeys.[<xref rid="ref31" ref-type="bibr">31</xref>] Every 4 weeks intravitreal VTE injection was also demonstrated to be safe in cynomolgus monkeys after 13 weeks of administration.[<xref rid="ref32" ref-type="bibr">32</xref>]</p><p>Julien <italic>et al</italic>.[<xref rid="ref33" ref-type="bibr">33</xref>] studied the different effects of intravitreal injections of ranibizumab and VTE on retinal and choroidal tissues of monkey eyes. Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of VTE, and they concluded that the ranibizumab permeated the retina through intercellular spaces, whereas VTE was taken up by neuronal and retinal pigment epithelium (RPE) cells. VTE induced protein complex formation and more hemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of VTE remain to be investigated.</p></sec><sec id="sec2-3"><title>Clinical</title><sec id="sec3-1"><title>Neovascular age-related macular degeneration</title><p>VTE is currently being evaluated for two eye diseases: Neovascular (wet) AMD and diabetic macular edema (DME). VTE has undergone phase I and II clinical trials in wet AMD, and is presently in phase III clinical testing. The phase I study, known as Clinical Evaluation of Anti-angiogenesis in the Retina Intravitreal Trial (CLEAR-IT) 1, consisted of two parts. It was designed to evaluate the safety, tolerability, and biological effects of intravitreal VTE in patients with neovascular AMD. Overall, in the CLEAR-IT 1, part 1 and part 2 studies, intravitreal injection of up to 4 mg of VTE was well-tolerated with no ocular inflammation seen.[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>] CLEAR-IT 2 was designed based upon the phase I study results showing clinically meaningful improvement in visual acuity (VA) with single doses of 0.5, 2.0, and 4.0 mg and sustained clinical activity beyond 1 month. At the analysis of CLEAR-IT 2 study, the most common adverse events were those typically associated with intravitreal injections and appeared to be related to the injection procedure. There was no relationship between VTE dose and the occurrence of any particular ocular adverse event. No adverse consequences of increased intraocular pressure were reported.[<xref rid="ref35" ref-type="bibr">35</xref>] In the phase I and II studies, intravitreal injections of VTE caused no drug-related systemic adverse effects.[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>] In the phase II AMD study, there were no drug-related serious adverse events and no systemic adverse events were mentioned by the investigators as being related to study drug administration.[<xref rid="ref36" ref-type="bibr">36</xref>] Phase III based upon the preliminary phase II study results which indicated that doses of 0.5 and 2 mg monthly produced substantial gains in VA and a single 2-mg dose had a sustained improvement in VA when compared to 2 mg monthly for at least 8 weeks, the phase III program was designed to evaluate these dosing schedules when compared to the standard dosing schedule for ranibizumab 0.5 mg monthly. Two identical, noninferiority phase III studies are currently under way to evaluate VTE for wet AMD, known as VIEW 1 and VIEW 2. Both will compare VTE with the monoclonal antibody fragment ranibizumab using a noninferiority design. Both are randomized, double-masked, active-controlled, efficacy and safety studies with a primary endpoint of the proportion of patients treated with VTE who maintains vision at the end of 1 year, compared to ranibizumab patients.[<xref rid="ref24" ref-type="bibr">24</xref>] In VIEW 1 and VIEW 2 were considered, continued monthly VTE achieved superior outcomes compared with every 8-week dosing.[<xref rid="ref37" ref-type="bibr">37</xref>] In the treatment-naive eyes included in the VIEW 1 and VIEW 2 trials, VTE and ranibizumab treatment resulted in clinically equivalent visual outcomes.[<xref rid="ref30" ref-type="bibr">30</xref>] VTE treatment for patients with exudative AMD, who were incomplete responders to multiple ranibizumab injections (TURF trial) which published by Wykoff <italic>et al</italic>.[<xref rid="ref38" ref-type="bibr">38</xref>] employed every other month dosing after the first 3-monthly doses, but was designed to permit pro re nata (PRN) dosing at the intervening months, supporting the observation that VTE does not maintain maximal retinal deturgescence for 2 months in many patients and indicative of the refractory exudative nature of the study eyes. In TURF trial, 72% eyes required retreatment at both PRN visits, and 79% PRN retreatments were required. Furthermore, VTE 2.0 mg treatment maintained mean VA improvements previously achieved with high-dose 2.0-mg ranibizumab injections in recalcitrant wet AMD patients and significant anatomic improvement and was required monthly in most patients.</p></sec><sec id="sec3-2"><title>Diabetic macular edema</title><p>The initial phase I study of VTE in DME, CLEAR-IT DME, was an exploratory study of the safety, tolerability and biological effect of a single intravitreal administration of 4 mg VTE in patients with DME at two centers involving five patients.[<xref rid="ref25" ref-type="bibr">25</xref>] According to the study, the maximum change from baseline in key outcome measures at 6 weeks included reductions in center retinal thickness, with a mean reduction of &#x02212;115.4 &#x003bc;m and a median reduction of &#x02212;118 &#x003bc;m (<italic>P</italic> &#x0003c; 0.03). Macular volume was reduced by a mean of &#x02212;1 &#x003bc;m³ and a median of &#x02212;0.6 &#x003bc;m³ (<italic>P</italic> &#x0003c; 0.04). The Early Treatment Diabetic Retinopathy Study best corrected VA (BCVA) letters improved by a mean of 6.8 and a median of 9 (<italic>P</italic> &#x0003c; 0.03) and no serious ocular adverse events were reported.[<xref rid="ref25" ref-type="bibr">25</xref>] Based on the results of a phase I study, a 52 weeks, multicenter, randomized, double-masked, active-controlled phase II clinical trial was conducted. The primary aim of the DME and VTE: Investigation of Clinical Impact (DA VINCI) study was to assess the safety and efficacy of intravitreal VTE in comparison with focal/grid laser photocoagulation in patients with DME. The primary end point results of the DA VINCI study (week 24) revealed that treatment with intravitreal VTE produced a statistically significant improvement in VA when compared with macular laser treatment. It also showed that VTE was well-tolerated, and its ocular adverse events were consistent with those seen with other intravitreal anti-VEGF agents. The DA VINCI study group has also published the results of different doses and dosing regimens of VTE with laser photocoagulation in eyes with DME after 52 weeks. Assessment of the changes in BCVA and mean changes in CRT at 24 and 52 weeks revealed that significant gains in BCVA from baseline, achieved at week 24, were maintained or improved at week 52 in all VTE groups.[<xref rid="ref39" ref-type="bibr">39</xref>]</p></sec></sec></sec><sec sec-type="conclusion" id="sec1-5"><title>Conclusion</title><p>VTE presents a potential exciting new addition to the current VEGF antagonists available for the treatment of retinal vascular diseases such as neovascular AMD and DME. Results from clinical trials with VTE have been favorable and comparable to other anti-VEGF agents. Due to its longer half-life, VTE may also decrease the frequency of injections for retinal vascular diseases patients. These results could be attributed to the stronger and prolonged binding of VTE to the VEGF-A receptor compared to other available antagonists.</p></sec>

Long term refractive and structural outcome following laser treatment for zone 1 aggressive posterior retinopathy of prematurity

<sec id="st1"><title>Aim:</title><p>To report the long term refractive, visual and structural outcome post-laser for zone 1 aggressive posterior retinopathy of prematurity (AP-ROP).</p></sec><sec id="st2"><title>Materials and Methods:</title><p>A retrospective analysis was performed of refractive status of premature infants with zone 1 AP-ROP who underwent laser photocoagulation from 2002 to 2007 and followed up till 2013. Once the disease regressed, children were followed up six monthly with detailed examination regarding fixation pattern, ocular motility, nystagmus, detailed anterior segment and posterior segment examination, and refractive status including best corrected visual acuity.</p></sec><sec id="st3"><title>Results:</title><p>Forty-eight eyes of 25 infants were included in the study. Average follow-up was 6.91 years (range, 3.8-9.5years) after laser treatment. Astigmatism was noted in 43 out of 48 eyes (89.6%). Two eyes had simple myopia whereas three eyes had no refractive error.</p></sec><sec id="st4"><title>Conclusion:</title><p>After successful laser treatment for zone 1 retinopathy of prematurity (ROP), 94% of our cases developed refractive error. Although most had a favorable anatomical and visual outcome, long-term follow-up even after a successful laser treatment in ROP was necessary.</p></sec>

<contrib contrib-type="author"><name><surname>Shah</surname><given-names>Parag K.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Ramakrishnan</surname><given-names>Minu</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Sadat</surname><given-names>Bani</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Bachu</surname><given-names>Sandeep</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Narendran</surname><given-names>V.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kalpana</surname><given-names>N.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Retinopathy of prematurity (ROP) is a potentially blinding vasoproliferative retinopathy seen in premature infants with low birth weight. Blindness from ROP is a very significant problem, with an incidence of 8% in developed countries[<xref rid="ref1" ref-type="bibr">1</xref>] and 40% in the developing countries.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Evolution of laser treatment for ROP was associated with better structural and functional outcome compared to that for eyes treated with cryotherapy.[<xref rid="ref3" ref-type="bibr">3</xref>] There are multiple reports of use of various lasers for threshold ROP with good structural and long-term refractive outcome.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] However, there are no reports on long-term refractive outcome of zone 1 disease. Our data represents long term refractive and structural outcome for zone 1 ROP.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>Materials and Methods</title><p>It is an interventional retrospective case series of premature infants with zone 1 aggressive posterior ROP (AP-ROP) who underwent laser photocoagulation, during the study period from 2002 to 2007. Data was analyzed in 2013. The study was approved by the ethics committee of our institute. All infants weighing &#x02264;1800 g or with gestational age &#x02264;34 weeks and those infants weighing &#x0003e;1800 g with an unstable course were screened for ROP. All infants with zone 1 AP-ROP, which regressed after laser treatment, were included in the study. Laser used was diode 810 nm (Oculight SLx, Iridex Co, LA, USA). Power settings were titrated to achieve a gray-white burn of moderate intensity, and spots were applied in a confluent manner to cover the entire avascular retina from the ridge to ora serrata. All lasers were performed under topical anesthesia with an anesthetist on standby. Treated eyes were followed up closely, often weekly or more frequently, for signs of regression or need for further laser. Once ROP regressed, children were followed up six monthly with detailed examination regarding fixation pattern, ocular motility, nystagmus, detailed anterior segment and posterior segment examination, and refractive status including best corrected visual acuity (BCVA). Spherical equivalent (SE) was calculated for all eyes with compound myopic astigmatism using the formula SE = Sphere + &#x000bd; Cylinder. High myopia was defined as spherical correction &#x02265; &#x02212;6 diopter (D). Astigmatism was calculated in plus cylinder form and classified as with-the-rule (WTR) 75&#x000b0;-105&#x000b0;, against-the-rule (ATR) 0&#x000b0;-15&#x000b0; and 165&#x000b0;-180&#x000b0; and oblique (OBL) 16&#x000b0;-74&#x000b0; and 106&#x000b0;-164&#x000b0;. Astigmatism was defined as cylindrical correction &#x02265;1 D whereas high astigmatism was defined as cylindrical correction &#x02265;2 D. We defined good visual outcome as &#x02265;6/12 on Snellen's chart.[<xref rid="ref4" ref-type="bibr">4</xref>] Spherical equivalent of the refractive error and the degree and axis of astigmatism were evaluated.</p></sec><sec sec-type="results" id="sec1-3"><title>Results</title><p>Forty-eight eyes of 25 infants with zone 1 AP-ROP were included in the study, of which 15 were males and 10 females. Mean birth weight was 1509.6 g (range, 850-2080 g) and mean gestational age was 32 weeks (range, 28-35 weeks). Average post-conceptional age at laser was 35.6 weeks. The average laser spots were 3261 spots (range, 800-6416). One patient needed additional cryotherapy to control the progression. Mean follow-up period was 6.91 years (range, 3.8-9.5 years). Three eyes had no refractive error. Two eyes had simple myopia. The average spherical equivalent in right eye was &#x02212; 6.14 D (range, &#x02212;18.5-1.25 D) and in left eye was &#x02212; 5.11 D (range, &#x02212;18.75-0.5 D). Overall astigmatism was seen in 43 out of 48 eyes (89.6%). All 43 children had a myopic astigmatism; 36 had with-the-rule (WTR) astigmatism, five eyes had against-the-rule (ATR) astigmatism, and two eyes had oblique astigmatism (16&#x000b0;-74&#x000b0; and 106&#x000b0;&#x02013;164&#x000b0;) [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Type of astigmatism</p></caption><graphic xlink:href="OJO-7-116-g001"/></table-wrap><p>Thirty-nine out of 43 eyes (90.7%) had astigmatism &#x02265;1 D, of which 21 (48.8%) had astigmatism &#x02265;2 D. The average astigmatism in right eye was &#x02212; 2.08 D (range, &#x02212;4.0-&#x02212;0.5 D) and in left eye was &#x02212; 1.78 D (range, &#x02212;4&#x02013;&#x02212;0.5 D).</p><p>Anisometropia &#x02265;1 D was seen in 12 eyes (27.9%) whereas eight eyes (18.6%) had anisometropia &#x02265;2 D. The visual outcome was good with BCVA of 6/6 in five eyes and &#x02265;6/12 in 39 eyes (81%) on Snellen's chart.</p><p>Two cases had poor visual outcome. One infant had disc pallor in both eyes with nystagmus and another infant with birth weight of 1690 g and gestational age of 31 weeks developed very high myopia (SE both eyes: &#x02212;18.5 D) with poor visual outcome. One child required additional cryotherapy with laser to control the vascular process and ended up having nystagmus with pale disc. This child had a birth weight of 1230 g and gestational age of 29 weeks. Coming to structural outcome, one eye developed fibrous proliferation with disc dragging while two eyes developed lamellar cataract.</p></sec><sec sec-type="discussion" id="sec1-4"><title>Discussion</title><p>Our study presented the visual outcome and refractive status over a period of 6.91 years in infants treated with laser photocoagulation for zone 1 ROP. Majority of our patients had favorable anatomical and visual outcomes.</p><p>This study showed that most of the children had a myopic refraction, with mean spherical equivalent in right eye was &#x02212;6.14 D (range, &#x02212;18.5-1.25 D) and in left eye was &#x02212; 5.11 D (range, &#x02212;18.75-0.5 D). The visual outcome was good, with BCVA &#x02265;6/12 on Snellen's chart seen in 81% children. In a study using diode laser for threshold ROP by Yang <italic>et al</italic>.,[<xref rid="ref4" ref-type="bibr">4</xref>] 65.5% eyes achieved &#x02265;6/12 vision. Of all eyes, 77% were myopic with mean SE of &#x02212;3.87 D and 16.7% of them had myopia &#x02265;6 D over a mean follow-up of 7.8 years. McLoone <italic>et al</italic>.[<xref rid="ref5" ref-type="bibr">5</xref>] reported similar results with 50% of their laser-treated eyes being myopic and 35% having myopia &#x0003e;4 D with diode laser in children with threshold ROP with a mean follow up of 11 years.</p><p>In a study using argon laser photocoagulation for threshold ROP by Ospina <italic>et al</italic>.,[<xref rid="ref8" ref-type="bibr">8</xref>] at a follow-up of 5 years, 30 eyes (71.4%) had a BCVA of 20/40 or better. In all, 26 (62%) eyes were myopic and the overall mean spherical equivalent was &#x02212;4.95 D. In a study with 1 year follow-up of patients with laser-treated threshold ROP by Dhawan <italic>et al</italic>. in 2008,[<xref rid="ref6" ref-type="bibr">6</xref>] mean refractive error with SE &#x02212;4.71 D was seen, with myopia occurring in 80.43% of eyes.</p><p>It is known that most babies with ROP subsequently become myopic.[<xref rid="ref9" ref-type="bibr">9</xref>] This myopic tendency is augmented by both cryotherapy and laser treatment, but its mechanism is controversial. Knight Nonan and O&#x02019;Keefe in a three-year nonrandomized retrospective study demonstrated lower myopia following laser treatment.[<xref rid="ref10" ref-type="bibr">10</xref>] This was confirmed by other studies, the most important being the 10-year prospective nonrandomized trial by Ng <italic>et al</italic>.[<xref rid="ref3" ref-type="bibr">3</xref>] and Kent <italic>et al</italic>.,[<xref rid="ref11" ref-type="bibr">11</xref>] which compared both the treatments and found less myopia in the laser-treated eyes. Cryotherapy-treated eyes had shallower anterior chamber depth and thicker lenses with increased axial length compared to laser-treated eyes. Cryotherapy causes larger areas of chorioretinal adhesion and destruction of normal choroidal architecture, which changes the scleral structure, making it susceptible to stretching, leading to high myopia with cryotherapy than with laser. The greater incidence of myopia in ROP is mostly associated with lens thickness and lens power with lesser contribution from corneal steepness and axial length and a more forward position of the lens center. Evidence of altered anterior segment development in ROP, as shown by the increased lens thickness with shallow anterior chamber depth and maintenance of anterior segment depth, leads to high myopia in these infants.[<xref rid="ref12" ref-type="bibr">12</xref>] Also, it has been shown that spontaneously regressed ROP has lesser myopia compared to treatment groups.[<xref rid="ref13" ref-type="bibr">13</xref>] Only one study by Kieselbach <italic>et al</italic>.[<xref rid="ref7" ref-type="bibr">7</xref>] showed predominant hypermetropia seen post laser photocoagulation for ROP. Wani <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] had unfavorable structural outcome in 7.4% eyes and poor visual outcome (visual acuity &#x0003c;20/40) in 47% eyes. They found zone I disease as the significant risk factor for unfavorable structural outcome (<italic>P</italic> &#x0003c; 0.0001). Katoch D[<xref rid="ref15" ref-type="bibr">15</xref>] <italic>et al</italic>. in a retrospective review of 36 infants (69 eyes) with Type 1 pre-threshold ROP treated with laser, reported myopia in nearly one-fourth of the eyes. Harder <italic>et al</italic>.[<xref rid="ref16" ref-type="bibr">16</xref>] compared refractive outcome between intravitreal bevacizumab and conventional retinal laser group and found that at a one-year follow-up, bevacizumab group led to less myopization and less astigmatism.</p><p>In our study, overall high astigmatism of &#x02265;2.0 D was seen in 48.8% eyes. WTR astigmatism was seen in 36 eyes (83.7%) and ATR in five eyes (11.6%) and two eyes (4.6%) had oblique astigmatism. A study by Davitt <italic>et al</italic>.[<xref rid="ref17" ref-type="bibr">17</xref>] in patients under Early Treatment for ROP (ETROP) study revealed that astigmatism was not influenced by zone of acute phase ROP, presence of plus disease, or retinal residua. Most often the eyes had WTR astigmatism, similar to our study. In our study, 43% developed astigmatism of &#x02265;1 D and 20% had &#x02265;2 D. It has been reported that most preterm infants have WTR astigmatism.[<xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] Several studies have shown that there is no difference in the prevalence of astigmatism between cryotherapy and laser photocoagulation groups.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref20" ref-type="bibr">20</xref>] The only difference is that patients receiving cryotherapy are more likely to have ATR astigmatism than those receiving laser photocoagulation.[<xref rid="ref20" ref-type="bibr">20</xref>] The one case that required additional cryotherapy developed ATR astigmatism.</p><p>In our study, anisometropia &#x02265;1 D was seen in 12 eyes (27.9%) whereas eight eyes (18.6%) had anisometropia &#x02265;2 D. In the study by Yang <italic>et al</italic>.,[<xref rid="ref4" ref-type="bibr">4</xref>] anisometropia was seen in 46.7% of patients. The presence of anisometropia was a significant risk factor associated with poor visual outcome &#x02264;6/15 in laser-treated ROP.</p><p>Various types of lasers with various delivery systems have been tried for ROP treatment with good success.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] Complications observed with laser treatment include corneal edema, anterior segment ischemia, vitreous hemorrhage, posterior synechiae, cataract, and macular ectopia. Others include abnormal liquefied vitreous with fibrillar condensation,[<xref rid="ref24" ref-type="bibr">24</xref>] fibrovascular organization,[<xref rid="ref25" ref-type="bibr">25</xref>] and subretinal haemorrhages.[<xref rid="ref26" ref-type="bibr">26</xref>] Also, changes in retinal vessel diameter,[<xref rid="ref27" ref-type="bibr">27</xref>] slight constriction of the peripheral visual fields,[<xref rid="ref28" ref-type="bibr">28</xref>] and angle closure glaucoma[<xref rid="ref29" ref-type="bibr">29</xref>] have been documented. Our case series showed none of the major complications, except fibrous proliferation in one case which was more due to progression of disease and lamellar cataract in both eyes of one child.</p><p>The main limitation of our study is that it is retrospective and we have not assessed progression of refractive error over the follow-up period; only the final follow-up refraction was used for analysis. Since all eyes were treated, there was no comparative group to assess refractive and visual outcome with untreated eyes. Thus pre-existing myopia and laser-induced myopia could not be classified separately.</p><p>In conclusion, majority of our patients treated with laser photocoagulation for zone 1 AP-ROP had favorable anatomical and visual outcome at an average follow-up of 6.91 years. Anisometropia and advanced refractive error are common causes of impaired visual function in these patients and emphasize the need for a long-term follow-up even after a successful laser treatment in ROP.</p></sec>

Influence of visual angle on pattern reversal visual evoked potentials

<sec id="st1"><title>Purpose:</title><p>The aim of this study was to find whether the visual evoked potential (VEP) latencies and amplitude are altered with different visual angles in healthy adult volunteers or not and to determine the visual angle which is the optimum and most appropriate among a wide range of check sizes for the reliable interpretation of pattern reversal VEPs (PRVEPs).</p></sec><sec id="st2"><title>Materials and Methods:</title><p>The present study was conducted on 40 healthy volunteers. The subjects were divided into two groups. One group consisted of 20 individuals (nine males and 11 females) in the age range of 25-57 years and they were exposed to checks subtending a visual angle of 90, 120, and 180 minutes of arc. Another group comprised of 20 individuals (10 males and 10 females) in the age range of 36-60 years and they were subjected to checks subtending a visual angle of 15, 30, and 120 minutes of arc. The stimulus configuration comprised of the transient pattern reversal method in which a black and white checker board is generated (full field) on a VEP Monitor by an Evoked Potential Recorder (RMS EMG. EPMARK II). The statistical analysis was done by One Way Analysis of Variance (ANOVA) using EPI INFO 6.</p></sec><sec id="st3"><title>Results:</title><p>In Group I, the maximum (max.) P100 latency of 98.8 &#x000b1; 4.7 and the max. P100 amplitude of 10.05 &#x000b1; 3.1 &#x003bc;V was obtained with checks of 90 minutes. In Group II, the max. P100 latency of 105.19 &#x000b1; 4.75 msec as well as the max. P100 amplitude of 8.23 &#x000b1; 3.30 &#x003bc;V was obtained with 15 minutes. The min. P100 latency in both the groups was obtained with checks of 120 minutes while the min. P100 amplitude was obtained with 180 minutes. A statistically significant difference was derived between means of P100 latency for 15 and 30 minutes with reference to its value for 120 minutes and between the mean value of P100 amplitude for 120 minutes and that of 90 and 180 minutes.</p></sec><sec id="st4"><title>Conclusion:</title><p>Altering the size of stimulus (visual angle) has an effect on the PRVEP parameters. Our study found that the 120 is the appropriate (and optimal) check size that can be used for accurate interpretation of PRVEPs. This will help in better assessment of the optic nerve function and integrity of anterior visual pathways.</p></sec>

<contrib contrib-type="author"><name><surname>Kothari</surname><given-names>Ruchi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Smita</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Ramji</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Shukla</surname><given-names>A. K.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Bokariya</surname><given-names>Pradeep</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Visual system is well adapted to the processing and coding of pattern information. Human visual cortical activity as measured by visual evoked potentials (VEP) is highly sensitive to the sharpness and density of contours of the evoking stimulus.</p><p>The preferred stimulus for VEP is a checkerboard pattern of black and white squares. The patterned stimuli are widely preferred because response to a pattern is much larger and bears a closer relationship to the act of seeing. Checks help to explore the function of striate cortex (area 17) because local spatial frequency analyzers are presumably present there.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Since one of the primary functions of human visual system is to analyze contours and edges, the use of patterned stimuli seems to have an added advantage in providing more information in this regard.</p><p>The size of the individual checks is usually reported in terms of visual angle in minutes of arc (&#x003b2;) which is expressed as &#x003b2; = tan^-1(W/2D) &#x000d7; 120 where W is width of checks in millimeters and D is distance of the pattern from the corneal surface in mm.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>It is already known that retina can be divided into central foveal, para-foveal, and peripheral region. The fovea subtends 5&#x000b0; of visual angle while para-foveal area subtends 8&#x000b0;. Smaller size of pattern elements is thought to be optimal for foveal stimulation and larger sized patterns stimulate both fovea and extra-foveal region. Thus, by selecting the appropriate pattern element size one can predominantly stimulate fovea or peripheral retina. So when a checkerboard pattern is used, by altering one of its most important attribute that is the visual angle, one can better understand the mechanisms of visual processing.</p><p>The influence of altered visual angle on the latency and amplitude of major positive component of pattern reversal visual evoked potentials (PRVEP) and other components namely, N75 and N145 is not well-understood and there is scanty data on how and to what extent they are modified so we made a systematic study of effects of different check sizes in terms of visual angle on the latencies and amplitude of the parameters of PRVEPs.</p><p>For this we performed an evaluation of visual evoked potential to pattern reversal stimulation (VEP-PR) in two groups of healthy subjects with normal visual acuity at baseline. The purpose of this study was to determine the visual angle which is the optimum and most appropriate among a wide range of check sizes for the reliable interpretation of PRVEPs and accurate assessment of optic nerve function and integrity of anterior visual pathways.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>Materials and Methods</title><p>The study population consisted of 40 healthy volunteers consisting of 19 males and 21 females. They were divided into two groups. One group consisted of 20 individuals (nine males and 11 females) in the age range of 25-57 years and they were exposed to checks subtending a visual angle of 90, 120, and 180 minutes of arc. Another group comprised of 20 individuals (10 males and 10 females) in the age range of 36-60 years and they were subjected to checks subtending a visual angle of 15, 30, and 120 minutes of arc. Each of the subjects was given thorough eye examination as a preliminary measure to exclude any ocular pathology. No subject had a history of relevant neurological or heart disease or of drug abuse.</p><sec id="sec2-1"><title>Inclusion Criteria</title><p> <list list-type="bullet"><list-item><p>Visual acuity at least 6/6 (with or without corrective glasses)</p></list-item><list-item><p>Normal pupillary size (2-4 mm) and reactions</p></list-item><list-item><p>Normal Fundus and optic disc</p></list-item><list-item><p>Intra-Ocular pressure &#x0003c; 21 mm Hg (as measured by the Non contact tonometer).</p></list-item></list> </p><p>This project was approved by the Institutional Ethics committee and written informed consent was taken from the volunteers before the study.</p></sec><sec id="sec2-2"><title>Stimulus</title><p>The stimulus configuration comprised of the transient pattern reversal method in which a black and white checker board is generated (full field) on a VEP Monitor (colour 14&#x0201d;) by an electronic pattern regenerator inbuilt in an Evoked Potential Recorder (RMS EMG.EP MARK II). The rate of pattern reversal (1.7 Hz), the luminance (59 cd/sqm) and contrast level (80%) was kept constant for all the recordings in all the cases.</p><p>The different sizes of the checks used were 15, 30, 90, 120, and 180 minutes of arc.</p><p>Checks of 8 &#x000d7; 8 subtending a visual angle of 120 minutes are the most commonly used in the neurophysiological laboratories, so it was kept as a reference for evaluation of the effects on VEP parameters. To determine the impact of check size on neuromagnetic visual cortical responses, visual evoked fields to pattern-reversal stimulation with central occlusion in ten subjects were recorded in the past.[<xref rid="ref4" ref-type="bibr">4</xref>] It was reported that magnitude of cortical activation during visual contrast processing is check size-dependent and the 120 minutes checks are optimum for studies on neuromagnetic visual cortical functions using central-occluded stimulation. The corresponding neuronal activation demonstrated a short refractory period less than 0.16 s. This also supports our contention of keeping 120 minutes visual angle as a reference.</p><p>Standard disc electro encepgalogram (EEG) electrodes were placed on the scalp areas after preparing the skin by degreasing and abrading with a conducting jelly or electrode paste (RMS recording paste) rubbed lightly into the area with a cotton swab. The standardized methodology as recommended by the International Federation of Clinical Neurophysiology (IFCN) Committee and International Society for Clinical Electrophysiology of Vision (ISCEV)[<xref rid="ref5" ref-type="bibr">5</xref>] was followed. As per 10-20 International System of EEG placements, the reference electrode (Fz) was placed 12 cm above the nasion, the ground electrode (Cz) at the vertex and the active electrode (Oz) at approximately 2 cm above the inion. The electrode impedance was kept below 5 K&#x003a9;.</p><p>The recording was done in a quiet, darkened room with a constant temperature (27-30&#x000b0;C) in the Neurophysiology unit.</p><p>The subjects were seated comfortably at a distance of one meter away from the screen of the VEP monitor. They were exposed to full-field monocular stimulation for the left and right eyes separately with the subjects wearing corrective glasses, if any during the test. The signals recorded were filtered (low cut and high cut frequency filter) through a band spread of 2 &#x02212; 100 Hz. The sensitivity was kept at 2 &#x003bc;V. The sweep duration was maintained at 300 ms. Responses to 200 stimuli were amplified and averaged for each eye, which were then analyzed by inline computer having automatic artifact rejection mechanism. An average of two trials with well-defined PRVEPs were obtained for all check sizes in all subjects.</p><p>The typical VEP elicited by a pattern reversal is a negative- positive-negative complex that is recorded maximally in the mid-occipital region with reference to mid-frontal region. The components of VEP are termed as N75, P100, and N145 to indicate their polarity and approximate latency (in msec.). The absolute latencies of the peaks of positive wave P100 and the negative waves N75 and N145 were recorded along with the peak to peak amplitude of N75-P100.</p><p>The influence of visual angle on latencies and amplitude of PRVEP components was assessed by One Way Analysis of Variance (ANOVA) using the statistical software EPI INFO 6. <italic>P</italic> &#x0003c;0.05 was taken to represent a significant difference.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>Results</title><p>A total of 80 eyes of 40 subjects were investigated for full field pattern reversal VEPs with visual stimuli as checkerboard patterns with checks of 5 visual angles-15, 30, 90, 120, and 180 minutes of arc. The mean age of subjects in Group I was 46.5 &#x000b1; 6.77 years (range 25-57 years). The mean age of subjects in Group II was 47.05 &#x000b1; 9.01(range 36-60 years).</p><p>The effect of visual angle on P100 waveform of the two groups under study has been represented in [Tables <xref ref-type="table" rid="T1">1</xref> and <xref ref-type="table" rid="T2">2</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Influence of visual angle on P100 waveform of Group I subjects (<italic>n</italic>=40 eyes)</p></caption><graphic xlink:href="OJO-7-120-g001"/></table-wrap><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Influence of visual angle on P100 waveform of Group II subjects (<italic>n</italic>=40 eyes)</p></caption><graphic xlink:href="OJO-7-120-g002"/></table-wrap><p>In Group I, the maximum mean P100 latency of 98.8 &#x000b1; 4.7 (with a range of 90.30&#x02013;109.30 msec) was obtained with checks of 90 minutes and the minimum mean P100 latency of 97.51 &#x000b1; 4.26 msec (with a range of 89.10&#x02013;107.85 msec) was observed with checks of 120 minutes. Further, the maximum mean P100 amplitude of 10.05 &#x000b1; 3.1 &#x003bc;V (with a range of 3.37&#x02013;14.32 &#x003bc;V) was obtained with checks of 90 minutes and the minimum mean P100 amplitude of 6.38 &#x000b1; 2.52 &#x003bc;V (with a range of 3.10 &#x02212; 12.86 &#x003bc;V) was obtained with checks of 180 minutes.</p><p>In Group II, the maximum mean P100 latency of 105.19 &#x000b1; 4.75 msec (with a range of 98.40-117.50 msec) was observed with checks of 15 minutes and the minimum mean P100 latency of 97.64 &#x000b1; 3.25 msec (with a range of 90.3-103.8 msec) was observed with checks of 120 minutes. The minimum mean P100 amplitude of 7.12 &#x000b1; 2.57 &#x003bc;V (with a range of 3.9-15.46 &#x003bc;V) was obtained with checks of 180 minutes and the maximum mean P100 amplitude of 8.23 &#x000b1; 3.30 &#x003bc;V (with a range of 3.69-15.55&#x003bc;V) was obtained with checks of 15 minutes.</p><p>The effects of visual angle on N75 and N145 have been tabulated in [Tables <xref ref-type="table" rid="T3">3</xref> and <xref ref-type="table" rid="T4">4</xref>]. In Group I, it was observed that the mean N75 latency was shortest with 67.66 &#x000b1; 5.15 msec (with a range of 58.75-78.10) for 180 minutes and the most prolonged duration of 70.42 &#x000b1; 4.26 msec (with a range of 60.90-78.10) for the visual angle 90 minutes. On the other hand, the mean N145 latency was found to be the longest with 138.62 &#x000b1; 9.60 msec (with a range of 125.30&#x02013;159.40) for 180 minutes checks and was the shortest with 134.27 &#x000b1; 10.88 msec (with a range of 116.30&#x02013;159.60) for 90 minutes checks.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Influence of visual angle on N75 and N145 of Group I subjects (<italic>n</italic>=40 eyes)</p></caption><graphic xlink:href="OJO-7-120-g003"/></table-wrap><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Influence of Visual angle on N75 and N145 of Group II subjects (<italic>n</italic>=40 eyes)</p></caption><graphic xlink:href="OJO-7-120-g004"/></table-wrap><p>In Group II, it was found that mean N75 latency was shortest with 68.02 &#x000b1; 4.53 msec (with a range of 60.00&#x02013;79.40) for 120 minutes and the most prolonged duration of 76.58 &#x000b1; 5.50 msec. (with a range of 75.00&#x02013;88.80) for the visual angle 15 minutes. Likewise, mean N145 latency was also found to be the longest with 143.34 &#x000b1; 7.61 msec (with a range of130.00&#x02013;159.10) for 15 minutes checks and was the shortest with 134.13 &#x000b1; 7.03 msec (with a range of 124.40-150.3) for 120 minutes checks.</p><p>On statistical analysis the difference between means of P100 latency for the visual angles 15 and 30 minutes with reference to its value for 120 minutes (8 &#x000d7; 8) was statistically highly significant (<italic>P</italic> &#x0003c; 0.001). When the mean value of P100 amplitude for 120 minutes (8 &#x000d7; 8) was compared with that of checks of 90 and 180 minutes, the difference of means was found to be statistically highly significant (<italic>P</italic> = 0.003). For the N75 latency, statistical significance (<italic>P</italic> = 0.02) was obtained for the difference in means of all the visual angles (15, 30, 90, 120, and 180 min. of arc). For N145 latency, the difference in means between 120, 15, and 30 minutes checks was statistically significant (<italic>P</italic> &#x0003c; 0.05).</p></sec><sec sec-type="discussion" id="sec1-4"><title>Discussion</title><p>The present work was conducted to study the effect of altered visual angle of the checkerboard pattern on the various components of PRVEPs.</p><p>Considerable evidence appears to support the notion that visual system processes information along multiple parallel channels. The optic tract starts from optic chiasma and terminates in the lateral geniculate body (LGB). From LGB, visual information is transmitted to striate area 17 via two principal pathways- Magnocellular or M pathway which is sensitive to low spatial frequency (large checks) and Parvocellular or P pathway more sensitive to high spatial frequency (small checks). Thus the specific range and degree of operation of each channel is a function of the size of the visual stimulus presented.</p><p>PRVEP is a very important non-invasive and highly objective tool in detecting abnormalities of visual system. It is useful not only for clinical neurophysiologist or ophthalmologist but also for neurologists and neurosurgeons, since many of the neurological disorders present with visual abnormalities. They may detect those abnormalities of the optic nerves which are poorly visualized by magnetic resonance imaging (MRI) and reflect subclinical involvement of the CNS even before the disease clinically manifests. The test is relatively inexpensive and can be repeated numerous times with high reliability. With proper understanding of its limitations and appreciation for its qualities, it will always remain one of the simple, harmless and invaluable tests to diagnosis abnormalities of the visual pathway.</p><sec id="sec2-3"><title>Clinical utilities of pattern reversal VEPs include the following</title><p> <list list-type="bullet"><list-item><p>Preferable in optic nerve lesions</p></list-item><list-item><p>More sensitive than MRI or physical examination in prechiasmatic lesions and very useful in detecting an anterior pre-chiasmatic visual conduction disturbance</p></list-item><list-item><p>Objective and reproducible test for optic nerve function</p></list-item><list-item><p>Abnormality persists over long periods of time</p></list-item><list-item><p>Inexpensive as compared with to MRI</p></list-item><list-item><p>Under certain circumstances, may be helpful to positively establish optic nerve function in patients with subjective complaint of visual loss; normal VEP excludes significant optic nerve disorder.</p></list-item></list> </p><p>PRVEPs to full-field stimulation are best suited to evaluate anterior visual pathways. The transient pattern VEP is a straightforward investigation which could be performed within 10-15 minutes in total and requires the patient to fixate for only about 30-60 seconds at any one time. It thus entails less cooperation than conventional kinetic or computerised static perimetry considered as a gold standard in detecting and monitoring diseases like glaucoma and therefore has distinct advantages with regard to that group of patients who have difficulty in performing a field investigation. It has therefore been suggested that VEP technique is a potentially useful tool in the early detection of functional deficits in diseases like glaucoma and its longitudinal assessment.</p><p>It has been well documented that VEP is primarily a reflection of activity originating in the central 2-6 degrees of visual field.[<xref rid="ref6" ref-type="bibr">6</xref>] It clearly implies that the conventional visual evoked potential response is dominated by the central macular response which is in agreement with the principal finding of our study that is the checks of 2&#x000b0; (120 minutes) produced the shortest P100 latency with substantially sound P100 amplitude in both the groups of our study. This observation is further indicative of optimal evoked response to a visual angle of 120 minutes.</p><p>Previous researchers[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] using different stimulation techniques have described an optimum check size for humans between 10-30 minutes of arc because maximum amplitude VEP was seen with checks which subtend visual angles in this range.[<xref rid="ref10" ref-type="bibr">10</xref>] According to them, when the check size becomes smaller or larger than this; the VEP shows a corresponding drop in amplitude.</p><p>In our present study also, for the visual angle of 15 minutes, a high value of mean P100 amplitude of 8.23 &#x000b1; 3.30 &#x003bc;V (with a range of 3.69-15.55) was observed. When the angle increased to 30 minutes from this value, a drop in P100 amplitude was obtained with the exception of an increase seen with the pattern of 90 minutes which showed maximum amplitude.</p><p>On the other hand it has also been reported that in some subjects the P100 amplitude continues to increase as check size increases and finally levels off.[<xref rid="ref11" ref-type="bibr">11</xref>] This is presumably thought to be due to &#x0201c;switchover&#x0201d; from contrast specific VEPs with small checks to luminance specific VEPs to large checks. Large checks may evoke small signals in some individuals. This appears to have some relationship to the inter-subject variability in luminance contributions to large checks.</p><p>Studies of adult pattern VEP have shown that the latency of major components varies as a function of pattern element size. The results have been quite variable and contradictory at times. VEPs with longer latencies using small checks have been observed by some[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>] whereas some have suggested that as check size increases above 30 minutes, the latency of P100 also increases.[<xref rid="ref10" ref-type="bibr">10</xref>] Contrary to this we have noticed a slight P100 delay as visual angle decreased from 120 upto 15 minutes.</p><p>However, our data is not in agreement with a study conducted to determine the optimum stimulus conditions for the detection of optic nerve damage using two check sizes (12 and 48 minutes). They found that the largest number of VEP abnormalities in terms of P100 delay and reduction in amplitude, were with the large checks.[<xref rid="ref14" ref-type="bibr">14</xref>]</p><p>But our observations are in consonance with a recently conducted study by Nakamura <italic>et al</italic>.[<xref rid="ref13" ref-type="bibr">13</xref>] on pattern reversal visual evoked magnetic field and potential. In that study, 7 healthy subjects were exposed to half-field stimuli with or without central occlusion with check sizes of 15, 30, 60, 90 and, 180 minutes of visual arc and simultaneously their pattern reversal visual evoked magnetic fields (VEF) and VEP were recorded. They have also reported that the latencies for the smaller checks were significantly longer than those for the larger checks.</p><p>The effect of visual angle using checks of 17, 35, 70, 144, and 288 minutes has been studied in the past where the values of P100 latency showed a U-shaped function with check size rather than a perfect linear relationship.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><p>In case of N75 latency, the present study reveals a linear rise (as per Statistical analysis by ANOVA) with corresponding decrease of visual angle from 180&#x02013;15 minutes. A reduction in check size was encountered with prolongation of N75 and P100 latency by some earlier workers[<xref rid="ref16" ref-type="bibr">16</xref>] but the relationship was not a linear one. The N75 latency was found by others[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] to have a significant relationship with a decrease in latency with increment of check size as we also have noted.</p><p>Modifications of the components of PRVEPs with changes in check size of the stimulating pattern had been studied in 11 healthy subjects by some previous workers where they have used 8 different check sizes ranging between 10 and 90 minutes of arc. They reported significant inverse linear relationship of latency and amplitude of N75 with the logarithm of the check size, while the P100 and N145 latencies showed significant curvilinear relationships, with minimal latencies at check sizes around 35 minutes.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>Another study[<xref rid="ref19" ref-type="bibr">19</xref>] on the effect of check size on PRVEPs in healthy adult volunteers revealed that as the check size increased, P100 amplitude together with N75 latency had a linear relationship. No significant relationships were found for P100 and N145 latencies. They concluded that N75 wave originated mainly from the activity of the foveola, whereas the more eccentric regions contribute more to the formation of P100, and the interaction of both regions elicited the N145 wave.</p><p>As to the N145 latency, one previous study[<xref rid="ref18" ref-type="bibr">18</xref>] revealed significant exponential decrement with an increase in check size again differing from our results. N145 latencies in our study displayed a parabolic relationship with visual angle. Maximum latency was observed for the pattern of 15 minutes and the second highest value was for 180&#x02032;. The U shaped modification of N145 latency implies presence of its spatial tuning and suggests that it originates in the cortex since visual cortical neurons possess this kind of feature.</p><p>Since the minimum mean P100 latency in both the groups of our study was observed with checks of 120 minutes and also considerable P100 amplitude was obtained with this check size, we consider this visual angle as the most suitable for optimal foveal stimulation. It further fosters our selection of this angle as the reference at our neurophysiology unit.</p></sec></sec><sec sec-type="conclusion" id="sec1-5"><title>Conclusion</title><p>It is evident from our study that the variation in visual angle subtended by the checks of the checkerboard pattern significantly influences the latency and amplitude of the PRVEPs. Investigation of the effect of altering the size of stimulus (visual angle) indicates that the best visual evoked responses are obtained when the central macular area of retina is stimulated. This can be achieved by employing an appropriate and optimal check size which we in our study have proposed to be of 120 minutes of arc. It would help in accurate interpretation of PRVEPs and better assessment of the optic nerve function and integrity of anterior visual pathways.</p></sec>

Spectral domain optical coherence tomography characteristics in diabetic retinopathy

<sec id="st1"><title>Purpose:</title><p>To report the appearance of diabetic retinopathy lesions using spectral domain optical coherence tomography (SD-OCT).</p></sec><sec id="st2"><title>Materials and Methods:</title><p>A total of 287 eyes of 199 subjects were included. All the subjects underwent complete ophthalmic examination including SD-OCT.</p></sec><sec id="st3"><title>Results:</title><p>The appearance of various lesions of diabetic retinopathy and the retinal layers involved were reported. In subjects with macular edema the prevalence of incomplete PVD was 55.6%.</p></sec><sec id="st4"><title>Conclusion:</title><p>SD-OCT brings new insights into the morphological changes of the retina in diabetic retinopathy.</p></sec>

<contrib contrib-type="author"><name><surname>Gella</surname><given-names>Laxmi</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Raman</surname><given-names>Rajiv</given-names></name><xref ref-type="aff" rid="aff2">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Rani</surname><given-names>Padmaja Kumari</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Tarun</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Diabetic retinopathy (DR) is a major cause of blindness in the working age group worldwide and remains one of the most serious complications of diabetes mellitus.[<xref rid="ref1" ref-type="bibr">1</xref>] Identification of early changes in cases of DR is important which can help in the diagnosis and management of disease. Newer imaging techniques like spectral domain optical coherence tomography (SD-OCT) with their better resolution are available which aid in assessment of morphological changes.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Ophthalmoscopy, fundus photography and fluorescein angiography (FA) are the common tools to diagnose DR and diabetic macular edema (DME). There is an increasing demand for high-resolution imaging of the ocular tissue to improve the early diagnosis and management of DR. This study aims to report the appearance of various lesions in DR using SD-OCT.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>Materials and Methods</title><p>Two hundred and eighty-seven eyes of 199 subjects were included in this study. The study sample includes subjects with diabetes mellitus with or without clinical evidence of DR and normal healthy subjects. This study was approved by institutional review board. Informed consent was obtained from all the subjects after explanation of the nature of the study and details of the procedure. All the subjects underwent a comprehensive eye examination and detailed fundus examination. The severity of DR was graded based on International Clinical Diabetic Retinopathy Disease Severity Scale.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>All the patients underwent SD-OCT (Copernicus, Optopol Technologies, Zawierci, Poland). The programs used for the present study were Asterisk scan and 3D scan protocols. For the purpose of this study we used a scan length of 7 mm with 6 B-scans and 3000 A-scans per B-scans through the center of the fovea for the asterisk scan protocol. 3D scan protocol was used with 7 mm scan length with 50 B-scans and 1000 A-scans per B-scan. The structural changes in cases of subjects with different stages of DR were analyzed on grey scale images based on hyper and hypo reflectivity and also the location of the lesions.</p><p>We classified the stages of posterior vitreous detachment (PVD) based on the SD-OCT findings. The absence of PVD was diagnosed when the posterior hyaloid was not detected on SD-OCT scan of the macula. Incomplete PVD was diagnosed when the posterior hyaloid remained partially attached to the macula on at least one SD-OCT scan. Complete PVD was diagnosed when posterior hyaloid is visible in the vitreous cavity without any attachment to the macula on all the scans on SD-OCT. Complete PVD was also confirmed with the indirect ophthalmoscopic examination if the posterior hyaloid echo was not visible in the SD-OCT scan.</p></sec><sec sec-type="results" id="sec1-3"><title>Results</title><p>The mean age of the study sample was 55.4 &#x000b1; 8.9 years. The mean duration of diabetes mellitus from diagnosis was 112.1 + 76.6 months. <xref ref-type="table" rid="T1">Table 1</xref> shows the quantitative parameters of various lesions in different stages of DR. Most frequently found lesions in SD-OCT are hard exudates and hemorrhages followed by cystic spaces.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Distribution of lesions in eyes with various stages of DR</p></caption><graphic xlink:href="OJO-7-126-g001"/></table-wrap><p><xref ref-type="table" rid="T2">Table 2</xref> shows qualitative parameters of these lesions in terms of SD-OCT appearance and layers involved. Four morphological patterns of hemorrhages seen on SD-OCT were oval-shaped hyperreflective echos surrounded by hyporeflectivity, medium to high reflective echos causing shadow in the inner retina, high reflective echos at retinal nerve fiber layer and ganglion cell layer causing shadow in case of flame-shaped and organized high reflective membrane at vitreoretinal interface causing shadow in case of subhyaloid hemorrhage.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Appearance of diabetic retinopathy lesions on SD-OCT and layers involved</p></caption><graphic xlink:href="OJO-7-126-g002"/></table-wrap><p>Prevalence of incomplete PVD was more in subjects with DR [<xref ref-type="fig" rid="F1">Figure 1</xref>]. In subjects with macular edema the prevalence of incomplete PVD was higher i.e, 55.6% and the no PVD was found in 44.4% and we did not see any cases in macular edema with complete PVD.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>PVD status in various stages of diabetic retinopathy</p></caption><graphic xlink:href="OJO-7-126-g003"/></fig></sec><sec sec-type="discussion" id="sec1-4"><title>Discussion</title><p>In our study we reported appearances of the DR lesions in SD-OCT. Microaneurysms are saccular outpouchings from the sides of cellular capillaries, both on the arteriolar and the venular side. On SD-OCT, they appear as very small medium to high reflective echos causing minimal shadow in the inner retinal layers [<xref ref-type="fig" rid="F2">Figure 2a</xref>]. Hard exudates are lipoprotein deposits and these appear as very high reflective echos [<xref ref-type="fig" rid="F2">Figure 2b</xref>] on SD-OCT which cast a shadow on the posterior layers making us difficult to visualize the underlying layers. These hard exudates are present in inner and outer, nuclear and plexiform layers.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Lesions on SD-OCT. (a) Microaneurysm, (b) hard exudates causing shadowing, (c) cystic spaces</p></caption><graphic xlink:href="OJO-7-126-g004"/></fig><p>Cystic spaces appear as optically empty spaces [<xref ref-type="fig" rid="F2">Figure 2c</xref>] and these involve the layers from outer nuclear layer (ONL) to the ganglion cell layer (GCL). Histopathologic studies have suggested that the development of macular edema is initiated by fluid accumulation within Muller cells. If the accumulation continues, or remains chronic, then at some point death of the Muller cells occurs and may result in the formation of large cystoid cavities, or cystoid macular edema.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Hemorrhages can be located pre-, intra- or subretinally. Various patterns of the hemorrhages can be observed by SD-OCT. In our study we observed 4 different patterns. Most commonly observed pattern was oval shaped hyper reflective echo surrounded by hypo reflective echo in the inner retinal layers causing shadow and this involves ONL, outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and GCL [<xref ref-type="fig" rid="F3">Figure 3a</xref>]. This pattern of appearance may be due to the resolving stage of hemorrhages. Another pattern was medium reflective echos causing shadowing in the inner retina. On ophthalmoscopy when the hemorrhages are present in the retinal nerve fiber layer (RNFL) they appear as flame shaped and in SD-OCT these appear as high reflective spots at the RNFL layer causing shadowing. These should not be misinterpreted as the normal blood vessels in the retina which also appear as high reflective echos in the RNFL causing shadowing. In the cases of subhyaloid hemorrhage, the SD-OCT shows the presence of a high reflective lesion consistent with blood which is localized and causing shadowing of the underlying retinal layers which is similar to the findings reported in the case reported by Punjabi <italic>et al</italic>.[<xref rid="ref5" ref-type="bibr">5</xref>] Due to the collection of blood in the most dependent position of the subhyaloid space with the gravity, the horizontal level of the blood is seen as the straight line in SD-OCT, beyond which it causes shadowing on the inner retinal layers. In <xref ref-type="fig" rid="F3">Figure 3b</xref> small arrow shows a thin bright line above the internal limiting membrane that approximates the organized blood. This could possibly be fibrous tissue that forms a capsule around the blood, but the exact nature of this bright band is unknown.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Lesions on SD-OCT. (a) Intraretinal hemorrhage, (b) pre-retinal hemorrhage causing shadow, (c) cotton wool spots</p></caption><graphic xlink:href="OJO-7-126-g005"/></fig><p>On SD-OCT cotton wool spots appear as hyperreflective, nodular or elongated lesion in the RNFL [<xref ref-type="fig" rid="F3">Figure 3c</xref>] and also involving the GCL, which cast shadow on the posterior layers. This appearance is assumed to reflect focal swelling of the nerve fibers,[<xref rid="ref6" ref-type="bibr">6</xref>] which is due to intracellular fluid and organelles accumulated secondary to interrupted axoplasmic flow.</p><p>Only in two eyes the SD-OCT was able to pick up the characteristic appearance of capillary non-perfusion (CNP) area. SD-OCT showed a thinning of inner retinal layers [<xref ref-type="fig" rid="F4">Figure 4a</xref>] than the normal retina. This might be due to the reason that the inner retinal layers may be particularly at risk to hypoxic insult because they are supplied with oxygen from the retinal vasculature, which is relatively sparse compared with the choroidal circulation, which supplies most of the outer retina.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Lesions on SD-OCT. (a) Neovascularization, (b) thinning of retinal layers in capillary non-perfusion area</p></caption><graphic xlink:href="OJO-7-126-g006"/></fig><p>Proliferative DR is characterized by either neovascularization of the disc or elsewhere. Preretinal neovascularization on SD-OCT can be detected as a preretinal high reflective membrane occasionally causing shadow [<xref ref-type="fig" rid="F4">Figure 4b</xref>]. This appearance is due to the presence of some amount of fibroglial tissue.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p>SD-OCT brings new insights into morphological changes of the retina in DR. It enhances the ability to exactly identify the epiretinal membranes (ERM), vitreomacular traction and the posterior hyaloid status. In our study incomplete PVD i.e, with macular attachment was the most frequent condition found in subjects with macular edema (55.6%) which was similar to the study results of Gaucher <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] On SD-OCT, the posterior hyaloid was thin and minimally reflective. The high prevalence of incomplete PVD might have been induced by the DME itself. The breakdown of inner blood-retinal barrier in cases of DME might have lead to the accumulation of cytokines or other mediators in the posterior vitreous cortex, which might have triggered the PVD. Another reason might be that the incomplete PVD with macular attachment may directly contribute to the development of DME.</p><p>In subjects with DR, secondary ERM can develop due to the epimacular proliferating fibroglial tissue. It appears as a hyperreflective band on the inner surface of the retina. In the starting stage it has a global retinal adherence later it causes tractional force on the retina leading to macular edema and due to it discontinuous attachment to the retinal surface causes internal limiting membrane folds.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>Conclusion</title><p>Structural abnormalities in diabetic retinopathy can be detected and characterized by SD-OCT. It compliments FA in understanding the pathology and managing it accordingly.</p></sec>

Outcome of rhegmatogenous retinal detachment repair by scleral buckling: The experience of a tertiary referral center in Scotland

<sec id="st1"><title>Purpose:</title><p>The primary aim of this study is to report the outcome of patients with rhegmatogenous retinal detachment (RRD) who underwent scleral buckling (SB) surgery.</p></sec><sec id="st2"><title>Methods:</title><p>This is a retrospective noncomparative case series study of all patients who underwent RRD repair by primary SB between March 2008 and February 2009. Patient demographics, visual outcome, complications, and failure rates were identified and recorded.</p></sec><sec id="st3"><title>Results:</title><p>A total of 65 patients underwent RRD repair by SB, with a mean age of 44.44 years. Results showed that the primary outcome (primary anatomical success following index surgery) was 90.77%, while the secondary outcome (anatomical success following repeat surgery) was 98.46%.</p></sec><sec id="st4"><title>Conclusion:</title><p>The study showed a high-success rate of SB in phakic eyes both in terms of postoperative best-corrected visual acuity and complication rates. We recommend the continued use of this technique in selected cases of RRD.</p></sec>

<contrib contrib-type="author"><name><surname>Shankar</surname><given-names>Vikas</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Lim</surname><given-names>Lik Thai</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ah-Kee</surname><given-names>Elliott Yann</given-names></name><xref ref-type="aff" rid="aff2">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Hammer</surname><given-names>Harold</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Rhegmatogenous retinal detachment (RRD) is primarily treated either by external approach with scleral buckling (SB), pars plana vitrectomy (PPV) or both. SB has been falling out of favor over the last two decades and is quickly becoming a lost art of vitreoretinal surgery. At present, the trend is toward PPV with advances such as small-incision techniques and wide-field viewing systems. In this study, we looked at the results of SB procedures performed at a tertiary center for treatment of RRDs. The main objective of the study was to study the outcome of RRD repair using the SB technique. We also sought to compare the surgical outcome with national standards and to identify the causes of failure and complications.</p></sec><sec sec-type="methods" id="sec1-2"><title>Methods</title><p>This is a retrospective noncomparative case series study of the outcomes of SB technique for RRD repair performed from 1^st March 2008 to 28^th February 2009 at Gartnavel General Hospital, Glasgow. Inclusion criteria included RRD requiring surgery using SB. Patients with any previous RRD surgery, pseudophakic patients, aphakic patients, patients with giant retinal tears, posterior breaks, proliferative vitreo-retinopathy (PVR) grade C, retinal detachment (RD) with vitreous hemorrhage, RD with choroidal detachment and patients who had a history of strabismus surgery were excluded. The study included mostly adults with the age of presentation ranging from 15 to 75 years with a mean age of 47.44 years. Cases were identified from the operating theatre logbook at Gartnavel General Hospital. Case notes were then retrieved with the help of the medical records department. Patient demographics, visual outcome, complications and failure rates were identified and recorded. The data collection sheet was completed, and results analyzed. All the patients had documented posterior vitreous characterized by a Weiss ring detected on fundoscopy.</p></sec><sec sec-type="results" id="sec1-3"><title>Results</title><p>The total number of primary SB repair surgery performed during the 12-month period was 65 patients of which 38 were female. The age of presentation varied from 15 to 75 years with a mean age of 47.44 years. The right eye was involved in 40 patients. Five surgeons were involved with Surgeon 1 performing 30 surgeries. The duration of symptoms ranged from 1 day to 3 months. Three patients presented within 24 h, 12 within 1-2 days, 18 within 3-7 days, while 32 presented with symptoms of &#x0003e;7 days duration. Forty-three patients (66%) underwent surgery within 2 days of the presentation. Twenty-two patients had surgery after 3 or more days. The maximum waiting time for surgery was 6 weeks because the patient had a previous operation on the other eye for RRD. More than half of the patients were myopes (54%), of which 23% were high myopes (&#x0003e;&#x02212;6 D) (<italic>n</italic> = 15) and 31% were low myopes (<italic>n</italic> = 20), emmetropia in 24 (37%) and hypermetropia in 6 (9%). History of trauma was elicited in four patients including one patient who was a rugby player presenting with retinal dialysis. Treated RD in the fellow eye was seen in three patients. Presenting symptoms are summarized in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Presenting symptoms</p></caption><graphic xlink:href="OJO-7-130-g001"/></table-wrap><p>The presenting visual acuity (VA) was 6/6 in 16 (24.67%) patients, 6/9-6/18 in 25 (38.5%) patients, 6/24-6/36 in 8 (12.3%) patients, 6/60 and less in 13 patients, hand movements (HMs) in three patients. Two patients were amblyopic in the affected eye with VA 6/60 and counting fingers.</p><p>Of the 41 patients with VA better than 6/18, 37 patients had macula-on RD at presentation. Seven patients had macula-on RD by ultrasound, in which the detached retina was folded over the front of the macula. The rest (32%) had macula-off RD (<italic>n</italic> = 21). All the patients who underwent an operation were phakic. Sixteen patients (25%) had retinal dialysis on presentation. The refractive status of most of the patients with dialysis was emmetropia (<italic>n</italic> = 11) with the rest having low myopia (<italic>n</italic> = 5). The RDs resulting from dialysis was found in 16 cases (24.6%) and those resulting from tears/breaks in 21 cases (32.3%), while holes were responsible in 28 cases (43.1%). Two patients had suspicious breaks, but no clearly defined breaks/holes were identified. It was interesting to note that there is area specific predilection for the tears, dialysis and holes in this series of RD, highlighted in Figures <xref ref-type="fig" rid="F1">1</xref>&#x02013;<xref ref-type="fig" rid="F3">3</xref>. Moreover, some patients had more than one quadrant involved.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Frequency of quadrant involvement in tears/breaks (UT = upper temporal; UN = upper nasal; LT = lower temporal; LN = lower nasal; RE = right eye; LE = left eye)</p></caption><graphic xlink:href="OJO-7-130-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Frequency of quadrant involvement in holes</p></caption><graphic xlink:href="OJO-7-130-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Frequency of quadrant involvement in dialysis</p></caption><graphic xlink:href="OJO-7-130-g004"/></fig><p>General anesthesia was used in 64 patients while one patient with near term pregnancy was done under local (sub-tenon) anesthesia. The variations in surgical technique used are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Variations in surgical techniques used</p></caption><graphic xlink:href="OJO-7-130-g005"/></table-wrap><p>The buckles used were mostly segmental in 59 patients, and radial buckles were performed in five, while one patient had a circumferential buckle. All buckles were made of silicone. Prophylactic treatment with cryotherapy in the fellow eye was required in 15 patients for suspicious areas, while one patient had bilateral detachment. The buckles when placed radially were at 10 o&#x02019;clock, 11 o&#x02019;clock, and 2 o&#x02019;clock hours in three patients. The choice of buckles used, is shown in <xref ref-type="table" rid="T3">Table 3</xref>.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Choice of buckles used</p></caption><graphic xlink:href="OJO-7-130-g006"/></table-wrap><p>The intraoperative complications included vitreous hemorrhage (<italic>n</italic> = 1), sub-retinal hemorrhage, while draining sub-retinal fluid (SRF) (<italic>n</italic> = 4), choroidal hemorrhage (<italic>n</italic> = 1), and tyre displaced posteriorly (<italic>n</italic> = 1). Fifty-eight patients had no complications intraoperatively [<xref ref-type="table" rid="T4">Table 4</xref>].</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Complications of SB surgery</p></caption><graphic xlink:href="OJO-7-130-g007"/></table-wrap><p>Postoperative complications included raised intraocular pressure (<italic>n</italic> = 4), re-detachment (<italic>n</italic> = 6) and PVR grade C with vitreous hemorrhage and new breaks in one patient. Epiretinal membrane developed in two patients. Residual SRF at macula was observed in two patients [<xref ref-type="table" rid="T4">Table 4</xref>]. Squint (hypertropia) developed in one patient, but was successfully treated with Fresnel prism and subsequently resolved after 2 months.</p><sec id="sec2-1"><title>Follow-up</title><p>Postsurgical follow-up is vital in that it ensures that the retina is in place and at the same time, it also helps to identify unsuccessful cases for repeat surgery. Anatomical success was defined as a reattached retina at the last postoperative visit. The shortest postoperative follow-up period was 2 weeks, while the longest postoperative follow-up period was 6 months. Forty-two patients were followed-up for 2 months after which they were discharged. Eleven patients were discharged at 3 months. Six patients were followed-up for 4-6 months. The postoperative best-corrected VA (BCVA) was included for 58 patients and is shown in <xref ref-type="table" rid="T5">Table 5</xref>.</p><table-wrap id="T5" position="float"><label>Table 5</label><caption><p>Postoperative corrected visual acuity of follow-up patients</p></caption><graphic xlink:href="OJO-7-130-g008"/></table-wrap><p>The follow-up results excluded the six patients who underwent repeat surgery. One patient failed to attend for further follow-up and another one attended follow-up in another hospital.</p><p><xref ref-type="fig" rid="F4">Figure 4</xref> shows the presenting VA, and VA after one successful operation and subsequent repeat surgery for those who were unsuccessful after the first operation. Out of the 65 patients, six needed repeat surgery with vitrectomy for persistent RD. Five of the six patients had good visual outcome and only one developed PVR.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Graph illustrating the presenting visual acuity, and visual acuity after one successful operation and subsequent repeat operations for those who were unsuccessful after the first operation</p></caption><graphic xlink:href="OJO-7-130-g009"/></fig></sec><sec id="sec2-2"><title>Repeat surgery</title><p>Six patients who underwent repeat surgery for the persistent detachment were aged between 15 and 63 years, with a mean age of 35.16 years. All patients were operated within 6 days of presentation. Four of the six patients were emmetropic, while the other two were low-myopes. The presenting VA was 6/9-6/18 in three patients, 6/24-6/36 in two patients, and HMs in one patient. Four of the six patients had retinal dialysis (3 in 7-8 o&#x02019;clock). One patient had retinal holes (3 holes 6-8 o&#x02019;clock) and another patient had a horseshoe retinal tear (10 o&#x02019;clock). Two patients had macula-off RD, while three were macula-on RD with the retina folded in front of the macula. The final visual outcome after repeat surgery was 6/6 in two patients, 6/9 in one patient, while two patients achieved 6/12 VA improvement. The patient who had a horseshoe retinal tear did not attend for follow-up.</p></sec></sec><sec sec-type="discussion" id="sec1-4"><title>Discussion</title><p>Scleral buckling is a very effective procedure in selected cases of RRD such as in uncomplicated phakic patients. Unfortunately, this technique is performed less frequently due to the introduction of PPV in early 1970s. However, SB has multiple advantages over PPV, including reducing the risk of cataract formation and endophthalmitis. It has faster visual rehabilitation compared to PV, which requires intra-vitreal gas or silicone oil.[<xref rid="ref1" ref-type="bibr">1</xref>] Furthermore, in certain cases like retinal dialysis, SB is the treatment of choice, with a better prognosis and success rate.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>However, SB technique is more difficult to learn and teach compared to PV. It is also demands more surgical expertise compared to PPV, which has the advantage of intraoperative visualization of retinal breaks.</p><sec id="sec2-3"><title>Success</title><p>In our study, the primary outcome (primary anatomical success following index surgery) is far better than that reported in the national average, although the secondary outcome (anatomical success following repeat surgery) is more or less the same as that reported by the national average.[<xref rid="ref3" ref-type="bibr">3</xref>] The postoperative BCVA was 6/6 in 37 patients (56%). Thirteen patients achieved 6/9-6/18, while four had VA between 6/24 and 6/36. Thus, 76.9% patients achieved a VA better than 6/18 postoperatively after the primary procedure. The VA of &#x0003c;6/60 was seen in three patients. Of those, two had preexisting amblyopia. One patient was left with VA of HM only. Moreover, one patient failed to follow-up, while six patients required repeat surgery.</p></sec><sec id="sec2-4"><title>Postoperative complications</title><p>In our study, only one patient experienced double vision (1.5%) due to exotropia. This was treated conservatively by prism and resolved in 2 months. In a study of 821 patients who underwent SB, 12 developed diplopia lasting &#x0003e;3 months. Six of the SB operations were vertical, three horizontal, and three oblique. Examination of the 12 patients showed seven cases of hypertropia, two cases of hypertropia associated with esotropia, one case of hypertropia with exotropia, and two cases of exotropia. The SB was removed in all 12 patients; binocular single vision was restored in six cases. Secondarily, prism correction restored binocular vision in three additional patients. Strabismus surgery was necessary for the remaining three patients.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Buckle-related complications following surgical repair of retinal dialysis has been reported in a study of 28 cases. Anatomical success was achieved with a single procedure in 26 cases (92.9%). Compared to our study, the primary success rate for related cases was lower at 75%. However, the secondary success rate is almost similar.[<xref rid="ref2" ref-type="bibr">2</xref>] Postoperative complications were seen in 20 cases (71.4%), with complications attributable to the buckle noted in 19 cases (67.9%).[<xref rid="ref2" ref-type="bibr">2</xref>] Buckle-related complications included exposure (<italic>n</italic> = 7; 25%), strabismus (<italic>n</italic> = 5; 17.9%), and infection (<italic>n</italic> = 3; 10.7%).[<xref rid="ref3" ref-type="bibr">3</xref>] In contrast, the complications attributable to SB in our study, were minimal with only one patient developing a strabismus, which was treated with prisms. None required removal of buckle, although the follow-up of the patient is shorter.</p><p>The case has been reported of a patient with visual field defect after retinal reattachment using the encircling procedure for RRD. However, it improved after relaxation of the buckle, which restored the ocular blood flow.[<xref rid="ref5" ref-type="bibr">5</xref>] The report suggested that the choroidal circulation disturbance, which was found following the encircling procedure, had a plausible role in the development of the visual field defect.[<xref rid="ref5" ref-type="bibr">5</xref>] We did not notice any visual field related complications in our series.</p><p>Sub-macular fluid was seen in two patients (3%) in our case series, one of which resolved in the follow-up. The incidence, pattern, duration, and clinical consequences of persistent, localized sub-macular fluid after SB surgery for RD has been reported in the literature in a prospective observational cohort series.[<xref rid="ref6" ref-type="bibr">6</xref>] Ninety-eight patients had an optical coherence tomography (OCT) scan of the macula preoperatively and at 6 weeks postoperatively. Of the 98 patients recruited in the study, 54 (55%) had SRF on OCT 6 weeks after surgery. Fluid was associated with delayed visual recovery. Of those with SRF, 78% had persistent fluid at 6 months, and resolution of fluid took a median of 10 months and was associated with an improvement in vision.[<xref rid="ref6" ref-type="bibr">6</xref>] The study concluded that persistent SRF 6 weeks following SB, occurs in approximately half of patients. Furthermore, this may persist for many months and cause delayed visual recovery.[<xref rid="ref6" ref-type="bibr">6</xref>] In our case series, two patients had residual SRF, which was absorbed in the subsequent follow-up of 2 months.</p><p>A retrospective, noncontrolled case series study was conducted in 93 patients (93 eyes) to evaluate the outcome of patients who had previous macula-off RRD treated with SB.[<xref rid="ref7" ref-type="bibr">7</xref>] Patients with low-grade myopia (&#x0003c;&#x02212;6 D) regained significantly better mean postoperative VA as compared with high myopia (&#x0003e;&#x02212;6 D) and emmetropic eyes (0 to + 3 D) (ANOVA, <italic>P</italic> &#x0003c; 0.001). In this series, SRF drainage procedure did not affect postoperative visual outcome. Multivariate logistic regression analysis revealed that the duration of macular detachment was the only variable affecting the visual outcome.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>A study of eyes with primary, uncomplicated, macula-off RD repaired with SB, concluded that eyes achieve excellent postoperative VA if repaired within the first 10 days of macular detachment.[<xref rid="ref8" ref-type="bibr">8</xref>] Patient age did not significantly affect anatomical outcomes.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p>In a large case series of 186 eyes, 82% achieved retinal reattachment with one SB procedure and with a median final VA of 20/40 at 20 years of follow-up.[<xref rid="ref9" ref-type="bibr">9</xref>] An additional 30 eyes (13%) achieved retinal reattachment after one or more additional vitreo-retinal procedures, with a median final VA of 20/50. Eleven eyes (5%) developed RD at the 20-year follow-up examination, with a final VA in all eyes of no light perception. This study may serve as a basis for comparison with the long-term results of other surgical techniques used in the treatment of primary RRDs.[<xref rid="ref9" ref-type="bibr">9</xref>] Our success rate is comparable to the above study, as we have achieved success after one procedure of 90.77% and 98.46% after two procedures.</p><p>The National RD Audit, UK reported primary reattachment in 82% and the final reattachment as 91%.[<xref rid="ref10" ref-type="bibr">10</xref>] In a randomized controlled trial of 546 patients, favorable surgical outcomes for primary RRDs with SB alone was achieved in 97.8% of the patients.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>From the results of our study, we observed that there are few factors that may be contributing toward the failure of the minority of cases reported in this study. Patients with dialysis were noted to have a higher incidence of failure rate after first surgery. The sample size of our study is too small to draw a significant correlation between success rate and duration of symptoms of RRD. However, the latter appears to have some bearing on the overall success of a first RRD repair using the SB technique. Persisting RD will eventually develop into PVR, which can further reduce the chance of a successful first-time operation.</p></sec></sec><sec sec-type="conclusion" id="sec1-5"><title>Conclusion</title><p>Scleral buckling is still a very effective technique to treat selected cases of RD, and as such should not be a neglected skill for present and future vitreo-retinal surgeons, despite in the advent of advancing techniques and technology of PPV.</p></sec>

Bilateral familial vertical Duane Syndrome with synergistic convergence, aberrant trigeminal innervation, and facial hypoplasia

<p>A 5-year-old girl presented with bilateral familial vertical Duane retraction syndrome with alternating esotropia, elevation deficit, Marcus gunn phenomenon, and facial hypoplasia. Abnormal adducting downshoots on attempting abduction suggestive of a synergistic convergence were noted. Hypothesis suggests aberrant innervations or peripheral anatomic connections between inferior and medial recti.</p>

<contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Malvika</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Om Prakash</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Vohra</surname><given-names>Vishal</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Vertical Duane syndrome, a rare entity, has been described as limitation of the affected eye on elevation or depression associated with globe retraction and narrowing of the palpebral fissure.[<xref rid="ref1" ref-type="bibr">1</xref>] Synergistic convergence, described as simultaneous bilateral adduction on attempted gaze into the field of action of the lateral rectus muscles, and facial hypoplasia have yet not been described in association with vertical Duane.[<xref rid="ref2" ref-type="bibr">2</xref>]</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 5-year-old girl presented with bilateral drooping of upper eyelids and inward deviation of either eye, with occasional crossing, since birth. There was no history of delayed milestones or any prior systemic illnesses. No prior history existed of spectacle prescription, occlusion therapy or surgical correction. Her father, three paternal uncles, and grandmother also had similar drooping. Her siblings were unaffected.</p><p>Ocular examination revealed a moderate compensatory head posture (chin elevation, right face turn, and mild head tilt to the right was noted). Mild facial hypoplasia of the right side with flattening of the right cheek and a broad nasal bridge was also present [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Visual acuity was 6/9 in both eyes (oculus uterque, OU). Cycloplegic refraction showed +0.5 Diopter sphere OU with an acceptance of 6/6. Gross stereopsis was absent, confirmed on titmus fly test. In primary gaze, on prism bar cover test, a large angle alternating esotropia of &#x0003e;90 PD, with V pattern, was noted at 33 and 6 m. Nystagmus was absent.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Bilateral severe congenital ptosis in a 5-year-old girl with facial hypoplasia Convergence is normal</p></caption><graphic xlink:href="OJO-7-135-g001"/></fig><p>She had bilateral, severe upper lid ptosis of 5 mm with poor levator action (4 mm) and absent Bells phenomenon. MRD 1 and 2 was 2 mm and 5 mm, respectively OU. There was limitation of elevation of both eyes [<xref ref-type="fig" rid="F2">Figure 2</xref>]. The left eye showed mild down-shoot in abduction. The right eye assumed a downward and adducted position on attempted dextro-depression suggestive of synergistic convergence. Convergence was normal in the both eyes [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Narrowing of the palpebral fissure on both sides on adduction was noted [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The child also demonstrated Marcus Gunn jaw winking phenomenon on left side while chewing. Anterior segment, pupillary, and posterior segment examination were unremarkable. A diagnosis of bilateral familial vertical Duane with synergistic convergence, aberrant trigeminal innervation with bilateral severe complicated congenital ptosis with superior rectus weakness, and facial hypoplasia was made. Magnetic resonance imaging (MRI) brain and orbit was reported to be normal.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Nine gaze depiction of ocular motility shows limited elevation in both eyes. The right eye assumes a downward and adducted position on attempted dextrodepression suggestive of synergistic convergence whereas the left eye showed mild down-shoot in abduction</p></caption><graphic xlink:href="OJO-7-135-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Classic narrowing of palpebral aperture on adduction of either eye is seen, typical of bilateral Duane retraction syndrome</p></caption><graphic xlink:href="OJO-7-135-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>Vertical retraction syndrome or the vertical variant of Duane has long been described. DeRespinis <italic>et al</italic>. in 1993, in their review of patients with classic Duane retraction syndrome, reviewed this rare variation.[<xref rid="ref1" ref-type="bibr">1</xref>] They described these patients to exhibit some degree of limitation of the affected eye on elevation or depression associated with globe retraction and narrowing of the palpebral fissure. Although not specifically mentioned by the authors, this is presumed to be on horizontal eye movements since palpebral aperture changes occur normally with vertical eye movements.</p><p>Since then many cases of spontaneous or familial vertical retraction, mostly unilateral, have been described.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Combined horizontal and vertical retraction syndrome has also been reported where the classical horizontal restrictions and retraction are concurrently seen with the vertical variant.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] Familial and bilateral vertical retraction syndrome was first described by Khodadoust and von Noorden.[<xref rid="ref6" ref-type="bibr">6</xref>] The syndrome is usually unilateral and sporadic. However, numerous cases of familial transmission have been reported with most being bilateral secondary to an autosomal-dominant inheritance pattern.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Our patient presented with features of bilateral, familial vertical retraction with narrowing of palpebral apertures on adduction suggesting a diagnosis of Duane Syndrome. Although one may be tempted to consider congenital fibrosis of the extraocular muscles (CFEOM) to be the more likely diagnosis, features of globe retraction and palpebral aperture narrowing, point more in favor of Duane retraction. Association with blepharoptosis is seen here and has been frequently associated with Duane.[<xref rid="ref1" ref-type="bibr">1</xref>] Also association of Duane retraction syndrome is seen with facial hypoplasia which has only once been described.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Vertical retraction is also seen to be associated with atypical strabismus and various congenital abnormalities. Synergistic divergence is a well-established clinical condition in which abduction arises on attempted adduction, leading to simultaneous abduction of both eyes on lateral gaze. Synergistic Convergence, a very rare entity, is the opposite of Synergistic Divergence. It was first described by Kim <italic>et al</italic>. in a patient with congenital fibrosis of extra ocular muscles[<xref rid="ref2" ref-type="bibr">2</xref>], as bilateral deficit of abduction associated with simultaneous bilateral adduction on attempted gaze into the field of action of the lateral rectus muscles. Later Christina Pieh <italic>et al</italic>. reported such an occurrence as an isolated finding.[<xref rid="ref8" ref-type="bibr">8</xref>] Their patient had no globe retraction, ptosis, no extraocular muscle hypoplasia, and no family history to suggest the diagnosis of CFEOM or Duane as in our case. Aberrant innervation during embryogenesis was suggested as the underlying pathomechanism. It could be due to a pattern of aberrant innervation similar to that in Duane syndrome, which leads to synergistic divergence (type IV). However, instead of aberrant oculomotor nerve fibers, a mis-wiring of abducens motor neurons to the medial rectus muscle was suspected. If most of the abducens nerve fibers were misdirected to the medial rectus muscle, intended abduction would lead to adduction of the eye, and the co-contraction of the lateral and medial rectus muscles would result in globe retraction and eyelid fissure narrowing. An unusual congenital sixth-nerve palsy similar to congenital third-nerve palsies was considered as a cause of mis-wiring since congenital third-nerve palsies often occur with anomalous re-innervation and no other neurologic or systemic abnormalities. However, they also suggested that a misdirection of regenerated nerve fibers to a muscle originally innervated by a different cranial nerve is less probable than a primary aberrant abducens nerve innervation. Again in 2011, Jain <italic>et al</italic>. reported the existence of synergistic convergence as an ocular motor anomaly where on attempted abduction or on attempted horizontal gaze, both the eyes converge.[<xref rid="ref9" ref-type="bibr">9</xref>] It was reported in association with kyphoscoliosis and brain stem dysplasia in two sisters.</p><p>Our case has features of globe retraction evidenced on narrowing of palpebral aperture on adduction and elevation deficit suggestive of vertical variant of Duane syndrome. Rarity lies in bilateral and familial nature of the case. It is unique in being associated with synergistic convergence, which, to the best of authors&#x02019; knowledge, has not been reported to be associated with the Duane retraction syndrome, let alone being associated with the vertical variant. Also authors differ slightly in defining synergisitic convergence as adduction downshoots on attempted abduction resulting from aberrant connections (anatomic or innervational) between inferior rectus (IR) and medial rectus (MR) muscles. IR over action secondary to superior rectus weakness, results in heightened innervations on attempted abduction where depressing function of IR is most prominent. This is also translated to the MR muscle which hypothetically bears aberrant (anatomic or innervational) connections to inferior rectus. This results in synergistic convergence.</p><p>Recent reports including imaging findings revealed that an accessory extraocular muscle or a fibrous band appeared to cause restriction on elevation and globe retraction in their cases.[<xref rid="ref10" ref-type="bibr">10</xref>] We hypothesize that in our case, the deficiency of elevation was either due to bands in association with both inferior recti or aberrant innervations of medial rectus from branch of third nerve supplying inferior rectus. This can then result in adducting down shoots (field of action of medial rectus superimposed with inferior pull by inferior rectus) on attempted abduction (where vertical action of inferior rectus is highest). The reported case is atypical and the balance scale may bend toward a diagnosis of CFEOM with atypical findings of globe retraction on adduction. But the abnormal downshoots on attempted abduction and globe retraction are more commonly seen in Duane syndrome.</p><p>Synergistic convergence in itself is a very rare feature and a full understanding of its etiopathogenesis is beyond the scope of current literature reports. We recognize CFEOM and vertical Duane syndrome as being a continuum of extraocular muscle abnormalities and the diagnosis is an amalgamation of all relevant associations.</p></sec>

Resistant retinoblastoma in a 23-year-old patient

<p>Retinoblastoma is a very rare disease in adults. We are reporting a rare case of resistant retinoblastoma in 23-year-old patient. A 23-year-old male patient presented with loss of vision in the right eye over one-month duration. Examination showed an epiretinal membrane in the right macula in addition to a white mass located inferiorly and associated with vitreous seeds. The diagnosis of retinoblastoma was established. In order to save the patient's life and to preserve the eye and vision, he was treated with chemotherapy, focal therapy, and radioactive Iodine¹²⁵ plaque therapy. The tumor was resistant for treatment and recurred two years after plaque therapy, and enucleation showed well-differentiated retinoblastoma. Retinoblastoma may present in adults, and it was resistant to both chemotherapy and plaque radiation therapy in our case.</p>

<contrib contrib-type="author"><name><surname>Yousef</surname><given-names>Yacoub A.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Istetieh</surname><given-names>Jihad</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Nawaiseh</surname><given-names>Ibrahim</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Al-Hussaini</surname><given-names>Maysa</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Alrawashdeh</surname><given-names>Khalil</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Jaradat</surname><given-names>Imad</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Sultan</surname><given-names>Iyad</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib><contrib contrib-type="author"><name><surname>Mehyar</surname><given-names>Mustafa</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Retinoblastoma (RB) is the most common intraocular malignancy in childhood and infancy, and 90% of cases are diagnosed before the age of 5 years.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Since RB is rare in adults, it is not usual to be considered in the differential diagnosis of an intraocular mass in adults.[<xref rid="ref4" ref-type="bibr">4</xref>] Search of published peer-reviewed literature showed 26 reported cases of retinoblastoma presenting in adult age,[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] all of which were managed by enucleation [<xref ref-type="table" rid="T1">Table 1</xref>]. Herein we present a case of intraocular retinoblastoma in adult patient that was resistant to both chemotherapy and plaque radiation therapy and ended with enucleation.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Reported cases of retinoblastoma in adults</p></caption><graphic xlink:href="OJO-7-138-g001"/></table-wrap></sec><sec id="sec1-2"><title>Case Report</title><p>A 23-year-old Jordanian male patient, medically free, presented with gradual painless visual loss in the right eye associated with floaters for one month prior to presentation. His best corrected visual acuity (BCVA) was 20/400 in the right eye and 20/20 in the left eye. Slit lamp examination of the anterior segment was within normal limit and intraocular pressure was 16 mmHg in the right eye and 15 mmHg in the left eye. Fundus examination showed right inferotemporal white opaque retinal tumor associated with large feeding vessel, vitreous seeds within 3.0 mm from tumor margin, and an epiretinal membrane (ERM) at the temporal part of macula [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Left fundus exam was normal. B-scan ultrasonography showed solitary solid intraocular mass with central calcification (thickness = 3.5 mm) with no optic nerve invasion. Similarly, computed tomography (CT) revealed an inferior solid mass (3.5 &#x000d7; 9.5 &#x000d7; 3.5 mm) with intratumoral calcifications, located 6.0 mm from the optic disc with no signs of extra ocular extension [<xref ref-type="fig" rid="F1">Figure 1</xref>]. According to the clinical features, diagnosis of unilateral intra-ocular retinoblastoma; international intraocular retinoblastoma (IIRC) group C was established, and the patient was managed accordingly by six cycles (four weeks apart) of carboplatin 500 mg/m² on day one, vincristine 1.50 mg/m² on day one, and etopside 150 mg/m² on days one and two. Consolidating laser therapy (810 laser therapy) was applied after the second cycle. Post chemotherapy, the patient was followed with regular exam every four weeks and was treated with 810 laser therapy for the residual tumor as needed. Seven months post chemotherapy, plaque radiation therapy (Iodine¹²⁵) was applied for tumor progression associated with adjacent vitreous seeds [<xref ref-type="fig" rid="F2">Figure 2</xref>]. On follow up, the tumor was inactive but was associated with persistent retinal detachment and ERM. The tumor did not show any signs of growth over the 12-month period post plaque therapy; therefore, retinal detachment (RD) was fixed by pars plana vitrectomy (PPV). Intraoperatively, RD was persistent tractional RD with epiretinal membrane dragging over calcified tumor. RD was repaired by three port PPV and membrane peeling (no drainage) then filled with silicon oil, which was removed 6 months later. Cytopathologic examination of the fluid aspirated during the vitrectomy was negative for malignant cells. Four months post silicon oil removal, the retina was flat but the patient required cataract surgery. Twelve months after PPV, BCVA was counting finger closely with very hazy fundus view. B-scan showed new dome-shaped tumor suggestive of recurrence [<xref ref-type="fig" rid="F2">Figure 2</xref>]; therefore enucleation was done. Histopathology of the enucleated eye showed differentiated retinoblastoma, displaying Homer-Wright rosettes and Flexner-Wintersteiner rosettes with no choroidal or optic nerve invasion with no signs of previous retinoma [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Fundus photos of the right eye with retinoblastoma. Poor vision was secondary to the epiretinal membrane (ERM) in the macula (a), due to traction caused by white opaque retinal tumor inferior to the macula (b). The tumor was associated with seeds in the vitreous cavity (c), and had calcifications shown as hyperdensity on computed tomography (CT scan) (d)</p></caption><graphic xlink:href="OJO-7-138-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Tumor features after treatment. The tumor looks fibrotic with no activity 12 months after radioactive iodine125 plaque therapy (a), as well as 9 months after pars plana vitrectomy (PPV) (b). More than 12 months after PPV, B-scan showed new tumor growth (c). Histopathology study after enucleation showed well-differentiated retinoblastoma, displaying Homer-Wright rosettes and Flexner-Wintersteiner rosettes</p></caption><graphic xlink:href="OJO-7-138-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>The differential diagnosis of amelanotic intraocular tumor in adults includes amelanotic melanoma, metastasis, hemangioma, lymphoma, leukemia, endophthalmitis, panophthalmitis, and inflammatory diseases of retina as well as retinoma and retinoblastoma.[<xref rid="ref9" ref-type="bibr">9</xref>] Retinoblastoma is extremely rare in adults, and more than 90% of cases are diagnosed before the age of 5 years,[<xref rid="ref2" ref-type="bibr">2</xref>] making diagnosis of retinoblastoma in adults challenging.</p><p>Large opaque white tumors with vitreous seeds can be easily identified as retinoblastoma mainly when associated with calcifications detected by ultrasound and/or CT scan and fluorescein angiography finding of diffuse tumor hyperfluorescence and dilated retinal feeder vessels,[<xref rid="ref10" ref-type="bibr">10</xref>] but small solitary lesions may be difficult to diagnose clinically. Calcification, which is a characteristic of retinoblastoma in children, is less common in adult-onset retinoblastoma.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Retinoma is a benign retinal tumor that is characterized by translucent, grey elevated mass and is frequently associated with cottage cheese calcification and retinal pigment epithelium hyperplasia.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>] It carries RB1 gene mutation causing genome instability making it premalignant lesion[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref15" ref-type="bibr">15</xref>] that requires close observation due to the risk of malignant transformation to retinoblastoma, and genetic testing and counseling is needed if available. Retinoma was observed in 1.8-15.6% of patients with retinoblastoma.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>]</p><p>Our patient had ophthalmic features that are more consistent with RB than retinoma and include opaque white color of the tumor, absence of both retinal pigment epithelium hyperplasia and chorioretinal atrophy, diffuse hyperfluorescence in fluorescein angiography, and responsiveness to radiation therapy because unlike RB, retinoma does not change with radiation therapy.[<xref rid="ref17" ref-type="bibr">17</xref>] There is no documented normal fundus exam for our patient. Therefore, it was a possible theory that our patient had pre-existing retinoma that transformed to retinoblastoma due to accumulation of oncogenic mutations,[<xref rid="ref18" ref-type="bibr">18</xref>] but the pathology did not show any signs suggestive of previous retinoma in our patient. Of interest, 10 out of the 26 previously reported cases of retinoblastoma in adults were associated with retinoma [<xref ref-type="table" rid="T1">Table 1</xref>].</p><p>Even the tumor in our case was initially responsive to chemotherapy with focal therapy; residual tumor could not be eliminated without radiation therapy (Iodine¹²⁵ Radiaoactive Plaque). The tumor was very resistant even to radiation therapy and recurred after more than one year of stability, and therefore, enucleation could not be avoided.</p><p>This report highlights the fact that the presence of an amelanotic mass lesion in the fundus of an adult should raise the suspicion of retinoblastoma. Retinoblastoma in adults may be more resistant to therapy than retinoblastoma in children, and enucleation is recommended mainly if the other eye had good vision.</p></sec>

Paediatric choroidal neovascular membrane secondary to toxoplasmosis treated successfully with anti-vascular endothelial growth factor

<p>The purpose of this report was to evaluate the role anti-VEGF in management of CNVM secondary to ocular toxoplasmosis. Young female diagnosed as a case of bilateral ocular toxoplasmosis presented with complaints of diminution of vision in the right eye. Fundus examination showed an active CNVM adjacent to toxoplasmosis scar. In view of active CNVM, patient was administered intravitreal ranibizumab. A total of 2 injections of intravitreal ranibizumab were given. Fundus showed a scarred CNVM adjacent to the toxoplasma scar with no clinical signs of activity. Anti-VEGF therapy has been successfully used to improve visual and anatomical outcome in juxtafoveal (deleted subfoveal)CNVM secondary to toxoplasmosis.</p>

<contrib contrib-type="author"><name><surname>Mathur</surname><given-names>Gaurav</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>George</surname><given-names>Amala Elizabeth</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Sen</surname><given-names>Parveen</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Pediatric choroidal neovascular membranes (CNVM) are uncommon but important cause of visual impairment. These may be idiopathic or may be seen secondary to trauma, infection, inflammation or retinal dystrophies. The limited published data on their natural history, as well as delay in their presentation make the management of pediatric CNVMs difficult. There is no uniform consensus on the management of these cases because of lack of randomized or controlled clinical trials. CNVMs have been seen in patients with toxoplasmic chorioretinitis.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] These patients have been managed by observation, anti-parasitic and/or anti-inflammatory medication, laser photocoagulation, surgical excision or photodynamic therapy, with variable outcomes.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Very few reports have been published for treating inflammatory CNVM successfully with anti-vascular endothelial growth factor (anti-VEGF).[<xref rid="ref8" ref-type="bibr">8</xref>] We report a case of congenital toxoplamosis that developed CNVM and was successfully treated with anti-VEGF therapy in a 13 year old.</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 13-year-old female diagnosed to have bilateral toxoplasmosis presented to us with complaints of diminution of vision in the right eye since 5 days. On examination vision in the right eye was 6/15, N10 and in the left eye was 6/36, N10. Anterior segment examination revealed grade 1 cells in the right eye with no other obvious abnormality. Left eye anterior segment was within normal limits. Fundus examination of the right eye revealed a juxtafoveal scarred toxoplasmosis lesion with well-defined punched-out margins. There was a presence of subretinal hemorrhage with subretinal fluid at the foveal edge of the scar suggestive of CNVM. Fundus fluorescein angiography (FFA) revealed hyper fluorescence of the lesion at the edge of the scar with active leakage in late stages of the angiogram. Optical coherence tomography (OCT) also revealed a juxtafoveal CNVM superotemporal to fovea and subretinal fluid superior and subfoveal [Figures <xref ref-type="fig" rid="F1">1a</xref> and <xref ref-type="fig" rid="F2">2a</xref>]. Left eye fundus revealed a punched out toxoplasmosis scar at the macula with no signs of activity. In view of the active CNVM in the right eye the patient was advised intravitreal ranibizumab (RBZ) 0.05 ml under general anesthesia. The patient underwent the same along with antiparasitic treatment for toxoplasmosis chorioretinitis which included oral clindamycin 300 mg four times a day and oral sulfamethoxazole and trimethoprim twice daily dosage for 2 weeks along with oral steroids in tapering dose.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) FFA shows active leakage from the CNVM adjacent to toxoplasma scar (b) FFA shows persisting leakage from the CNVM at 2 month follow up (c) FFA shows scarred CNVM adjacent a toxoplasmosis scar with scar staining (d) FFA shows scarred CNVM with no active leakage</p></caption><graphic xlink:href="OJO-7-141-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>(a) OCT image along the vertical axis shows an active CNVM superior to fovea with surrounding sub-retinal hemorrhage. A small pigmentation is seen within the lesion causing shadowing. Sub-retinal fluid is seen at the fovea. Foveal depression is maintained (b) OCT image through the same location as the previous visit shows a partially regressed CNVM with minimal subretinal fluid and retinal thickening at the inferior edge of the lesion (c) Vertical OCT scan shows a well defined hyper reflective scarred CNVM with no evidence of activity (d) OCT scan shows scarring CNVM with complete resolution of SRF. Note the thinning of IS-OS junction at fovea</p></caption><graphic xlink:href="OJO-7-141-g002"/></fig><p>One month post intravitreal injection vision in the right eye improved to 6/12, N6. Anterior segment was within normal limits. Fundus examination showed reduced subretinal hemorrhage with regressing CNVM which was confirmed on OCT with decreasing subretinal fluid. FFA still showed active leakage from the CNVM [Figures <xref ref-type="fig" rid="F1">1b</xref> and <xref ref-type="fig" rid="F2">2b</xref>]. In view of the persisting active CNVM patient was administered a second dose of ranibizumab intravitreal injection and was followed up after a month. On her next visit, 1 month post second injection, vision in the right eye further improved to 6/9, N6. Fundus showed a scarred CNVM adjacent to the toxoplasma scar with resolving subretinal hemorrhage with no clinical signs of activity. OCT showed scarred a CNVM with no subretinal fluid [Figures <xref ref-type="fig" rid="F1">1c</xref> and <xref ref-type="fig" rid="F2">2c</xref>]. At her next follow up, 1 month later she had maintained stable vision with no signs of activity in the CNVM [Figures <xref ref-type="fig" rid="F1">1d</xref> and <xref ref-type="fig" rid="F2">2d</xref>]. On her last visit, 10 months post injection, vision in the right eye was 6/9, N6 and fundus showed a scarred CNVM. OCT showed a scarred subretinal lesion.</p><sec id="sec2-1"><title>Comment</title><p>Anti-VEGF therapy has been successfully used to improve visual outcome in patients with subfoveal and juxtafoveal CNVM. It reduces concomitant tissue destruction to the neurosensory retina and choroid as seen in other treatment modalities like laser photocoagulation. Because active infectious retinitis may be obscured by hemorrhage and because intravitreal injections may reactivate chorioretinitis, concurrent therapy with oral anti-toxoplamosis medicine is also recommended in these cases.[<xref rid="ref8" ref-type="bibr">8</xref>] Though photodyanamic therapy (PDT) has been shown to be effective by other authors[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] we did not advise it because cooperation during the procedure was questionable due to the younger age group of the patient. In our case only two injections resulted in resolution of the neovascular membrane and stabilization of visual acuity. The reduced number of treatments to achieve resolution of fluid and involution of CNVMs can be explained by the relative good health of the RPE pump in patients in younger age group compared with the RPE in adults with age-related macular degeneration.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] We used adult dosage in this patient since the patient was 13 year old; adult dose in such cases has also been used in other case reports.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] We also believe that fewer injections given on PRN basis may reduce the risk of systemic adverse effects also which may be a subject of concern especially in the paediatric age group. In our case, we did not observe any short-term adverse ocular or systemic side effects secondary to the treatment with intravitreal anti-VEGF agents.(Deleted reference9) However, long-term studies will be required to establish the safety of these injections in this age group.</p></sec></sec>

A case of submacular cysticercosis treated by pars plana vitrectomy in Kuwait

<p>We report a case of submacular cysticercosis in the left eye of an Indian patient living in Kuwait. Though he was systemically asymptomatic, his magnetic resonance imaging showed multiple cysts in the brain. The patient underwent pars plana vitrectomy (PPV), and the cyst was removed in total through a retinotomy over the cyst. He had another small cyst in the periphery that was also removed. He was treated with oral albendazole and systemic steroids after the surgery to treat his neurocysticercosis. He developed rhegmatogenous retinal detachment after the surgery, and the retina was successfully reattached with scleral buckling and PPV procedure. His final best corrected visual acuity improved from counting fingers at 1/2 m at presentation to 20/400. This case report shows that the ocular cysticercosis may be seen among expatriates working in the Middle East. It is important to rule out the presence of neurocysticercosis in such patients as well as multiple cysts in the affected eye. However, the functional outcome of surgical treatment of submacular cysticercosis can be disappointing.</p>

<contrib contrib-type="author"><name><surname>Wani</surname><given-names>Vivek B.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Niranjan</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Uboweja</surname><given-names>Anil K.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kazem</surname><given-names>Mahmood A.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Human cysticercosis is caused by the larval form of the tapeworm, <italic>Taenia solium</italic>. Ocular involvement is common in endemic areas, and its presence in the subretinal space has been reported.[<xref rid="ref1" ref-type="bibr">1</xref>] However, its presence in the submacular area is rare, and treatment of submacular cysticercosis can be difficult.[<xref rid="ref2" ref-type="bibr">2</xref>] Pars plana vitrectomy (PPV) offers a safe method of approach to submacular cysticercosis, but the visual results can still be disappointing.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] We are reporting a case of submacular cysticercosis in a young Indian male working in Kuwait who was successfully treated by a vitreoretinal procedure.</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 24-year-old Indian male patient presented with a history of diminished vision in the left eye of 20 days duration in January 2010. He had no other ocular or systemic complaints.</p><p>The best corrected visual acuity (BCVA) was counting fingers at 1/3 m in the left eye. The left eye showed few small keratic precipitates with no cells in the anterior chamber (AC). The fundus examination of the left eye showed oval subretinal translucent cystic lesion of three disc diameters at the posterior pole with its nasal edge under the fovea [<xref ref-type="fig" rid="F1">Figure 1</xref>] and a white dense lesion near surface in the center. The cyst showed undulating movements upon throwing light on the lesion. The right eye was normal. Optical coherence tomography (OCT) examination by using Stratus OCT (Carl Zeiss Meditec, Dublin, CA, USA) showed the presence of well-demarcated cyst wall on the vitreal side with scolex attached to the wall of the cyst on the vitreal side [<xref ref-type="fig" rid="F2">Figure 2</xref>]. His physical, blood count and stool examinations were normal. However, magnetic resonance imaging (MRI) of the brain showed nearly 50 small cysts spread in cerebral cortex, basal ganglia, midbrain, thalamus, and cerebellum.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Preoperative photograph of left eye fundus. Vertical arrow: Probable site of entry of the cysticercosis from the choroid showing retinal pigment epithelial disturbance. Horizontal arrow: Shows area of serous retinal detachment</p></caption><graphic xlink:href="OJO-7-144-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Optical coherence tomography of the cyst with the larva</p></caption><graphic xlink:href="OJO-7-144-g002"/></fig><p>The patient underwent three port 20G PPV in which, after removal of the posterior hyaloid, a small retinotomy was created by endocautery in the superotemporal area and the cyst was extracted in whole by engaging it with a flute needle. Another small cyst of &#x0003c;1 DD was seen superiorly, at 12 o&#x02019;clock meridian near the ora, which was removed with the vitrectomy cutter. At the end of the vitrectomy, fluid-air exchange, endolaser and injection of perfluoropropane gas (C₃F₈) 14% were performed. He received antiparasitic treatment with steroids for neurocysticercosis as advised by the neurophysician. The histopathology examination of the cyst confirmed the diagnosis of cysticercosis.</p><p>Two months after surgery, he developed localized superonasal retinal detachment due to two tears in the periphery, which was successfully treated with scleral buckling with PPV with endolaser and sulfur hexafluoride (SF₆) 20% injection. One year after surgery, the BCVA in the left eye had improved to 20/400. The retina was attached in the left eye, but the macular area showed atrophic changes [<xref ref-type="fig" rid="F3">Figure 3</xref>] and he was free of any systemic illness.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Postoperative photograph 1-year later</p></caption><graphic xlink:href="OJO-7-144-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>Human beings are definitive hosts for <italic>T. solium</italic> and pigs are the intermediate hosts. Humans get the infection when they ingest undercooked pork meat containing the cysts. The larvae in the cysts attach to the human gut and develop into adult tapeworms that shed proglottids containing tapeworm eggs into the human feces. The feces may contaminate the food source of the pigs and eggs ingested by the pigs develop into larvae that pierce the intestinal wall and enter the blood stream and end up in various tissues including the muscles where they develop into cysts. However, the human cysticercosis infection occurs when, humans ingest eggs of <italic>T. solium</italic>. This could be due to contamination of food by food handlers, who are harboring adult <italic>T. solium</italic> or auto ingestion of eggs due to poor hygienic practice or contamination of food due to use of human waste or contaminated water to grow vegetables and fruits. When humans ingest the eggs of <italic>T. solium</italic>, the eggs develop into larvae which pierce the gut and enter the blood stream and enter various tissues where they develop into cysts producing the clinical syndrome of cysticercosis. As pigs are not reared in Middle East, it is extremely rare to see systemic or ocular cysticercosis in this region and hence there are only few reports about ocular and systemic cysticercosis from the Middle East.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] All the cases reported from the region, are among expatriate patients from India or other endemic countries.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Cysticercosis can occur in those who have never eaten pork and even in vegetarians, as the infection occurs due to consumption of food or water contaminated with fecal matter.[<xref rid="ref4" ref-type="bibr">4</xref>] Our patient was free from any neurological symptoms, but his MRI showed presence of neurocysticercosis. Neurocysticercosis can be coexisting in up to 24% of the cases.[<xref rid="ref4" ref-type="bibr">4</xref>] Hence, it is important to perform MRI examination to rule out neurocysticercosis. However, the subretinal cysticercosis should be surgically removed first and then start the antiparasitic treatment for neurocysticercosis. Otherwise, death of the larva in the eye due to antiparasitic drugs can enhance the inflammation and lead to loss of vision in the affected eye.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p>Our patient showed few keratic <bold>precipitates</bold> with no reaction in the AC or vitreous. Wender <italic>et al</italic>. reported AC inflammation in 7 of the 22 cases and mild to severe vitritis in all the 22 cases they reported.[<xref rid="ref9" ref-type="bibr">9</xref>] They observed that the degree of inflammation in the eye depended upon time interval between onset of symptoms and intactness of the cyst.[<xref rid="ref9" ref-type="bibr">9</xref>] The inflammation in our case might have been mild as the patient presented early due to the decreased vision caused by submacular cysticercosis. Wender <italic>et al</italic>. do not mention about the presence of any submacular cysticercosis in their case series.[<xref rid="ref9" ref-type="bibr">9</xref>]</p><p>The OCT examination of our patient showed a clearly demarcated hypereflective cyst wall on the vitreal side with the scolex protruding in the cyst producing a comet shaped reflection. These OCT findings have been well-described by Sinha <italic>et al</italic>.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>Visual results after removal of the cysticercosis from the vitreous and subretinal location other than the macula are satisfactory.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] The removal of the submacular cyst by external route with an opening in the sclera and choroid at the site of the cyst can be disastrous, as the area is difficult to reach.[<xref rid="ref2" ref-type="bibr">2</xref>] Hence, its removal by vitrectomy offers the best chances of some visual recovery. However the visual acuity after its successful removal by vitrectomy can still be disappointing.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Sharma <italic>et al</italic>. reported five cases of submacular cysticercosis, and none of them achieved postoperative vision of better than 20/400.[<xref rid="ref4" ref-type="bibr">4</xref>] We speculate that it could be due to damage to the foveal photoreceptors caused by the toxins released by the cyst in the submacular space.</p><p>In summary, we are reporting a case of submacular cysticercosis successfully treated by PPV in Kuwait. The patient had another smaller cyst in the periphery in the same eye. The patient was found to have subclinical neurocysticercosis by radiological examination.</p></sec>

Vitreous occlusion of tube implant in a phakic patient with traumatic glaucoma

<p>Tube implants or glaucoma drainage devices have become an important method of intraocular pressure reduction when treating complex cases of traumatic glaucoma. However, it is not uncommon to have complications associated with tube implants. The optimal treatment of patients who have undergone glaucoma implant surgery complicated by vitreous incarceration is uncertain. If vitreous is present or is able to prolapse into anterior chamber, as in aphakic or pseudophakic patient without an intact posterior capsule, a concurrent anterior vitrectomy is usually performed. In such cases, pars plana vitrectomy has been found to be more effective in several studies. However, there are no set guidelines for management of such a case in a phakic eye and the management can be more challenging especially when there is no obvious deficiency in posterior capsule, zonular dialysis, or loose vitreous gel in the anterior chamber prior to or during tube implantation. We describe a case of 14-year-old phakic patient with traumatic glaucoma without vitreous gel in anterior chamber whose tube implant became occluded by vitreous resulting in increased intra ocular pressure. This is the first documented report of vitreous incarceration in a phakic patient and its successful management.</p>

<contrib contrib-type="author"><name><surname>Dubey</surname><given-names>Suneeta</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Pegu</surname><given-names>Julie</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Agarwal</surname><given-names>Manisha</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Agrawal</surname><given-names>Anugya</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Tube implants or glaucoma drainage devices (GDD) have become an important method of intraocular pressure reduction when treating complex cases of traumatic glaucoma.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] However, GDD implantation is not without complications like hypotony, choroidal effusion, suprachoroidal hemorrhage, endophthalmitis, tube exposure and its occlusion by blood, fibrin, iris, or vitreous. Since there is no set guidelines for management of such complications, treatment needs to be individualized. Here, we report an unforeseen complication of vitreous incarceration following Ahmed Glaucoma Valve (AGV) implantation in a case of traumatic glaucoma.</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 14-year-old male presented to us in 2008 following blunt trauma to right eye with cricket ball with complaints of severe pain, redness, and marked diminution of vision. There was no history of any systemic disease or blood disorders.</p><p>On examination, the left eye was essentially within normal limits. The vision in the right eye was decreased to perception of light with an Intraocular pressure (IOP) of 50 mm Hg. There was presence of corneal epithelial edema and diffuse hyphema. The pupil was irregular, semi dilated, and sluggishly reacting to light with multiple sphincter tears present superiorly. The rest of the anterior segment view was hazy on day 1. B scan showed signs of vitreous hemorrhage. Treatment was initiated in the form of tablet acetazolamide, timolol maleate 0.5%, cycloplegics, topical, and systemic corticosteroids. On day 4, hyphema had cleared sufficiently and examination showed presence of mild inferior iridodonesis [<xref ref-type="fig" rid="F1">Figure 1</xref>]. However, there was no obvious zonular dehiscence or vitreous prolapse. The IOP was still high at 46 mm Hg. Consequently, the treatment was stepped up to maximal medical treatment (MMT). At the end of third week, vision had improved to 6/18; however, IOP still remained uncontrolled. Gonioscopy revealed angle recession extending to 180 degrees. The vitreous hemorrhage had significantly cleared by then and fundus examination revealed a cup disc ratio of 0.5:1. In view of uncontrolled IOP on MMT, we decided to intervene surgically. AGV implantation in the superotemporal quadrant proceeded routinely without any complications. On first post-operative day, IOP decreased to 8 mm Hg but on the fourth day, IOP again rose to 52 mm Hg with a band of vitreous blocking the tube, coming from the site inferiorly where mild iridodonesis was noticed [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Nd: YAG laser vitreolysis was attempted with Zeiss Visulas Yag II laser system. However, the IOP remained high. Vitreoretinal consultation was sought at this stage. Anterior vitrectomy was successfully performed using a 23G vitrectomy cutter via corneal approach followed by an attempt to disengage the vitreous incarcerated in tube using active suction and pulling the tag from lumen using end gripping forceps [<xref ref-type="fig" rid="F3">Figure 3</xref>]. At 3-years follow up, IOP was 16 mm Hg with well-formed bleb, vision was 6/9 with stable disc, and visual fields [<xref ref-type="fig" rid="F4">Figure 4</xref>]. So far, there have been no recurrences of vitreous incarceration or posterior segment complication [<xref ref-type="fig" rid="F5">Figure 5</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Figure showing area of inferior irididonesis with suspected zonular dehiscence, no vitreous prolapse</p></caption><graphic xlink:href="OJO-7-147-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Vitreous strand from behind the lens blocking the lumen of the tube</p></caption><graphic xlink:href="OJO-7-147-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>After vitrectomy, the tube was cleared of vitreous. Few vitreous strands were seen inferiorly in anterior chamber away from the tube</p></caption><graphic xlink:href="OJO-7-147-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Follow-up at 3 years showing patent tube and well formed anterior chamber</p></caption><graphic xlink:href="OJO-7-147-g004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>At last follow-up, bleb was well formed and functional</p></caption><graphic xlink:href="OJO-7-147-g005"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>Traumatic glaucoma encompasses a spectrum of disease which can include damage to lens, zonules, peripheral retina, choroid, and macula and needs to be evaluated on individual basis. The barrier to success of trabeculectomy in children include a thick and active Tenon's capsule, a rapid wound healing response, and high risk of bleb-related infections.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>The GDD, however, maintains a channel for aqueous flow in the setting of exuberant fibrovascular wound healing and improves the surgical prognosis. GDD surgery avoids the development of a thin, avascular, filtering bleb associated with trabeculectomy, and Mitomycin-C, which may minimize the risk of delayed infection.</p><p>We acknowledge that GDD implantation is not without complications.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] Desatnik <italic>et al</italic>. reported that vitreous incarceration in implant tubes occurred despite six of the series of eight eyes having previous anterior vitrectomy and concluded that anterior vitrectomy was insufficient in preventing vitreous occlusion in aphakic or pseudophakic eyes with deficient posterior capsule. However, in these eyes, the implant was either inserted into vitreous cavity or the vitreous was already present in the anterior chamber.[<xref rid="ref6" ref-type="bibr">6</xref>] Since there was no obvious zonular dehiscence or vitreous in anterior chamber before and during surgery in our case, we did not perform anterior vitrectomy primarily. Desatnik <italic>et al</italic>. also reported that even when vitreous is not present in anterior chamber preoperatively, it can prolapse through an open or absent post capsule, particularly if choroidal effusion or haemorrhage reduces the vitreous cavity volume and displaces residual vitreous gel forward into anterior chamber.[<xref rid="ref6" ref-type="bibr">6</xref>] However, the possibility of such an occurrence was remote in our case as there was minimal residual vitreous hemorrhage.</p><p>According to Y. Fernandez Barrientos <italic>et al</italic>., the presence of tube/stent modifies the physiological flow of aqueous with diversion of flow towards its lumen.[<xref rid="ref7" ref-type="bibr">7</xref>] The tube in our case was directed inferiorly towards the site of suspected zonular dehiscence, which could have attracted the vitreous towards its lumen especially in presence of low IOP. So, we recommend that the direction and placement of the tube should be away from the site of suspected zonular dehiscence.</p><p>The optimal treatment of patients who have undergone glaucoma implant surgery complicated by vitreous incarceration is uncertain. Nd: YAG vitreolysis has been described by various authors with variable results.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] We also attempted laser viteolysis without success. Combined glaucoma implant surgery and parsplana vitrectomy (PPV) has been reported to be safe and effective in controlling IOP and preventing tube occlusion with vitreous gel in aphakic and pseudophakic eyes without an intact posterior capsule and with vitreous prolapse in anterior chamber.[<xref rid="ref5" ref-type="bibr">5</xref>] However, in pseudophakic patients without loose gel in anterior chamber, a concurrent PPV is not recommended as primary procedure. Since our patient was phakic and having good visual acuity, we decided to perform anterior vitrectomy with thorough cutting of vitreous strands in and around the tube taking utmost care not to touch the clear lens instead of PPV. In complex glaucomas, the complications may arise with GDD, but if treated timely with vigilance, most of it can be managed with a favourable prognosis. The treatment, therefore, should be individualized depending on the extent of damage and the visual prognosis.</p></sec>

Functional spasm of accommodation: Changes on scheimpflug imaging

<p>Spasm of accommodation is a rare condition characterized by a sudden increase in myopia. It can occur in emmetropes, hypermetropes and myopes. All the physiological changes that occur in normal accommodation are exaggerated. Mostly the cause is functional disorder and the condition gradually improves. We hereby present the changes occurring in spasm of accommodation as seen on Scheimpflug imaging.</p>

<contrib contrib-type="author"><name><surname>Sukhija</surname><given-names>Jaspreet</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Dogra</surname><given-names>Mangat R.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Zadeng</surname><given-names>Thara</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ram</surname><given-names>Jagat</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Spasm of accommodation is characterized by intermittent and usually painful convergence, accommodation, and miosis in varying combinations with variable amount of time.[<xref rid="ref1" ref-type="bibr">1</xref>] Various etiologies have been implicated, including functional spasm of oculo-motor muscle, hysterical convergence spasm and emotional distress.[<xref rid="ref2" ref-type="bibr">2</xref>] The excessive accommodative tone usually causes pseudomyopia.[<xref rid="ref3" ref-type="bibr">3</xref>] We herein report scheimpflug imaging in a patient with spasm of accommodation.</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 17-year-old girl complained of sudden onset blurring of vision in both eyes following prolonged reading under a dim light. It was associated with mild headache and eye strain. She was a known case of myopia. Her refractive error was &#x02013;2 diopters in both eyes. Her records showed that her refractive error increased to &#x02013;4 dioptre sphere on the day of her complaint and she was prescribed spectacles on the same day. She was on salbutamol inhaler for bronchial asthma.</p><p>At presentation to us on the third day of her symptoms, visual acuity was 6/36 and 6/24 in the right and the left eye with &#x02013;4 dioptre spherical correction which she was wearing in both eyes. BCVA (Best corrected visual acuity) was 6/6 with manifest refraction of &#x02013;7 diopter sphere OD and &#x02013; 7.5/&#x02013;0.5 &#x000d7; 70 OS. Anterior segment examination revealed miosis, shallow anterior chamber in both eyes. Intra-ocular pressure on applanation tonometry was 14 and 16 mmHg in right and left eye, respectively. On Pentacam examination after dilatation with phenylepherine 5% drops, the anterior chamber depth was 1.64 mm OD and 1.57 mm OS. The crystalline lens thickness was 4.01 mm in the right eye and 4 mm in the left eye and the distance between the cornea and posterior pole of the lens was 5.65 mm OD and 5.57 mm OS [Figure <xref ref-type="fig" rid="F1">1a</xref> and <xref ref-type="fig" rid="F1">1b</xref>]. Cycloplegic refraction under cyclopentolate 1% done 1 day later showed &#x02013;1.5 dioptre sphere in the right and &#x02013;1.75 dioptre sphere in the left eye and visual acuity of 6/6 OU. At this time scheimpflug imaging showed an anterior chamber depth of 2.98 mm OD and 2.95 mm OS with a lens thickness of 3.7 mm in the right eye and 3.62 mm in the left eye and the distance between the cornea and posterior pole of the lens was 6.68 mm OD and 6.57 mm OS [Figure <xref ref-type="fig" rid="F2">2a</xref> and <xref ref-type="fig" rid="F2">2b</xref>]. No supraciliary effusion could be documented on ultrasound biomicroscope. A diagnosis of accommodative spasm was thus made clinically and morphological changes were documented by pentacam. The patient was put on cyclopentolate 1% drops once a day.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) Scheimpflug image of the right eye during accommodative spasm after dilatation with phenylepherine 5% drops objectively reveals shall anterior chamber along with lenticular parameters. (b) Scheimpflug image of the left eye showing a similar picture</p></caption><graphic xlink:href="OJO-7-150-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>(a) Scheimpflug image of right eye after dilatation with cyclopentolate 1% drops. Note the increase in the anterior chamber depth with a decrease in lens thickness and posterior movement of the crystalline lens. (b) Scheimpflug image of the left eye with similar findings</p></caption><graphic xlink:href="OJO-7-150-g002"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>Accommodative spasm is a condition where there is an involuntary, sustained accommodation in the absence of an accommodative stimulus. The anterior lens becomes more sharply curved, moving closer to the cornea.[<xref rid="ref4" ref-type="bibr">4</xref>] Baikoff <italic>et al</italic>. analyzed the anterior segment changes during accommodation using optical coherence tomography (OCT).[<xref rid="ref5" ref-type="bibr">5</xref>] They found that the anterior pole of crystalline lens moves forward during accommodation and there is a linear relationship between this movement and anterior radius of curvature. Accordingly the posterior curvature decreased proportionally. Controversy still exists as to the movement of the posterior lenticular surface during accommodation. Recent study by Strenk <italic>et al</italic>. reported no movement of the posterior lenticular surface during accommodation whereas Bolz <italic>et al</italic>. have showed posterior movement.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Koretz <italic>et al</italic>. showed that in young adults the posterior lens surface may tend to move posteriorly a short distance as well, so that total anterior segment length may increase slightly whereas in adults this surface moves forward adding to the decrease in anterior chamber depth.[<xref rid="ref4" ref-type="bibr">4</xref>] Tsorbatzoglou <italic>et al</italic>. found by partial coherence interferomtery that the decrease in ACD was greater than the increase in anterior segment length (ASL) and thus hypothesized that the crystalline lens move forward simultaneously during accommodation.[<xref rid="ref8" ref-type="bibr">8</xref>] Dubbelman <italic>et al</italic>. found significant anterior movement of the human lens during accommodation.[<xref rid="ref9" ref-type="bibr">9</xref>] In our young patient we observed forward movement of the posterior lenticular surface as well. It may be hypothesized that this anterior movement of the posterior surface of the crystalline lens occurs in accommodative spasm which contributes to the increase in accommodative amplitude. A myopic patient will appear to be more myopic, a hyperopic patient will appear to be less hyperopic and an emmetropic patient will appear to be a myopic.</p><p>Scheimpflug imaging is an important tool in objectively quantifying the changes in accommodative spasm.</p></sec>

Ocular findings in a case of periorbital giant congenital melanocytic nevus

<p>Giant congenital melanocytic nevus (GCMN) is a large melanocytic nevus that rarely occurs in the periorbital region. Various systemic, as well as ophthalmic associations, have been reported with GCMN. However, there is only one case report describing ophthalmic findings in periorbital GCMN. We describe the ocular findings in a case of periorbital GCMN.</p>

<contrib contrib-type="author"><name><surname>Raina</surname><given-names>Usha K.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Seth</surname><given-names>Anisha</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Anika</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Batta</surname><given-names>Supriya</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<sec sec-type="intro" id="sec1-1"><title>Introduction</title><p>Giant congenital melanocytic nevus (GCMN) is usually defined as a melanocytic lesion present at birth that will reach a diameter &#x02265;20 cm in adulthood.[<xref rid="ref1" ref-type="bibr">1</xref>] Its incidence is estimated in &#x0003c;1:20,000 newborns. Periorbital region is a rare site of occurrence of congenital hairy melanocytic nevus, especially the giant form. We describe the ocular findings in a 6 years old girl, with a periorbital GCMN.</p></sec><sec id="sec1-2"><title>Case Report</title><p>A 6-year-old girl presented with redness and watering associated with a decrease in vision in the left eye for 1 week. There was no history of preceding trauma or fever and no history of taking any treatment. She had a giant pigmented hairy nevus involving both the lids on the left side and the nose, present since birth and gradually increased in size with age [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Her best-corrected visual acuity in the right eye was 20/20, while in the left eye was 20/100.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Clinical photograph of the patient showing giant hairy congenital melanocytic nevus involving the nose and the left upper and lower lids</p></caption><graphic xlink:href="OJO-7-153-g001"/></fig><p>On ocular examination, she was orthophoric, and the ocular movements were full and free. The right eye was within normal limits. The left eye showed diffuse conjunctival congestion suggestive of viral conjunctivitis and conjunctival melanosis in the superonasal quadrant [<xref ref-type="fig" rid="F2">Figure 2</xref>]. There was also a paracentral macular corneal opacity, 0.5 cm in diameter with superior pannus; and iris melanosis between 11 and 12 o&#x02019;clock [<xref ref-type="fig" rid="F3">Figure 3</xref>]. There was no evidence of any cells or flare in the anterior chamber. The left eye optic disc was hyperemic with dilated and tortuous vessels whereas the rest of the retina was normal [<xref ref-type="fig" rid="F4">Figure 4</xref>]. The intraocular pressure measured by Goldmann applanation tonometer was 14 mm of mercury in both eyes. Hertel's exophthalmometry was 16 mm in both eyes at the bar reading of 92 mm.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Slit lamp photograph of the patient showing conjunctival congestion and conjunctival melanosis in the supero-nasal quadrant</p></caption><graphic xlink:href="OJO-7-153-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Slit lamp photograph of the patient showing paracentral macular corneal opacity (red arrow), superior pannus (yellow arrow) and iris melanosis (white arrow)</p></caption><graphic xlink:href="OJO-7-153-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Fundus photograph of the left eye showing optic disc hyperemia and dilated and tortuous vessels</p></caption><graphic xlink:href="OJO-7-153-g004"/></fig><p>The patient was started on topical antibiotic and lubricants, and she improved within a week. Retinoscopy under 2% homatropine revealed no refractive error in the right eye, whereas the left eye had an astigmatism of &#x02212; 2 DC at 180&#x000b0;. However, the left eye vision did not improve with refraction, indicating anisometropic amblyopia. Magnetic resonance imaging (MRI) scan of brain and orbits was done and was within the normal limits. The patient was referred to the plastic surgery department for further management.</p></sec><sec id="sec1-3"><title>Comment</title><p>Despite its rarity, GCMN is important because it may be associated with severe complications such as malignant melanoma, affect the central nervous system (neurocutaneous melanosis), and have major psychosocial impact on the patient due to its unsightly appearance, especially if it is present on the face. Other rare systemic associations reported are diffuse lipomatosis, hypertrophy of cranial bones, atrophy of limbs, skeletal asymmetries, scoliosis, urinary tract anomalies, capillary vascular malformations, cafe-au-lait spots, ectopic mongolian spots, fibroepithelial polyps and CNS malformations like arachnoid cysts, choroid plexus papilloma, cerebellar astrocytoma, type I Arnold-Chiari and Dandy-Walker malformation.[<xref rid="ref1" ref-type="bibr">1</xref>] Ophthalmic findings reported in association with GCMN include ectopic lacrimal gland, intraocular choristoma, anterior staphyloma, ankyloblepharon and iris melanosis.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Giant congenital melanocytic nevus is mostly found on the trunk, followed by the limbs and the head. Multiple smaller congenital nevi called satellite lesions may also be seen in the vicinity of the main nevus.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Periorbital region is an uncommon site for the occurrence of congenital hairy melanocytic nevus, especially involving both lids (called divided nevus), with roughly 40 cases reported in the literature.[<xref rid="ref5" ref-type="bibr">5</xref>] The giant form is the rarest, and there are very few cases of periorbital giant congenital hairy melanocytic nevus reported in the literature.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref6" ref-type="bibr">6</xref>] Only one of them had associated ocular findings like ocular choristoma and anterior staphyloma.[<xref rid="ref3" ref-type="bibr">3</xref>] Our patient had conjunctival and iris melanosis as the ocular findings in periorbital GCMN.</p><p>Since, the MRI of brain and orbits was normal, the left fundus findings remain unexplained. It might be due to occult melanocytic deposits in the lepto-meninges (neurocutaneous melanosis) or left optic nerve sheath that are known to be missed on MRI, and can only be confirmed on autopsy.</p></sec>

ERRATUM

Could not extract abstract

Could not extract contributor

Oman Journal of Ophthalmology

<boxed-text position="float"><p><bold>Oman Journal of Ophthalmology 2014; Vol 7; Issue 2</bold></p><p><bold>Title: Progressive Painless Loss of Vision - What is the diagnosis?</bold></p><p><bold>Authors</bold></p><p><bold>Radha Shenoy, Badar Al Barwani, Rashid Al Saidi</bold></p><p>Correction:</p><p><bold>Figure 2</bold>: (a) Early phase fundus fluorescein angiogram right eye (b) Late phase fundus fluorescein angiogram right eye</p><p><bold>Figure 3</bold>: (a) Early phase fundus fluorescein angiogram left eye (b) Late phase fundus fluorescein angiogram left eye</p><p>Should read as</p><p><bold>Figure 2</bold>: (a) Early phase fundus fluorescein angiogram left eye (b) Late phase fundus fluorescein angiogram left eye</p><p><bold>Figure 3</bold>: (a) Early phase fundus fluorescein angiogram right eye (b) Late phase fundus fluorescein angiogram right eye</p><p>The error is regretted</p><p>- <italic>Editor, OJO</italic></p></boxed-text>

Interdisciplinary approach: A boon for ocular rehabilitation

Could not extract abstract

<contrib contrib-type="author"><name><surname>Sonune</surname><given-names>Shital J.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Deeksha</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Nirmal</surname><given-names>Narendra</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Mehta</surname><given-names>Sonal</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib>

Oman Journal of Ophthalmology

<p>The face and eyes not only reveal identity but also communicate the intent of our words. The loss of a vital bodily organ such as the eye is not only traumatic but produces grief and irreversible loss of function. As the so-called mirror of the soul has been the focus of many disciplines, hence successful rehabilitation of an individual who has lost an eye requires attention of several of specialists like Ophthalmologist, Psychologist, Plastic Surgeon and Maxillofacial Prosthodontist.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>It can be stated that where the work of a plastic surgeon ends, the work of maxillofacial prosthodontist begins. The demand for a maxillofacial prosthetic device for the rehabilitation of patients has intensified in the recent years.[<xref rid="ref2" ref-type="bibr">2</xref>] The art of making artificial eyes has been known to man for centuries.[<xref rid="ref3" ref-type="bibr">3</xref>] The two difficult challenges for maxillofacial prosthodontist are superior sulcus deformities from inadequate orbital volume and eyelid ptosis or laxity.</p><p>Implant retained ocular prosthesis would be the best approach to rehabilitate an anopthalamic eye, if not for economic and systemic factors. The next preferred technique is the custom-made ocular prosthesis. The advantages of customized ocular prosthesis is improved adaptation to underlying tissues, increased mobility of prosthesis, improved facial contours and control over the size of iris, pupil and color of the iris and sclera. As per literature fabrication of custom-made ocular prosthesis, it involves complex painting procedures, high skill and expertise of the dentist.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Hence, in this clinical report using patient's stock eye shell [<xref ref-type="fig" rid="F1">Figure 1</xref>], custom-made ocular prosthesis was fabricated with a simple, reversible and economical technique. An impression of the anopthalamic socket was made with a good quality alginate in first clinical step and orientation with trial of the wax pattern-stock eye assembly was done during the second step [Figures <xref ref-type="fig" rid="F2">2</xref> and <xref ref-type="fig" rid="F3">3</xref>]. In the third appointment, the prosthesis was inserted [Figure <xref ref-type="fig" rid="F4">4a</xref> and <xref ref-type="fig" rid="F4">b</xref>]. In this case, as the stock eye shell was already been selected for the patient by his ophthalmologist, the work of the maxillofacial prosthodontist was made much easier and simpler.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Unaesthetic look with existing stock eye shell</p></caption><graphic xlink:href="OJO-7-156-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Orientation of a wax sclera pattern for correct gaze</p></caption><graphic xlink:href="OJO-7-156-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Trial of wax pattern-stock eye assembly</p></caption><graphic xlink:href="OJO-7-156-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>(a and b) Superior natural appearance with customized ocular prosthesis</p></caption><graphic xlink:href="OJO-7-156-g004"/></fig><p>As it is quoted very well &#x0201c;coming together is a beginning, working together is a progress and achieving together is a success&#x0201d;, the work of an ophthalmologist and maxillofacial prosthodontist creates custom ocular prosthesis but what we give patients can be much more improved self-esteem and the confidence necessary for them to return to an independent and productive living. An interdisciplinary approach can work wonders for even the utterly hopeless cases; we only need to widen our approach.</p>

A 48-year-old female with sudden onset of painless diminution of vision in left eye

Could not extract abstract

<contrib contrib-type="author"><name><surname>Bhandari</surname><given-names>Akshay J.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Bangal</surname><given-names>Surekha V.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Padghan</surname><given-names>Dipti</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Pratik</surname><given-names>Gogri</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<p>A 48-year-old female came to ophthalmology outpatient department with sudden onset painless diminution of vision in left eye since last 4 days, which was not associated with trauma, floaters, flashes of light, blurring of vision or any other ocular complaints. On general examination patient's pulse was 76/min that was regularly regular, and blood pressure was found to be 200/120. On ophthalmic examination, the distant vision in left eye was finger counting close to face that was not improved with refraction and in right eye was 6/6. In left eye, anterior segment examination was normal except for relative afferent pupillary defect and early cortical cataract, while fundus examination revealed findings shown in Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Fundus photograph left eye day 3 after admission</p></caption><graphic xlink:href="OJO-7-158-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Fundus photograph right eye</p></caption><graphic xlink:href="OJO-7-158-g002"/></fig><sec id="sec1-1"><title>Questions</title><p> <list list-type="order"><list-item><p>Describe the findings in Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>?</p></list-item><list-item><p>What is the most likely diagnosis?</p></list-item><list-item><p>What all investigations will you do to confirm the diagnosis?</p></list-item></list> </p></sec><sec id="sec1-2"><title>Answers</title><p> <list list-type="order"><list-item><p>Pale white retinal background with cherry red spot at the fovea, thread like attenuated blood vessels with cattle-track appearance [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p></list-item><list-item><p>Grade II hypertensive retinopathy with arteriovenous crossing changes [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p></list-item><list-item><p>Routine blood investigations were normal except for raised erythrocyte sedimentation rate which was 56 mm at the end of 1 h and positive C-reactive protein. Rest of the blood investigations such as blood sugar, lipid profile, prothrombin time, bleeding time and clotting time was normal. Two-dimensional-echocardiography was done, which showed good systolic and diastolic function with no valvular pathology. Bilateral carotid Doppler was done, which showed a plaque of 5.2 &#x000d7; 1.2 mm size in the posterolateral aspect of left carotid artery in the carotid bulb [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p></list-item></list> </p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Carotid Doppler left common carotid artery</p></caption><graphic xlink:href="OJO-7-158-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>A central retinal artery occlusion is a blockage of the main artery to the retina. Like all nervous tissue, the retina requires large quantities of oxygen delivered by the bloodstream in order to function properly. Interruption of blood supply even for a few minutes impairs vision, and unless the blood supply is restored within 90 min, permanent damage to the retina with loss of part of the visual field results. In many instances, it is impossible to ascertain the exact pathophysiologic process responsible for central retinal artery obstruction.[<xref rid="ref1" ref-type="bibr">1</xref>] The main causes are emboli, intraluminal thrombosis, vasculitis, spasm, hypertensive arterial necrosis, dissecting aneurysm.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] These causes are intimately related to associated systemic abnormalities like diabetes mellitus (25%)[<xref rid="ref1" ref-type="bibr">1</xref>] hypertension (66%) and cardiac valvular disease (25%).[<xref rid="ref4" ref-type="bibr">4</xref>] The emboli are visible within the retinal arterial system in about 20-40% of eyes with central retinal artery occlusion. Emboli can originate from any part of the arterial system. The most common is cholesterol emboli from atherosclerotic deposits in carotid arteries. The other emboli are fibrin platelet thrombus and calcific emboli which usually originate from cardiac valves and cause more severe obstruction. Abnormalities in the cardiac valves or circulation as seen in infective endocarditis should be ruled out in every case of central retinal artery occlusion. Embolus from the heart is the most common cause of central retinal artery occlusion in patients younger than 40 years. In our case, the carotid Doppler revealed a plaque in left-sided carotid bulb from there it might have gone to central retinal artery. There are various triggering factors for atherosclerosis; in our case, the patient was found to be hypertensive. Treatment of central retinal artery occlusion can be categorized as conservative (ocular massage, pharmacological treatment, anterior chamber paracentesis) or invasive (catheterization of the proximal ophthalmic artery through the femoral artery with the infusion of thrombolytic agents). However, to date, no satisfactory therapy is available for patients with this disorder[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] and in most cases therapy is not successful.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>Conclusion</title><p>Central retinal artery occlusion is an ophthalmic emergency, causing acute painless loss of vision. There are various triggering factors for atherosclerosis; in our case, the patient was found to be hypertensive.</p></sec>

Pre-macular hemorrhage in chronic malaria

Could not extract abstract

<contrib contrib-type="author"><name><surname>Takkar</surname><given-names>Brijesh</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Chandra</surname><given-names>Parijat</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Rathi</surname><given-names>Anubha</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Azad</surname><given-names>Rajvardhan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<p>Sir/Madam,</p><p>Malaria is a common infectious disease in developing nations but rarely a cause of concern for ophthalmologists. We had such a unique case when a 28-year-old female presented with history of sudden onset painless blurred vision since 1 month in the right eye (RE) with mild spontaneous improvement since last 10 days. She was diagnosed chronic malaria with anemia 1 month back by a physician following complains of persistent fever and malaise. Peripheral blood film examination had confirmed the presence of parasites and hemolysis with hemoglobin recorded as 7.4 g/dl. Fever had subsided following a short course of chloroquine.</p><p>On examination best-corrected visual acuity (BCVA) was 6/18 and 6/6 in the right and left eyes, respectively. Both the eyes had normal anterior segment and IOP. Fundus examination of RE revealed resolving pre-macular hemorrhage overlying the fovea with hazy borders [<xref ref-type="fig" rid="F1">Figure 1a</xref>]. LE fundus was normal. High Definition Ocular Coherence Tomography (HDOCT) images (Cirrus; Zeiss Meditec Inc, Dublin, CA) of the right eye revealed a dense pre-foveal opacity with corresponding optical shadow [<xref ref-type="fig" rid="F1">Figure 1b</xref>]. Fluorescein angiography appeared to be normal in both the eyes as the lesion was present in the foveal avascular zone and no vascular anomaly was detected. She was advised regular follow-up and 15 days later RE BCVA had improved to 6/9p. The hemorrhage had decreased in size and become more defined [Figure <xref ref-type="fig" rid="F2">2a</xref> and <xref ref-type="fig" rid="F2">b</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) Fundus photograph of RE showing resolving pre-macular hemorrhage. (b) HDOCT line scan of RE macula revealing a dense pre-macular lesion with corresponding optical shadowing</p></caption><graphic xlink:href="OJO-7-159-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>(a) Fundus photograph of RE showing decreased size of the pre-macular hemorrhage. (b) HDOCT line scan of RE showing decreased size of the pre-macular hemorrhage and the optical shadow</p></caption><graphic xlink:href="OJO-7-159-g002"/></fig><p>Anterior ischemic optic neuropathy,[<xref rid="ref1" ref-type="bibr">1</xref>] retrobulbar neuritis[<xref rid="ref2" ref-type="bibr">2</xref>] and sub-macular bleed[<xref rid="ref3" ref-type="bibr">3</xref>] have been reported as a cause of vision loss with non-cerebral malaria. Anti-malarial drugs are also well-identified cause of retinopathy. Retinal hemorrhages can occur in cerebral malaria and have been identified as prognostic factors where they were commonly associated with vascular events.[<xref rid="ref4" ref-type="bibr">4</xref>] Our patient was not a case of cerebral malaria and vision loss occurred before initiation of the antimalarial regimne which was given for a very brief period, ruling out drug toxicity. A possible cause could be localized vascular obstruction in the retinal venous system similar to the previously reported choroidal vascular anomaly,[<xref rid="ref3" ref-type="bibr">3</xref>] though this could not be proved on angiography. Another likely explanation is anemic retinopathy induced by anemia of chronic malaria, which is well known to cause retinal vascular anomalies and pre-retinal hemorrhages.[<xref rid="ref5" ref-type="bibr">5</xref>] This is consistent with the course and the site of the bleed in our patient. Such cases can hence be followed up without intervention.</p><p>To the best of our knowledge, this is the first documented case of pre-macular hemorrhage in malaria, possibly related to anemic retinopathy.</p>

External ophthalmomyiasis

Could not extract abstract

<contrib contrib-type="author"><name><surname>Ranjan</surname><given-names>Ratnesh</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Arvind</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Oman Journal of Ophthalmology

<p>Sir,</p><p>Ophthalmomyiasis, infestation of ocular and orbital tissues with fly larvae (maggots), is a rare but relatively more common condition in under-developed world due to poor living and hygiene standard.[<xref rid="ref1" ref-type="bibr">1</xref>] Affected patients are usually unattended old persons and suffer from debilitating underlying conditions. It can also affect persons living or working in close proximity to livestock, children, and even non-compromised hosts rarely.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><sec id="sec1-1"><title>Case Report</title><p>A 72-year-old man was brought to the ophthalmology department with complaints of ulceration, severe pain, and discomfort over left eye for the last 7 days. According to his attendant, the patient was living alone in his village home. The patient was unable to walk properly and was living a sedentary life due to multiple joint-related discomforts. His mental status was normal and he gave a history of fall about 10 days back with minor trauma to left upper eyelid.</p><p>On initial examination, the patient was restless and was not able to open his left eye. There was a large excoriated ulceration over left eye with erythema and oedematous swelling of periorbital tissues. On thorough examination, it was only upper eyelid which was severely necrotized with numerous maggots moving to and fro in the necrotic tissue [Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>]. On retracting the upper eyelids with difficulty, the palpebral conjunctiva and other ocular tissues were intact. Orbital computed tomography (CT) scan revealed intact globe with no disturbance of periocular tissues and periorbital sinuses. Thus, orbital invasion was ruled out and the patient was admitted as a case of external ophthalmomyiasis.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Examination of left eye revealing large excoriated ulcer with surrounding erythema and edematous swelling in upper eyelid and central necrotized tissue with numerous motile maggots while examination of the anterior/posterior segments of the affected eye was unremarkable</p></caption><graphic xlink:href="OJO-7-160-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Magnified view showing maggots</p></caption><graphic xlink:href="OJO-7-160-g002"/></fig><p>On the day of admission, the patient was taken to the minor operation theater. After instillation of topical xylocaine (4%) drops, all necrotic tissues and over 100 maggots were removed. Maggots were removed by catching them with fine smooth forceps. During removal, maggots were tending to migrate into the deeper tissues and soft tissues of forehead. Daily dressing was done to remove all residual larvae until fourth day when they were no more. Every day, after removal of larvae, cleaning was done with betadine solution and antibiotic ointment was applied. Systemic antibiotic and analgesic were also prescribed for 5 days. Swelling and redness reduced gradually and granulation tissue started to appear in about a week [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Newly formed granulation tissue (9^th day image) following mechanical removal of maggots with necrotized tissue and daily dressing with betadine solution and application of antibiotic ointment</p></caption><graphic xlink:href="OJO-7-160-g003"/></fig></sec><sec sec-type="discussion" id="sec1-2"><title>Discussion</title><p>Ophthalmomyiasis is divided into orbital, internal, and external, based on site of larval infestation.[<xref rid="ref3" ref-type="bibr">3</xref>] Orbital and internal ophthalmic myiasis is caused by larva with invading habits leading to blinding manifestations. External ophthalmic myiasis refers to superficial infestations of ocular tissue such as conjunctiva and eyelids. But external ophthalmomyiasis caused by invading larva can also result in serious complications such as corneal ulcer, iridocyclitis, globe invasion, endophthalmitis, and even blindness.[<xref rid="ref4" ref-type="bibr">4</xref>] However, none of these complications were encountered in our patient and the invasion was limited to upper eyelid and soft tissue of forehead only.</p><p>There are three families of flies which cause ophthalmic infestation, i.e. Oestridae, Calliphiride, and Sarcophagidae.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref5" ref-type="bibr">5</xref>] Ophthalmomyiasis is usually caused by larvae of the sheep nose botfly (<italic>Oestrus ovis</italic>) while human botfly (<italic>Dermatobia hominis</italic>) is less commonly involved.[<xref rid="ref2" ref-type="bibr">2</xref>] All these flies are oviparous and eject their eggs on necrotic dead tissue, which hatch to larvae (maggots). If possible, exact taxonomic classification of these larvae gives idea about potential risk of intraocular complications. Removed larvae should be preserved in 70% alcohol and sent to specialist for examination.</p><p>Except mechanical removal of these larvae, there is no other therapy described.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref6" ref-type="bibr">6</xref>] In external myiasis, larvae can be removed by using fine forceps. Topical anesthetic is used to reduce the motility of migrating larvae.[<xref rid="ref1" ref-type="bibr">1</xref>] Severe orbital myiasis may require exenteration. However, in internal myiasis larvae can be destroyed by laser photocoagulation or removed by pars plana vitrectomy.[<xref rid="ref7" ref-type="bibr">7</xref>]</p></sec>

Recurrent microbial keratitis in eyes with keratoconjunctivitis sicca with coexisting ocular surface pathology

Could not extract abstract

<contrib contrib-type="author"><name><surname>Nanavaty</surname><given-names>Mayank A.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Shah</surname><given-names>Anish N.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Al-Sukkar</surname><given-names>Rima</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Lake</surname><given-names>Damian B.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib>

Oman Journal of Ophthalmology

<p>Sir/Madam,</p><p>The management of microbial keratitis (MK) in presence of keratoconjunctivitis sicca and other coexisting surface pathologies presents a formidable medical and surgical challenge. MK in such eyes is associated with poor outcomes.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Other variables with a significant association with poor outcomes are prior treatment with topical steroids,[<xref rid="ref4" ref-type="bibr">4</xref>] a delay in seeking treatment and failure to use fortified antibiotics.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>We performed a retrospective review of all eyes with keratoconjunctivitis sicca (KCS) coexisting with other surface pathologies such as mucosal scarring, previous transplants and/or eyelid disorders, which had recurrent MK (&#x0003e;1 discrete episode) and who were under regular follow-ups. The patients were identified from the microbiology register over 3 years period. The notes were reviewed in detail to study the clinical and microbiological profile.</p><p>Ten eyes (10 patients) were identified [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Average time interval between 2 episodes of MK was 5 months. Seven subjects experienced a total of 2 episodes of MK, while three patients had 3 episodes. <xref ref-type="table" rid="T1">Table 1</xref> shows the baseline characteristics and <xref ref-type="table" rid="T2">Table 2</xref> shows the characteristics of the eye at the time of the latest episode of MK. All had dryness of ocular surface and a degree of neurotrophia due to various reasons [<xref ref-type="table" rid="T1">Table 1</xref>]. All eyes had at least one corneal transplant except patient 2 [<xref ref-type="table" rid="T1">Table 1</xref>]. Five eyes had sutures <italic>in-situ</italic> at the time of recurrent MK [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Before the latest episode of MK, all eyes underwent at least one tarsorrhaphy procedure except one (patient no. 8, <xref ref-type="table" rid="T1">Table 1</xref>) that underwent several lid procedures for cicatricial entropion. All patients were on preservative free topical medications. Six eyes had BCVA of &#x02264;6/60 before any episode of MK compared to 7 following the latest episode due to residual scarring [<xref ref-type="table" rid="T2">Table 2</xref>]. (This concurs with Musch <italic>et al</italic>,[<xref rid="ref6" ref-type="bibr">6</xref>] who demonstrated poor visual outcomes in eyes with previous surgery/pathology and MK at a higher risk for poor visual outcome). Six eyes grew normal commensals (Coagulase negative <italic>Staphylococcus</italic>, <italic>Staphylococcus epidermidis</italic>, <italic>Staphylococcus aureus</italic>) on culture during the previous and the latest episodes of MK and 2 had no growth at all episodes [Tables <xref ref-type="table" rid="T1">1</xref> and <xref ref-type="table" rid="T2">2</xref>]. At the latest episode of MK, 9 had a BCL, 7 were using topical steroids, 7 were using topical lubricants, 4 had concurrent meibomian gland dysfunction and were taking oral Doxycycline, 8 had occluded eyelid puncti and 5 had sutures at the time of recurrent infection [<xref ref-type="table" rid="T2">Table 2</xref> and <xref ref-type="fig" rid="F1">Figure 1</xref>]. All patients on soft bandage contact lens (BCL) where using preservative free G. Chloramphinicol (Minims Chloramphinicol, Baush and Lomb, UK) 4 times a day. Additional tarsorraphies and eyelid procedures to correct residual exposure (blink lagophthalmos) led to prevention of recurrence of infection in all cases.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Montage of eyes in this study. (a) Traumatic lid loss and several eyelid procedures including medial and lateral tarsorraphy showing active microbial keratitis. (b) Keratoconjunctivitis sicca secondary torheumatoid arthritis, a corneal patch graft and active microbial keratitis. (c) Keratoconjunctivitis sicca secondary to icthyosis, tarsorraphies, corneal patch graft and active microbial keratitis. (d) Keratoconjunctivitis sicca secondary to rheumatoid arthritis, lateral tarsorraphy, corneal patch graft and active microbial keratitis (e) Cicatricial phemphigoid, previous corneal graft and active microbial keratitis (f) Patient 6 with atopy and history of herpetic keratitis, corneal graft with active keratitis (g) Corneal graft for PBK and active microbial keratitis. (h) Steven Johnson Syndrome and Graft Vs Host Disease with previous eyelid procedures showing active microbial keratitis.(i) Keratoconjunctivitis sicca secondary to rheumatoid arthritis, previous tarsorraphy and microbial keratitis with drug deposits.(j) Corneal graft for pseudophakic bullous keratopathy, keratoconjunctivitis siccas due to rheumatoid arthritis and active microbial keratitis</p></caption><graphic xlink:href="OJO-7-161-g001"/></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline characteristics of the patients in this study</p></caption><graphic xlink:href="OJO-7-161-g002"/></table-wrap><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Characteristics of the eye at the time of final episode of microbial keratitis</p></caption><graphic xlink:href="OJO-7-161-g003"/></table-wrap><p>Simple but important implications of this series are Use of even mild or low-dose topical steroid should be critically questioned in patient with KCS with coexisting surface pathology or any degree of corneal exposure in presence of corneal sutures, especially if they could be supplanted by other measures to reduce ocular surface inflammation, such as improved lid hygiene until the exposure is corrected surgically. All measures to promote corneal epithelial health and integrity, including topical lubricants and eyelid closure should be pursued aggressively. While soft BCL may prove useful in promoting epithelial healing and adhesion, they may also predispose to corneal infections by virtue of relative stasis of the tears and routine use of preservative free G Chloramphinicol with soft BCL does not eliminate the possibility of microbial keratitis. During episodes of active ocular surface inflammation, punctal occlusion should be discouraged until inflammation is controlled, to allow the drainage of tears with inflammatory cytokines.</p>

Descompresi&#x000f3;n microvascular en neuralgia del trig&#x000e9;mino: Reporte de 36 casos y revisi&#x000f3;n de la literatura

<sec id="st1"><title>Objetivo:</title><p>El prop&#x000f3;sito del presente trabajo es presentar los resultados de 36 pacientes con diagn&#x000f3;stico de neuralgia del trig&#x000e9;mino (NT), en los cuales se realiz&#x000f3; una descompresi&#x000f3;n microvascular (DMV).</p></sec><sec id="st2"><title>Material y M&#x000e9;todo:</title><p>Desde junio de 2005 a mayo de 2012, 36 pacientes con diagn&#x000f3;stico de NT fueron operados por el primer autor (AC), realizando una DMV. Se evalu&#x000f3;: Edad, sexo, tiempo de sintomatolog&#x000ed;a previo a la cirug&#x000ed;a, hallazgos intraoperatorios (a trav&#x000e9;s de los videos quir&#x000fa;rgicos), y resultados postoperatorios.</p></sec><sec id="st3"><title>Resultados:</title><p>De los 36 pacientes operados, 25 fueron mujeres y 11 varones. El promedio de edad fue de 48 a&#x000f1;os. El seguimiento postoperatorio fue en promedio de 38 meses. De los 36 pacientes, 32 (88%) evolucionaron sin dolor hasta la fecha. De los 4 casos con recurrencia de dolor, en dos pacientes se observ&#x000f3; como hallazgo intraoperatorio un conflicto venoso.</p></sec><sec id="st4"><title>Conclusi&#x000f3;n:</title><p>La DMV como tratamiento de la NT es un procedimiento efectivo y seguro. El hallazgo intraoperatorio de una &#x0201c;compresi&#x000f3;n&#x0201d; venosa podr&#x000ed;a indicar una evoluci&#x000f3;n postoperatoria desfavorable.</p></sec>

<contrib contrib-type="author"><name><surname>Campero</surname><given-names>Alvaro</given-names></name><email xlink:href="alvarocampero@yahoo.com.ar">alvarocampero@yahoo.com.ar</email><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Ajler</surname><given-names>Pablo</given-names></name><email xlink:href="pablo.ajler@hospitalitaliano.com.ar">pablo.ajler@hospitalitaliano.com.ar</email><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Campero</surname><given-names>Abraham Agust&#x000ed;n</given-names></name><email xlink:href="camperoa@asunt.org.ar">camperoa@asunt.org.ar</email><xref ref-type="aff" rid="aff2">2</xref></contrib>

Surgical Neurology International

<sec id="sec1-1"><title>INTRODUCCI&#x000d3;N</title><p>La descompresi&#x000f3;n microvascular (DMV) para el tratamiento de la neuralgia del trig&#x000e9;mino (NT), primeramente reportada por Gardner en el a&#x000f1;o 1962,[<xref rid="ref3" ref-type="bibr">3</xref>] fue popularizada por Jannetta a partir de 1967.[<xref rid="ref4" ref-type="bibr">4</xref>] La tasa de remisi&#x000f3;n del dolor luego de una DMV, en los primeros meses, es mayor al 80%,[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref25" ref-type="bibr">25</xref>] con una incidencia de recurrencia entre el 15 y 30%.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref25" ref-type="bibr">25</xref>] La mayor incidencia de recurrencia se da en los primeros 2 a&#x000f1;os luego de la cirug&#x000ed;a.[<xref rid="ref25" ref-type="bibr">25</xref>]</p><p>La NT es un desorden neurop&#x000e1;tico caracterizado por episodios de intenso dolor facial, originados por el nervio trig&#x000e9;mino.[<xref rid="ref11" ref-type="bibr">11</xref>] Basado en la hip&#x000f3;tesis que una compresi&#x000f3;n vascular es la causa de la NT primaria, la curaci&#x000f3;n sin necesidad de tratamiento m&#x000e9;dico se puede lograr, en la mayor&#x000ed;a de los casos, con una DMV.[<xref rid="ref11" ref-type="bibr">11</xref>] M&#x000e1;s a&#x000fa;n, muchas publicaciones muestran que cuanto mayor es la compresi&#x000f3;n vascular sobre el nervio, mejor es el resultado postoperatorio.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>] Como alternativa de la DMV existen t&#x000e9;cnicas menos invasivas (compresi&#x000f3;n con bal&#x000f3;n, termolesi&#x000f3;n por radiofrecuencia, radiocirug&#x000ed;a, etc.), mostrando no obstante una mayor tasa de recurrencia.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref22" ref-type="bibr">22</xref>]</p><p>El prop&#x000f3;sito del presente trabajo es presentar los resultados de 36 pacientes con diagn&#x000f3;stico de NT, en los cuales se realiz&#x000f3; una DMV.</p></sec><sec id="sec1-2"><title>MATERIAL Y M&#x000c9;TODO</title><p>Desde Junio de 2005 a Mayo de 2012, 36 pacientes con diagn&#x000f3;stico de NT primaria fueron operados por el primer autor (AC), realizando una DMV. Las historias cl&#x000ed;nicas y los videos quir&#x000fa;rgicos fueron analizados, evaluando: Edad, sexo, tiempo de sintomatolog&#x000ed;a previo a la cirug&#x000ed;a, hallazgos intraoperatorios (a trav&#x000e9;s de los videos quir&#x000fa;rgicos), y resultados postoperatorios.</p><p>Todos los pacientes de la presente serie llegaron a la consulta luego de realizar tratamiento m&#x000e9;dico con una o m&#x000e1;s drogas. Carbamazepina fue la medicaci&#x000f3;n m&#x000e1;s utilizada, combin&#x000e1;ndose en algunos casos con amitriptilina, pregabalina, gabapentin o baclofeno. En un caso, el paciente hab&#x000ed;a realizado una termolesi&#x000f3;n percut&#x000e1;nea, con buen resultado por 18 meses. La decisi&#x000f3;n quir&#x000fa;rgica fue por falta de eficacia del tratamiento m&#x000e9;dico en 31 casos (86%), por efectos adversos de la medicaci&#x000f3;n en 4 casos (11%), y por falta de eficacia del tratamiento m&#x000e9;dico y de la radiofrecuencia en 1 caso (2,7%).</p><sec id="sec2-1"><title>T&#x000e9;cnica Quir&#x000fa;rgica [<xref ref-type="fig" rid="F1">Figura 1</xref>].</title><fig id="F1" position="float"><label>Figura 1</label><caption><p>Fotos del abordaje. (a) se encuentra marcada la incisi&#x000f3;n, la ranura dig&#x000e1;strica y la ap&#x000f3;fisis mastoides. (b) foto luego de la disecci&#x000f3;n subperi&#x000f3;stica. (c) exposici&#x000f3;n dural. (d) apertura dural</p></caption><graphic xlink:href="SNI-5-441-g001"/></fig><p>El paciente es colocado en posici&#x000f3;n semisentada, con la cabeza flexionada y rotada hacia el lado del abordaje. La incisi&#x000f3;n es de aproximadamente 10 cm de longitud, vertical, 1 cm medial al borde posterior de la ranura dig&#x000e1;strica. Luego de una disecci&#x000f3;n subperi&#x000f3;stica, se realiza una craniectom&#x000ed;a de aproximadamente 3 cm de di&#x000e1;metro, exponiendo los m&#x000e1;rgenes de los senos transverso y sigmoideo. Se realiza una apertura dural de forma curva, siguiendo el borde interno del seno lateral. Despu&#x000e9;s de colocado el microscopio, se accede a la regi&#x000f3;n del nervio trig&#x000e9;mino reclinando el cerebelo en el borde entre su superficie tentorial y petrosa. Una vez visualizado, el nervio es expuesto en todo su recorrido cisternal, con especial inter&#x000e9;s en la zona de entrada (a nivel de la protuberancia). Una vez encontrado el conflicto vascular, se coloca el tefl&#x000f3;n separando el vaso del nervio. Se ilustra el presente trabajo con 9 casos con fotos intraoperatorias [Figuras <xref ref-type="fig" rid="F1">2</xref> a <xref ref-type="fig" rid="F10">10</xref>]. Finalmente la duramadre es cerrada en forma herm&#x000e9;tica.</p><fig id="F2" position="float"><label>Figura 2</label><caption><p>Compresi&#x000f3;n de arteria cerebelosa superior. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n (doble)</p></caption><graphic xlink:href="SNI-5-441-g002"/></fig><fig id="F3" position="float"><label>Figura 3</label><caption><p>Compresi&#x000f3;n de arteria cerebelosa anteroinferior. (a) vena en relaci&#x000f3;n con el nervio trig&#x000e9;mino. (b) compresi&#x000f3;n arterial (m&#x000e1;s proximal). (c) luego de colocado el tefl&#x000f3;n</p></caption><graphic xlink:href="SNI-5-441-g003"/></fig><fig id="F4" position="float"><label>Figura 4</label><caption><p>Compresi&#x000f3;n de arteria cerebelosa anteroinferior en paciente de 16 a&#x000f1;os de edad. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n (doble)</p></caption><graphic xlink:href="SNI-5-441-g004"/></fig><fig id="F5" position="float"><label>Figura 5</label><caption><p>Compresi&#x000f3;n de arteria cerebelosa anteroinferior en paciente que luego de la cirug&#x000ed;a present&#x000f3; sordera. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n. El asterisco muestra la marca de la compresi&#x000f3;n en el nervio</p></caption><graphic xlink:href="SNI-5-441-g005"/></fig><fig id="F6" position="float"><label>Figura 6</label><caption><p>Compresi&#x000f3;n venosa. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n. Se coloc&#x000f3; el mismo en la parte que la vena presentaba mayor contacto con el nervio</p></caption><graphic xlink:href="SNI-5-441-g006"/></fig><fig id="F7" position="float"><label>Figura 7</label><caption><p>Compresi&#x000f3;n venosa. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n; (b) luego de colocado el tefl&#x000f3;n. Se coloc&#x000f3; el mismo en la parte que la vena presentaba mayor contacto con el nervio. (c) imagen durante la re cirug&#x000ed;a; se observa la correcta colocaci&#x000f3;n del tefl&#x000f3;n. (d) luego de la rizotom&#x000ed;a del tercio externo del nervio trig&#x000e9;mino</p></caption><graphic xlink:href="SNI-5-441-g007"/></fig><fig id="F8" position="float"><label>Figura 8</label><caption><p>Compresi&#x000f3;n doble, de arteria cerebelosa superior y arteria cerebelosa anteroinferior. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n (se colocaron dos teflones)</p></caption><graphic xlink:href="SNI-5-441-g008"/></fig><fig id="F9" position="float"><label>Figura 9</label><caption><p>Compresi&#x000f3;n doble, de arteria cerebelosa superior y arteria basilar. (a) imagen antes de la colocaci&#x000f3;n del tefl&#x000f3;n. (b) luego de colocado el tefl&#x000f3;n (se colocaron dos teflones)</p></caption><graphic xlink:href="SNI-5-441-g009"/></fig><fig id="F10" position="float"><label>Figura 10</label><caption><p>Paciente con compresi&#x000f3;n de arteria cerebelosa anteroinferior, con recurrencia del dolor. Se re oper&#x000f3;. (a) imagen de la re cirug&#x000ed;a, donde se aprecia como el tefl&#x000f3;n se corri&#x000f3; de su lugar de origen. (b) luego de colocado un nuevo tefl&#x000f3;n</p></caption><graphic xlink:href="SNI-5-441-g010"/></fig></sec></sec><sec id="sec1-3"><title>RESULTADOS</title><p>De los 36 pacientes operados, 25 fueron mujeres y 11 varones. El promedio de edad fue de 48 a&#x000f1;os (m&#x000ed;nimo de 16 y m&#x000e1;ximo de 73 a&#x000f1;os). Desde el comienzo del dolor a la cirug&#x000ed;a, el tiempo promedio transcurrido fue de 6,6 a&#x000f1;os (m&#x000ed;nimo de 5 meses y m&#x000e1;ximo de 14 a&#x000f1;os).</p><p>Los hallazgos operatorios fueron los siguientes: (a) 22 casos con compresi&#x000f3;n de la arteria cerebelosa superior; (b) 10 casos con compresi&#x000f3;n de la arteria cerebelosa anteroinferior; (c) 2 casos con compresi&#x000f3;n venosa; (d) 2 casos con compresi&#x000f3;n doble (arteria cerebelosa superior + arteria cerebelosa anteroinferior en un caso y arteria cerebelosa superior + arteria basilar en otro caso).</p><p>El seguimiento postoperatorio fue en promedio de 38 meses (m&#x000e1;ximo de 76 meses y m&#x000ed;nimo de 11 meses). De los 36 pacientes, 32 (88%) evolucionaron sin dolor hasta la fecha. De los 4 pacientes (12%) con recurrencia de dolor, en dos pacientes se observ&#x000f3; como hallazgo intraoperatorio un conflicto venoso; uno de esos pacientes se re oper&#x000f3;, realizando una rizotom&#x000ed;a de V3, lo cual fue efectivo s&#x000f3;lo por 1 mes [<xref ref-type="fig" rid="F7">Figura 7</xref>]. El otro paciente con conflicto venoso, con recurrencia del dolor un mes posterior a la cirug&#x000ed;a, no regres&#x000f3; luego a la consulta [<xref ref-type="fig" rid="F6">Figura 6</xref>]. El tercer caso fue una paciente donde recurri&#x000f3; el dolor al cabo de 3 a&#x000f1;os de la cirug&#x000ed;a. Se volvi&#x000f3; a colocar carbamazepina, y hasta la fecha se encuentra sin dolor (lleva 9 meses). El cuarto caso fue una paciente con compresi&#x000f3;n de arteria cerebelosa anteroinferior, que recurri&#x000f3; el dolor luego de 16 meses de la cirug&#x000ed;a; no mejor&#x000f3; con medicaci&#x000f3;n. Se re oper&#x000f3;, constat&#x000e1;ndose que el tefl&#x000f3;n se hab&#x000ed;a corrido del lugar. Se coloc&#x000f3; un nuevo tefl&#x000f3;n, y hasta el momento se encuentra sin dolor (lleva 4 meses) [<xref ref-type="fig" rid="F10">Figura 10</xref>].</p><p>En cuanto a las complicaciones postoperatorias, la mortalidad fue del 0%. En relaci&#x000f3;n a la morbilidad de la t&#x000e9;cnica quir&#x000fa;rgica, dos pacientes (5,5%) presentaron una f&#x000ed;stula de l&#x000ed;quido cefalorraqu&#x000ed;deo por la herida quir&#x000fa;rgica, que se solucion&#x000f3; en ambos casos con un drenaje espinal colocado durante 7 d&#x000ed;as. Adem&#x000e1;s, un paciente (2,7%) present&#x000f3; en el postoperatorio sordera, la cual no mejor&#x000f3; con el correr del tiempo. No se observ&#x000f3; d&#x000e9;ficit postoperatorio del nervio facial en ning&#x000fa;n caso. Tampoco hubo en la presente serie infecci&#x000f3;n de la herida quir&#x000fa;rgica ni meningitis.</p></sec><sec id="sec1-4"><title>DISCUSI&#x000d3;N</title><p>La hip&#x000f3;tesis de un conflicto arteria-nervio como causa de la NT fue postulada originalmente por Dandy en el a&#x000f1;o 1934;[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref23" ref-type="bibr">23</xref>] dicha idea fue avalada y difundida tiempo despu&#x000e9;s por Jannetta, gracias a los hallazgos intraoperatorios a trav&#x000e9;s del microscopio.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>]</p><p>Si bien el primer tratamiento en la NT es la terapia m&#x000e9;dica, usualmente a trav&#x000e9;s de carbamazepina,[<xref rid="ref6" ref-type="bibr">6</xref>] la baja tasa de control del dolor a largo plazo, asociado a los efectos adversos de la propia medicaci&#x000f3;n hicieron que la mayor&#x000ed;a de los pacientes terminen recibiendo un tratamiento quir&#x000fa;rgico (percut&#x000e1;neo o microquir&#x000fa;rgico).[<xref rid="ref16" ref-type="bibr">16</xref>] En los &#x000fa;ltimos a&#x000f1;os se agreg&#x000f3; a las posibilidades terap&#x000e9;uticas la radiocirug&#x000ed;a.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref24" ref-type="bibr">24</xref>]</p><p>Lo m&#x000e1;s frecuente de ver es una compresi&#x000f3;n arterial, generalmente a trav&#x000e9;s de una arteria cerebelosa. Sin embargo, la compresi&#x000f3;n venosa ha sido reportada entre un 9 y 25%.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref16" ref-type="bibr">16</xref>] Incluso hay trabajos que hablan de un &#x000ed;ndice de recurrencia m&#x000e1;s alto en casos de compresi&#x000f3;n venosa.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref14" ref-type="bibr">14</xref>] En nuestra serie dos pacientes presentaron un conflicto venoso (5,5%); en dichos 2 pacientes se coloc&#x000f3; tefl&#x000f3;n entre la vena y el nervio, no siendo efectivo el procedimiento.</p><p>La ausencia de alg&#x000fa;n tipo de compresi&#x000f3;n vascular fue vista entre un 3 y 17%.[<xref rid="ref16" ref-type="bibr">16</xref>] En nuestros casos, todos presentaron alg&#x000fa;n tipo de conflicto vascular (94,5% compresi&#x000f3;n arterial y 5,5% compresi&#x000f3;n venosa), similar al trabajo de Tucer y colaboradores, donde una compresi&#x000f3;n vascular fue detectada en el 100% de los casos.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Rughani y colaboradores utilizaron el <italic>Nationwide Impatient Sample</italic> para obtener informaci&#x000f3;n sobre 3273 pacientes que se les realiz&#x000f3; una DMV en Estados Unidos; as&#x000ed;, evaluaron la relaci&#x000f3;n entre edad y morbilidad/mortalidad. La mortalidad fue de 0,13% para pacientes menores de 65 a&#x000f1;os, 0,68% para pacientes entre 65 y 75 a&#x000f1;os, y de 1,16% para pacientes mayores de 75 a&#x000f1;os.[<xref rid="ref8" ref-type="bibr">8</xref>] Si bien la DMV contin&#x000fa;a siendo una buena alternativa en pacientes de edad avanzada, es importante tener presente que el riesgo de complicaciones aumenta con los a&#x000f1;os. La opci&#x000f3;n de DMV en pacientes mayores de 65 a&#x000f1;os contin&#x000fa;a siendo viable, si el neurocirujano presenta la experiencia y habilidades necesarias, y si el paciente es totalmente informado del procedimiento, las alternativas y los riesgos.[<xref rid="ref20" ref-type="bibr">20</xref>] En nuestra serie 4 pacientes con m&#x000e1;s de 65 a&#x000f1;os fueron operados, los cuales presentaban una muy buena condici&#x000f3;n cl&#x000ed;nica, por lo cual se decidi&#x000f3; microcirug&#x000ed;a; el resultado fue satisfactorio en los cuatro casos.</p><p>En la presente serie de 36 pacientes no hubo ninguna muerte, dato que concuerda con las publicaciones sobre DMV.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] La morbilidad total fue del 8,3%, siendo dos los pacientes que presentaron f&#x000ed;stula de l&#x000ed;quido cefalorraqu&#x000ed;deo por la herida quir&#x000fa;rgica, las cuales se solucionaron sin cirug&#x000ed;a; y un caso de sordera, la cual fue permanente. Los resultados en cuanto a morbilidad son similares a los encontrados en la literatura.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref25" ref-type="bibr">25</xref>] Tucer y colaboradores, en un estudio con similar n&#x000fa;mero de casos de DMV (37 pacientes), mostraron como complicaciones 2 casos de infecci&#x000f3;n de la herida, un caso de perdida de audici&#x000f3;n (mejor&#x000f3; a los dos meses), un caso de psicosis y un caso de hipoestesia postoperatoria (mejor&#x000f3; a los 6 meses).[<xref rid="ref11" ref-type="bibr">11</xref>]</p></sec><sec id="sec1-5"><title>CONCLUSI&#x000d3;N</title><p>La DMV como tratamiento de la NT es un procedimiento efectivo y seguro. El hallazgo intraoperatorio de una &#x0201c;compresi&#x000f3;n&#x0201d; venosa podr&#x000ed;a indicar una evoluci&#x000f3;n postoperatoria desfavorable.</p></sec>

Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots

Could not extract abstract

<contrib contrib-type="author"><name><surname>Henderson</surname><given-names>Sam W</given-names></name><address><email>sam.henderson@adelaide.edu.au</email></address><xref ref-type="aff" rid="Aff1"/></contrib><contrib contrib-type="author"><name><surname>Baumann</surname><given-names>Ute</given-names></name><address><email>ute.baumann@acpfg.com.au</email></address><xref ref-type="aff" rid="Aff2"/></contrib><contrib contrib-type="author"><name><surname>Blackmore</surname><given-names>Deidre H</given-names></name><address><email>deidre.blackmore@csiro.au</email></address><xref ref-type="aff" rid="Aff3"/></contrib><contrib contrib-type="author"><name><surname>Walker</surname><given-names>Amanda R</given-names></name><address><email>mandy.walker@csiro.au</email></address><xref ref-type="aff" rid="Aff3"/></contrib><contrib contrib-type="author"><name><surname>Walker</surname><given-names>Rob R</given-names></name><address><email>rob.walker@csiro.au</email></address><xref ref-type="aff" rid="Aff3"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Gilliham</surname><given-names>Matthew</given-names></name><address><email>matthew.gilliham@adelaide.edu.au</email></address><xref ref-type="aff" rid="Aff1"/></contrib><aff id="Aff1"><label/>Australian Research Council Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, &#x00026; Waite Research Institute, University of Adelaide, PMB1, Glen Osmond, South Australia, 5064 Australia </aff><aff id="Aff2"><label/>Australian Centre for Plant Functional Genomics, South Australia, 5064 Australia </aff><aff id="Aff3"><label/>CSIRO Plant Industry, PO Box 350, Glen Osmond, South Australia 5064 Australia </aff>

BMC Plant Biology

<sec id="Sec1" sec-type="introduction"><title>Background</title><p>Grapevine (<italic>Vitis vinifera</italic> L.), used for wine, table grape and dried grape production, is an economically important crop plant that is moderately sensitive to salinity [<xref ref-type="bibr" rid="CR1">1</xref>]. Grapevine salt stress symptoms include reduced stomatal conductance, reduced photosynthesis [<xref ref-type="bibr" rid="CR2">2</xref>,<xref ref-type="bibr" rid="CR3">3</xref>] and leaf burn [<xref ref-type="bibr" rid="CR4">4</xref>], which are generally associated with increases in shoot chloride (Cl^&#x02212;) rather than sodium (Na⁺) concentrations [<xref ref-type="bibr" rid="CR3">3</xref>]. Reduced vigour [<xref ref-type="bibr" rid="CR5">5</xref>] and reduced yield [<xref ref-type="bibr" rid="CR6">6</xref>] are further effects of salt stress, with a strong positive correlation between the two [<xref ref-type="bibr" rid="CR5">5</xref>]. Certain non-<italic>vinifera Vitis</italic> spp. rootstocks are used commercially to constrain shoot Cl^&#x02212; accumulation and confer improved salt tolerance to grafted <italic>V. vinifera</italic> scions [<xref ref-type="bibr" rid="CR7">7</xref>,<xref ref-type="bibr" rid="CR8">8</xref>]. Despite a detailed understanding of the physiology of shoot Cl^&#x02212; accumulation in grapevine and other plants, the genes responsible for this process across the plant kingdom are not known [<xref ref-type="bibr" rid="CR9">9</xref>]. This is in contrast to the control of long-distance Na⁺ transport in plants where numerous reports have targeted known genes in order to improve the salt tolerance of plants, particularly cereals e.g. [<xref ref-type="bibr" rid="CR10">10</xref>-<xref ref-type="bibr" rid="CR13">13</xref>]. Due to extensive natural variation in the shoot Cl^&#x02212; exclusion capacity of <italic>Vitis</italic> spp. [<xref ref-type="bibr" rid="CR14">14</xref>,<xref ref-type="bibr" rid="CR15">15</xref>] grapevine represents an ideal model to identify candidate genes involved in controlling shoot Cl^&#x02212; exclusion.</p><p>Solutes travel from the roots to the shoot in the xylem. Physiological studies using radiotracers and fluorescent dyes in grapevine have indicated that the transfer of solutes to the xylem apoplast involves a symplastic step, and that rootstocks confer Cl^&#x02212; exclusion to a grafted scion by reducing net xylem loading of Cl^&#x02212; [<xref ref-type="bibr" rid="CR15">15</xref>,<xref ref-type="bibr" rid="CR16">16</xref>]. Patch clamp studies of xylem parenchyma protoplasts identified the passive quickly activating anion conductance (X-QUAC) as capable of catalysing the majority of Cl^&#x02212; flux to the xylem of barley roots [<xref ref-type="bibr" rid="CR17">17</xref>]. Cl^&#x02212; entry to the root xylem is down-regulated by abscisic acid (ABA), as demonstrated by ³⁶Cl^&#x02212; fluxes in excised roots and whole seedlings of barley [<xref ref-type="bibr" rid="CR18">18</xref>], and reduces X-QUAC of maize xylem parenchyma cells [<xref ref-type="bibr" rid="CR19">19</xref>]. Given that ABA rises in concentration in plant roots exposed to salt stress [<xref ref-type="bibr" rid="CR20">20</xref>], anion transporters expressed in cells that surround the root xylem, especially those that change activity when plants are salt treated are likely to be good targets to explore for improving our understanding how shoot Cl^&#x02212; exclusion is conferred.</p><p>There have been a limited number of studies that have provided insights to the genetic elements that control long-distance transport of Cl^&#x02212;. Like grapevine, <italic>Citrus</italic> spp. are moderately salt-sensitive woody perennial crops frequently grown on salt-excluding rootstocks. Brumos <italic>et al.</italic> [<xref ref-type="bibr" rid="CR21">21</xref>] compared the partial leaf transcriptomes of <italic>Citrus</italic> rootstocks Cleopatra mandarin (a good shoot Cl^&#x02212; excluder) and Carrizo citrange (a poor shoot Cl^&#x02212; excluder) exposed to NaCl and KCl stress using a cDNA microarray covering 6,875 putative unigenes. They concluded that a nitrate (NO₃^&#x02212;) transporter with homology to <italic>GmNRT1-2</italic> from soybean was differentially expressed between rootstocks and therefore was deemed a candidate gene for influencing Cl^&#x02212; movement. Using the same germplasm, Brumos <italic>et al.</italic> [<xref ref-type="bibr" rid="CR22">22</xref>] used quantitative PCR to measure root expression of three candidate genes for the control of long-distance Cl^&#x02212; transport derived from the literature. Candidates included a homolog of a cation chloride co-transporter (<italic>CcCCC1</italic>), <italic>CcICln1</italic> (a putative regulator of chloride channel conductance) and <italic>CcSLAH1</italic>, a homolog of the plant guard cell slow anion channels (SLAC) [<xref ref-type="bibr" rid="CR22">22</xref>]. Of these genes <italic>SLAH1</italic> was more highly expressed in the chloride accumulating rootstock under 90&#x000a0;mM NaCl stress. In guard cells, SLAC chloride channels meditate ABA induced passive Cl^&#x02212; efflux causing stomatal closure [<xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR24">24</xref>]. SLAC homologs (SLAH) in plant roots are therefore particularly interesting candidates for xylem loading of Cl^&#x02212;, but their role in roots remains uncharacterised. <italic>CCC</italic> was proposed to regulate retrieval of Na⁺, K⁺ and Cl^&#x02212; from the Arabidopsis root xylem but was not regulated transcriptionally by salt [<xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR25">25</xref>]. Furthermore, questions remain as to how CCC can act directly in xylem loading on the plasma membrane due to unfavourable electrochemical gradients [<xref ref-type="bibr" rid="CR9">9</xref>]. <italic>ICln1</italic> homologs from rat and <italic>Xenopus laevis</italic> elicit Cl^&#x02212; currents in voltage clamp experiments [<xref ref-type="bibr" rid="CR26">26</xref>]. In <italic>Citrus</italic>, <italic>ICln1</italic> exhibited strong repression in the Cl^&#x02212; excluder after application of 4.5&#x000a0;mM Cl^&#x02212; [<xref ref-type="bibr" rid="CR22">22</xref>]. However, <italic>ICln</italic> proteins from plants remain uncharacterised. Whilst these genes are good candidates for regulating Cl^&#x02212; transport in <italic>Citrus</italic>, analyses of entire root transcriptomes is likely to provide a more complete list of factors that mediate long-distance transport of Cl^&#x02212;.</p><p>Gene expression studies of <italic>V. vinifera</italic> have been greatly aided by the draft genome sequence of Pinot Noir inbred line PN40024 [<xref ref-type="bibr" rid="CR27">27</xref>,<xref ref-type="bibr" rid="CR28">28</xref>]. These studies have concentrated on berry development [<xref ref-type="bibr" rid="CR29">29</xref>,<xref ref-type="bibr" rid="CR30">30</xref>], leaf responses to heat stress [<xref ref-type="bibr" rid="CR31">31</xref>] and to UV radiation [<xref ref-type="bibr" rid="CR32">32</xref>]. The most comprehensive grapevine expression study to date compared the transcriptome of 54 samples representing different vegetative and reproductive organs at various developmental stages [<xref ref-type="bibr" rid="CR33">33</xref>]. Although abiotic stress was not analysed in this study, grapevine roots were found to express more organ-specific transcripts than leaves [<xref ref-type="bibr" rid="CR33">33</xref>]. This is consistent with findings from Tillett <italic>et al.,</italic> [<xref ref-type="bibr" rid="CR34">34</xref>] who compared large-scale EST libraries from roots and shoots of Cabernet Sauvignon and identified 135 root enriched transcripts. These findings indicate that shoot expression analyses of grapevine, while useful, might not give a complete picture of root gene expression patterns, and therefore studies into root responses to abiotic stresses are required. Two microarray studies have examined the effect of salinity stress on transcript levels of Cabernet Sauvignon shoot tips [<xref ref-type="bibr" rid="CR35">35</xref>,<xref ref-type="bibr" rid="CR36">36</xref>]. Increased levels of a transcript encoding a putative <italic>NRT</italic> were observed, while decreased expression of a chloride channel (<italic>CLC</italic>) with sequence similarity to Arabidopsis <italic>AtCLC-d</italic> was detected by two probe sets, but this was not statistically significant [<xref ref-type="bibr" rid="CR36">36</xref>].</p><p>We performed a comparative microarray of mRNAs derived from roots of salt stressed and control Cabernet Sauvignon, 140 Ruggeri and K51-40 rooted leaves as an unbiased method to identify candidates for long-distance transport of Cl^&#x02212;. We aimed to test the hypothesis that the differences in Cl^&#x02212; exclusion between rootstocks 140 Ruggeri and K51-40 could be due to expression differences in genes that encode membrane transport proteins which facilitate root-to-shoot Cl^&#x02212; translocation. The identification of genes that prevent excessive shoot Cl^&#x02212; accumulation in grapevine will facilitate continued rootstock development by providing genetic markers for rootstock breeding programs. Furthermore, this study will aid a greater understanding of plant Cl^&#x02212; homeostasis by using grapevine as a model species to elucidate genes that underpin the Cl^&#x02212; exclusion trait in plants in general.</p></sec><sec id="Sec2" sec-type="materials|methods"><title>Methods</title><sec id="Sec3"><title>Preparation of rooted-leaves</title><p>Grapevine, being a woody perennial crop, is challenging to use in controlled conditions experiments, especially where large amounts of material and multiple replicates are required. We therefore used the method of Schachtman and Thomas [<xref ref-type="bibr" rid="CR37">37</xref>] where leaves are excised from a parent plant and grown as rooted-leaves. This is consistent with previous studies of Cl^&#x02212; accumulation in vines, where it was demonstrated that root and leaf phenotypes acquired with this system are similar to field observations [<xref ref-type="bibr" rid="CR15">15</xref>,<xref ref-type="bibr" rid="CR16">16</xref>]. Rooted leaves were established from pot-grown grapevines of K51-40 (<italic>Vitis champinii</italic> X <italic>Vitis riparia</italic>), 140 Ruggeri (<italic>Vitis berlandieri</italic> X <italic>Vitis rupestris</italic>) and Cabernet Sauvignon (<italic>Vits vinifera</italic>) established from cuttings and maintained in a glasshouse as described previously [<xref ref-type="bibr" rid="CR15">15</xref>]. After approximately 3&#x000a0;weeks, rooted-leaves were transferred to aerated hydroponic tanks containing modified Hoagland Solution with the following nutrients (in mM) for a two-week pre-treatment period: KNO₃, 1.0; Ca(NO₃)₂&#x02009;&#x000b7;&#x02009;4H₂O, 1.0; MgSO₄&#x02009;&#x000b7;&#x02009;7H₂O, 0.4; KH₂PO₄, 0.2; H₃BO₃, 4.6&#x02009;&#x000d7;&#x02009;10^&#x02212;2; MnCl₂&#x02009;&#x000b7;&#x02009;4H₂O, 9.1&#x02009;&#x000d7;&#x02009;10^&#x02212;3; ZnSO₄&#x02009;&#x000b7;&#x02009;7H₂O, 7.6&#x02009;&#x000d7;&#x02009;10^&#x02212;4; CuSO₄&#x02009;&#x000b7;&#x02009;5H₂O, 3.2&#x02009;&#x000d7;&#x02009;10^&#x02212;4; Na₂MoO₄&#x02009;&#x000b7;&#x02009;2H₂O, 2.4&#x02009;&#x000d7;&#x02009;10^&#x02212;4; EDTA-Fe-Na, 7.1&#x02009;&#x000d7;&#x02009;10^&#x02212;2 (pH&#x000a0;6.5) [<xref ref-type="bibr" rid="CR15">15</xref>].</p></sec><sec id="Sec4"><title>Response of intact rooted-leaves to short term salinity</title><p>Rooted-leaves of K51-40, 140 Ruggeri and Cabernet Sauvignon were subjected to nutrient solution only (control) or to 50&#x000a0;mM Cl^&#x02212; (Na⁺: Ca²⁺: Mg²⁺ = 6:1:1) in nutrient solution for 4&#x000a0;days. At harvest, the rooted-leaves of each genotype were washed in de-ionised water, blotted dry with paper towel, weighed, then separated into lamina, petiole and roots. Fresh weights of all plant parts were also obtained. Samples were divided equally for RNA extraction and ion composition analysis. Samples for RNA extraction were snap frozen in liquid nitrogen and stored at minus 80&#x000b0;C. Root, petiole and lamina samples for ion analysis were weighed before being dried in an oven at 60&#x000b0;C and retained for Cl^&#x02212; analysis.</p><p>For stele and cortex expression studies roots were salt-treated and harvested as described above, lateral roots were removed from main roots and then cortex was stripped from stele of the main root using fine tweezers. Three biological replicates were harvested, each consisting of dissected tissue from three rooted-leaves. Tissue samples were immediately frozen in liquid nitrogen and stored at minus 80&#x000b0;C for RNA extraction.</p></sec><sec id="Sec5"><title>Ion analyses</title><p>Laminae, petiole and root samples were dried at 60&#x000b0;C for at least 72&#x000a0;h and ground to a fine powder using a mortar and pestle. Cl^&#x02212; concentration was measured by silver ion titration with a chloridometer (Model 442&#x02013;5150, Buchler Instruments, Lenexa, Kansas, USA) from extracts prepared by digesting 20&#x02013;100&#x000a0;mg dry samples in 4&#x000a0;mL of acid solution containing 10% (v/v) acetic acid and 0.1&#x000a0;M nitric acid overnight before analysis.</p></sec><sec id="Sec6"><title>RNA extraction</title><p>Frozen root tissues were ground to a fine powder in liquid nitrogen using a mortar and pestle. RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma, St. Louis, Missouri, USA) following the manufacturer&#x02019;s protocol. RNA was DNase I treated with Turbo DNA-free (Life Technologies, Carlsbad, California, USA) for 1&#x000a0;hour at 37&#x000b0;C to remove contaminating genomic DNA. RNA was precipitated at minus 80&#x000b0;C overnight in 5 volumes of 100% ethanol (v/v) and 1/10 volumes of 3&#x000a0;M NaOAC. After ethanol precipitation, RNA was resuspended in nuclease free water and analysed on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Only RNA samples with 260/280 and 260/230 absorbance ratios greater than 1.8 were used. RNA integrity was screened on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA) and only RNA samples with an RNA integrity number (RIN) above 8.5 were used.</p></sec><sec id="Sec7"><title>Microarray chip design, labelling and hybridisation</title><p>Custom 8x60K gene expression microarrays were designed using eArray (Release 7.6) (Agilent Technologies). Oligonucleotide probes (60-mers) were designed against 26,346 annotated <italic>V. vinifera</italic> transcripts from the 12x Genoscope build available from <ext-link ext-link-type="uri" xlink:href="http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/">http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/</ext-link>. The Agilent 60-mer probe format is considered more tolerant to sequence mismatches than 25-mers, and more suitable for analysis of polymorphic DNA sequences [<xref ref-type="bibr" rid="CR38">38</xref>]. Also, the use of a custom Agilent expression array enabled us to print a subset of probes for 90 putative anion transporters multiple times on the array (Additional file <xref rid="MOESM1" ref-type="media">1</xref>). This multi-probe approach increases the robustness of the expression values obtained when the probes for these genes are averaged. Probes that detect differential gene expression many times show a greater probability of genuine differential expression when the B-statistic probability (log-odds) of differential gene expression is calculated. The higher the B-statistic, the greater the chance that the gene is differentially expressed (B-statistic = 0 represents 50:50 chance of differential gene expression).</p><p>Twenty-two microarrays were used which consisted of 4 biological replicates for Cabernet Sauvignon (&#x000b1;50&#x000a0;mM Cl^&#x02212;), 4 biological replicates of K51-40 (&#x000b1;50&#x000a0;mM Cl^&#x02212;) and 3 biological replicates of 140 Ruggeri (&#x000b1;50&#x000a0;mM Cl^&#x02212;). Each biological replicate consisted of roots from 4 rooted-leaves pooled together. Single colour labelling, hybridisations and image analysis were performed at the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Australia).</p></sec><sec id="Sec8"><title>Functional annotation of genes</title><p>Gene functional annotation, which included InterPro descriptions, Gene Ontology terms and Arabidopsis orthologs, was obtained from BioMart at EnsemblPlants (<ext-link ext-link-type="uri" xlink:href="http://plants.ensembl.org/biomart/martview/">plants.ensembl.org/biomart/martview/</ext-link>)<italic>.</italic> Additional functional annotation was gathered from Grimplet <italic>et al.</italic> [<xref ref-type="bibr" rid="CR39">39</xref>], and this annotation was used for the tables and figures presented in this manuscript.</p></sec><sec id="Sec9"><title>Microarray data analysis</title><p>Scanned images were analysed with Feature Extraction Software 10.7.3 (Agilent Technologies, Santa Clara, California, USA) and the Cy3 median signal intensities for each spot on the arrays were imported into R for further processing. The data was log(2) transformed and quantile normalized. Since the microarray hybridizations were performed at different dates we observed batch effects that we corrected for with the ComBat package [<xref ref-type="bibr" rid="CR40">40</xref>]. The quality of the microarray hybridisation and reproducibility amongst biological replicates was validated using arrayQualityMetrics version 3.12.0 [<xref ref-type="bibr" rid="CR41">41</xref>]. Differentially expressed genes were identified using the Linear Model for Microarray Data (LIMMA) package [<xref ref-type="bibr" rid="CR42">42</xref>], and the Benjamini and Hochberg correction method was applied to account for multiple testing [<xref ref-type="bibr" rid="CR43">43</xref>]. To filter the probes, the probe sequences were blasted against the predicted cDNAs of the 12xV1 genome sequence at EnsemblPlants. Probes with an e-value &#x02265;1&#x000d7;10^&#x02212;10 and probes that showed no blast hit were excluded from the initial analyses. Gene expression changes were considered significant when a threshold fold change of greater than or equal to 1.41 was reached (log(2) FC &#x000b1;0.5) and a false discovery rate (FDR) corrected probability of <italic>P</italic> &#x02264;0.05. The raw data for the microarray are available at the Gene Expression Omnibus database (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>) under accession number GSE57770.</p><p>Hierarchical clustering and co-expression analysis was performed using Genesis 1.7.6 [<xref ref-type="bibr" rid="CR44">44</xref>] using tab delimited text files of the log(2) fold change values of gene expression of averaged probes. Transcripts and experiments were clustered using the average linkage method. Singular enrichment analysis was performed using Agrigo [<xref ref-type="bibr" rid="CR45">45</xref>]. At the time of writing, the Agrigo server is incompatible with 12xV1 <italic>V. vinifera</italic> gene IDs. Therefore transcripts that were differentially expressed (identified after filtering) were entered into the Agrigo server using the 12xV0 transcript ID&#x02019;s (Genoscope). The hypergeometric method with Hochberg (FDR) multi-test adjustment was used to identify statistically significant GO terms (<italic>P</italic> &#x0003c;0.05).</p></sec><sec id="Sec10"><title>Phylogenetic analyses</title><p><italic>V. vinifera</italic> protein sequences of interest were obtained from EnsemblPlants using the 12xV1 gene IDs. <italic>V. vinifera</italic> amino acid sequences were used as a query in a protein-protein BLAST (blastp) at the National Centre for Biotechnology Information (NCBI) against non-redundant protein sequences limited to <italic>Arabidopsis thaliana</italic> (taxid: 3702). Arabidopsis sequences with the best total score were reciprocally blasted at EnsemblPlants against the <italic>Vitis vinifera</italic> peptide database. Arabidopsis and grapevine sequences that were obtained using this approach were aligned using Clustal W2 [<xref ref-type="bibr" rid="CR46">46</xref>]. Phylogenetic trees were generated with Geneious 6.1.2 (Biomatters) using the neighbour-joining method and the Jukes-Cantor genetic distance model. A consensus tree was generated by re-sampling 1000 times using the bootstrap method. Branch lengths are proportional to the amount of divergence between nodes in units of substitutions per site. Gene identifiers for the protein sequences used are shown in Additional file <xref rid="MOESM2" ref-type="media">2</xref>, while the multiple sequence alignment is shown in Additional file <xref rid="MOESM3" ref-type="media">3</xref>.</p></sec><sec id="Sec11"><title>Quantitative real-time PCR (qRT-PCR)</title><p>One microgram of total RNA was reverse transcribed in a 20&#x000a0;&#x003bc;L reaction using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, California, USA). The procedure was modified from the manufacturer&#x02019;s to include an initial RNA denaturation step of 65&#x000b0;C for 5&#x000a0;minutes then incubation on ice for 1&#x000a0;minute, and cDNA synthesis step of 42&#x000b0;C for 1&#x000a0;hour. cDNA was diluted 1 in 5. Two microliters of cDNA was used as a template for PCR and qRT-PCR reactions. PCR targets were first amplified from cDNA using KAPA taq (KAPA Biosystems, Woburn, Massachusetts, USA) following manufacturer&#x02019;s procedures. Fragments of the correct size and target were confirmed by agarose gel and sequencing. PCR fragments, or linearised plasmid containing the PCR fragment, were serially diluted and used as a template for qRT-PCR in duplicate. Standard curves were generated using iCycler iQ optical system software version 3.1 (Bio-Rad), which also calculates the reaction efficiency of each primer pair using the formula E = 10^-1/slope. qRT-PCR was performed on a Bio-Rad iCycler. Reactions consisted of 250 nM forward and reverse primer, 1x KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems), and 2&#x000a0;&#x003bc;L of diluted cDNA. Reactions were performed in triplicate following a three-step protocol consisting of 40&#x000a0;cycles of the following: 95&#x000b0;C 15&#x000a0;sec, 56&#x000b0;C 20&#x000a0;sec, 72&#x000b0;C 10&#x000a0;sec (plus data acquisition). Melt curve analysis was performed by heating PCR products from 52&#x000b0;C to 92&#x000b0;C for 20&#x000a0;seconds increasing at 0.5&#x000b0;C per cycle with continuous fluorescence detection. Relative expression ratios were calculated using the primer pair efficiency and the formula described by Pfaffl [<xref ref-type="bibr" rid="CR47">47</xref>], with the geometric mean of <italic>VvActin1</italic>, <italic>VvUbiquitin-L40</italic> and <italic>VvElongation-factor-1-&#x003b1;</italic> used as the reference for normalisation [<xref ref-type="bibr" rid="CR48">48</xref>]. Normalised expression values were transformed to log(2) values for comparison with microarray data. Primers used for qRT-PCR are listed in Additional file <xref rid="MOESM4" ref-type="media">4</xref>. Primers were designed using Primer3 [<xref ref-type="bibr" rid="CR49">49</xref>]. Primers were designed to amplify single products from the target gene between 140 and 250&#x000a0;bp with an optimal GC content of 50% and, where possible, designed to span an intron to ensure that cDNA targets were amplified. Before their use, primers were screened for potential non-selective amplification using PrimerBLAST at NCBI against the Refseq RNA database limited to <italic>Vitis</italic> species.</p></sec></sec><sec id="Sec12" sec-type="results"><title>Results</title><sec id="Sec13"><title>Salt treatment, grapevine growth and ion accumulation</title><p>Following 4-days of 50&#x000a0;mM Cl^&#x02212; treatment, roots of 140 Ruggeri retained significantly more Cl^&#x02212; compared to those of Cabernet Sauvignon and K51-40 (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>A). Conversely, Cabernet Sauvignon and K51-40 petioles and laminae accumulated significantly more Cl^&#x02212; compared to 140 Ruggeri (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>B and C). K51-40 accumulated the highest amount of Cl^&#x02212; in aerial tissues under salt stress (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>B and C). Under control conditions, 140 Ruggeri also accumulated significantly less petiole and laminae Cl^&#x02212; than K51-40, indicating that the Cl^&#x02212; exclusion mechanism may be active in low Cl^&#x02212; conditions (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>B and C). Overall, the shoot Cl^&#x02212; accumulation of varieties can be expressed as 140 Ruggeri&#x02009;&#x0003c;&#x02009;Cabernet Sauvignon&#x02009;&#x0003c;&#x02009;K51-40.<fig id="Fig1"><label>Figure 1</label><caption><p> <bold>Differential chloride accumulation in tissues of different</bold> <bold><italic>Vitis</italic></bold> <bold>spp.</bold> Chloride concentration (% dry weight) in the roots <bold>(A)</bold>, petiole <bold>(B)</bold> and laminae <bold>(C)</bold> of hydroponically grown rooted-leaves under control (white bars) or 50&#x000a0;mM Cl^&#x02212; (black bars) conditions. Bars represent the mean&#x02009;&#x000b1;&#x02009;SEM of 4 biological replicates. Statistical significance was determined by one-way ANOVA with Bonferroni post-hoc test (<italic>P</italic> &#x0003c;0.05). CS&#x02009;=&#x02009;Cabernet Sauvignon, 140 R = 140 Ruggeri.</p></caption><graphic xlink:href="12870_2014_273_Fig1_HTML" id="MO1"/></fig></p></sec><sec id="Sec14"><title>Validation of microarray data using real-time quantitative PCR (qRT-PCR)</title><p>To validate the microarray expression data and further quantify mRNA expression levels, we measured the expression of 12 genes by qRT-PCR and compared the datasets. Expression ratios of genes from control and 50&#x000a0;mM Cl^&#x02212; treated samples were analysed by linear regression and an R² value of 0.88 was observed, indicating good correlation (Additional file <xref rid="MOESM5" ref-type="media">5</xref>a). Similarly, qRT-PCR and microarray ratios for 12 genes were compared between varieties under control conditions, which provided an R² value of 0.89, also demonstrating good correlation (Additional file <xref rid="MOESM5" ref-type="media">5</xref>b).</p></sec><sec id="Sec15"><title>Differentially expressed genes due to chloride stress</title><p>Following Cl^&#x02212; stress 1361 unique genes were differentially expressed in at least one grapevine variety (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>, Additional file <xref rid="MOESM6" ref-type="media">6</xref>). The number of differentially expressed genes due to Cl^&#x02212; treatment was positively correlated with Cl^&#x02212; accumulation in shoot tissues. The Cl^&#x02212; accumulator K51-40 had the highest number of Cl^&#x02212; responsive transcripts (817), followed by the intermediate accumulator Cabernet Sauvignon (511), while the Cl^&#x02212; excluder 140 Ruggeri had the least number of Cl^&#x02212; responsive transcripts (252) (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>). This correlation is consistent with findings in <italic>Citrus</italic> leaves when salt tolerant and sensitive rootstocks were compared after salt stress [<xref ref-type="bibr" rid="CR21">21</xref>].<fig id="Fig2"><label>Figure 2</label><caption><p> <bold>Transcriptomic response of</bold> <bold><italic>Vitis</italic></bold> <bold>spp. to 50&#x000a0;mM Cl</bold> ^{<bold>&#x02212;</bold>} <bold>treatment.</bold> Venn diagram showing the number of significantly differentially expressed unique transcripts predicted by the 12xV1 annotation of the <italic>V. vinifera</italic> genome in Cabernet Sauvignon, 140 Ruggeri and K51-40 roots under 50&#x000a0;mM Cl^&#x02212; stress. Significance was determined as <italic>P</italic> &#x0003c;0.05, &#x02265;1.41-fold change.</p></caption><graphic xlink:href="12870_2014_273_Fig2_HTML" id="MO2"/></fig></p></sec><sec id="Sec16"><title>Cluster analysis</title><p>The transcript profiles of Cabernet Sauvignon, 140 Ruggeri and K51-40 roots exposed to high Cl^&#x02212; were grouped by hierarchical clustering (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>). 140 Ruggeri and Cabernet Sauvignon had the most similar transcriptional response to Cl^&#x02212; in roots, while the Cl^&#x02212; includer K51-40 had a unique response (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>, top dendrogram). Gene clusters were examined by singular enrichment analysis (SEA) of gene ontology (GO) terms. Three clusters of interest showed enrichment of GO biological processes (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>). Other gene clusters showed no significant enrichment of GO terms.<fig id="Fig3"><label>Figure 3</label><caption><p> <bold>Hierarchical clustering of chloride responsive transcripts in grapevine roots.</bold> Transcripts (rows) that changed in response to 50&#x000a0;mM Cl^&#x02212; in at least one variety with a fold change&#x02009;&#x02265;&#x02009;&#x000b1;1.41 (<italic>P</italic> &#x0003c;0.05) were clustered. The response of each grapevine variety (columns) was also grouped (dendrogram above). Log(2) fold changes not statistically significant were set to 0. Clusters of interest are shown to the right of the heatmap, and contain genes that responded uniquely in each variety <bold>(A, B and C)</bold>. Expression profiles and enriched GO biological processes for each cluster are also shown to the right of the heat map. CS&#x02009;=&#x02009;Cabernet Sauvignon, 140 R =140 Ruggeri.</p></caption><graphic xlink:href="12870_2014_273_Fig3_HTML" id="MO3"/></fig></p><p>In 140 Ruggeri, Cl^&#x02212; treatment induced the expression of transcripts involved in abiotic stress tolerance (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>, Cluster A), including glutathione-S-transferases (GST) and heat shock proteins (HSP) (Additional file <xref rid="MOESM7" ref-type="media">7</xref>). Overexpression of GSTs in tobacco enhances growth under salt stress [<xref ref-type="bibr" rid="CR50">50</xref>], while HSPs act as molecular chaperones that help maintain correct protein conformation under abiotic stress [<xref ref-type="bibr" rid="CR51">51</xref>]. These unique trancriptional changes might enable 140 Ruggeri to perform better under salt stress relative to other grapevine genotypes.</p><p>In K51-40, Cl^&#x02212; treatment repressed genes involved in the hypersensitive response and flavonoid biosynthesis (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>, Cluster B; Additional file <xref rid="MOESM8" ref-type="media">8</xref>). Flavonoids have diverse roles in plants including scavenging of reactive oxygen species (ROS) and pathogen defence [<xref ref-type="bibr" rid="CR52">52</xref>]. Under salt stress, leakage of photosynthetic and respiratory electrons may react with oxygen, leading to ROS production and subsequent oxidative stress [<xref ref-type="bibr" rid="CR53">53</xref>]. Therefore the transcriptional regulation of flavonoid biosynthesis in K51-40 might prevent damage from excessive ROS production. In Cabernet Sauvignon, Cl^&#x02212; treatment repressed mitochondrial specific transcripts, such as NADH dehydrogenases, c-type cytochromes and pentatricopeptide repeat (PPR) domain proteins (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>, Cluster C; Additional file <xref rid="MOESM9" ref-type="media">9</xref>). Transcriptional repression of respiratory transcripts in Cabernet Sauvignon probably functions to reduce ROS production.</p><p>The stress-inducible phytohormone ABA restricts anion entry to the root xylem [<xref ref-type="bibr" rid="CR18">18</xref>] and inward anion currents (anion efflux) from xylem parenchyma protoplasts from barley [<xref ref-type="bibr" rid="CR17">17</xref>] and maize [<xref ref-type="bibr" rid="CR19">19</xref>]. We therefore investigated whether high Cl^&#x02212; treatment reduces the expression of genes likely to facilitate Cl^&#x02212; transport to aerial tissues of 140 Ruggeri. Only four membrane transporters were repressed in 140 Ruggeri upon Cl^&#x02212; treatment and none were predicted to facilitate anion movement across membranes (Additional file <xref rid="MOESM10" ref-type="media">10</xref>).</p></sec><sec id="Sec17"><title>Transcriptional differences between grapevine varieties under control conditions</title><p>Given that Cl^&#x02212; accumulation in shoot tissues was significantly different between grapevine varieties in the absence of salt stress (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>B and C), we hypothesised that there might be a difference in gene expression of anion transporters under control conditions. Under these conditions, 4527 genes were differentially expressed between 140 Ruggeri and K51-40 with approximately half (2310 genes) being lower in 140 Ruggeri (Additional file <xref rid="MOESM11" ref-type="media">11</xref>). Genes encoding 214 membrane integral proteins were expressed differently between roots of K51-40 and 140 Ruggeri (Additional file <xref rid="MOESM12" ref-type="media">12</xref>). Multigene families have been proposed as regulators of salt tolerance and anion homeostasis in plants, including <italic>NRT</italic>, <italic>ALMT</italic>, <italic>SLAH</italic> and <italic>CLC</italic> [<xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR54">54</xref>]. Members from these and other gene families encoding membrane proteins, as well as possible regulatory proteins, that were expressed differently between rootstocks, are summarised (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) and described below. As an alternative analysis, genes with a high B-statistic (log-odds) for differential expression between rootstocks are listed in Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>.<table-wrap id="Tab1"><label>Table 1</label><caption><p> <bold>Differentially expressed genes between contrasting rootstocks encoding putative solute transporters under control conditions</bold> </p></caption><table frame="hsides" rules="groups"><thead><tr><th> <bold>Probe ID</bold> </th><th> <bold>12xV1 blast hit</bold> </th><th> <bold>12xV0 gene ID</bold> </th><th> <bold>Arabidopsis homolog</bold> </th><th> <bold>Log(2) FC 140R - K51-40</bold> </th><th> <bold>p-value</bold> </th><th> <bold>Functional annotation</bold> </th></tr></thead><tbody><tr><td>CUST_15333_17284</td><td>VIT_02s0012g01270</td><td>GSVIVT01013161001</td><td>AT4G17870</td><td>1.41</td><td>1.11E-09</td><td>Abscisic acid receptor PYL1 RCAR11</td></tr><tr valign="top"><td>NG2_36172_20391</td><td>VIT_06s0080g00170</td><td>GSVIVT01036162001</td><td>AT1G08440</td><td>&#x02212;0.69</td><td>3.02E-04</td><td>Aluminum activated malate transporter 1</td></tr><tr valign="top"><td>CUST_44694_7793</td><td>VIT_06s0009g00450</td><td>GSVIVT01037570001</td><td>AT1G08440</td><td>0.78</td><td>1.90E-03</td><td>Aluminum activated malate transporter 1</td></tr><tr valign="top"><td>CUST_46237_21897</td><td>VIT_08s0105g00250</td><td>GSVIVT01011148001</td><td>AT3G11680</td><td>1.30</td><td>2.51E-04</td><td>Aluminum activated malate transporter 1</td></tr><tr valign="top"><td>CUST_8680_62299</td><td>VIT_11s0052g00320</td><td>GSVIVT01029283001</td><td>AT4G29900</td><td>&#x02212;0.70</td><td>3.31E-04</td><td>Calcium ATPase 10 (ACA10), plasma membrane</td></tr><tr valign="top"><td>NG2_12175_47390</td><td>VIT_07s0129g00180</td><td>GSVIVT01000123001</td><td>AT4G37640</td><td>0.58</td><td>8.86E-03</td><td>Calcium ATPase 2 (ACA2), plasma membrane</td></tr><tr valign="top"><td>CUST_16133_33172</td><td>VIT_07s0129g00110</td><td>GSVIVT01000116001</td><td>AT4G37640</td><td>0.66</td><td>1.65E-02</td><td>Calcium ATPase 2 (ACA2), plasma membrane</td></tr><tr valign="top"><td>NG2_35892_10569</td><td>VIT_06s0004g06570</td><td>GSVIVT01024741001</td><td>AT3G51860</td><td>1.44</td><td>5.00E-12</td><td>Calcium/proton exchanger CAX3</td></tr><tr valign="top"><td>CUST_50946_56104</td><td>VIT_02s0025g04520</td><td>GSVIVT01019868001</td><td>AT1G12580</td><td>0.70</td><td>2.43E-05</td><td>Calcium-dependent protein kinase 1 CPK protein kinase</td></tr><tr valign="top"><td>CUST_17465_49753</td><td>VIT_08s0032g00780</td><td>GSVIVT01022524001</td><td>AT2G38910</td><td>0.52</td><td>2.67E-02</td><td>Calcium-dependent protein kinase 20 CPK20</td></tr><tr valign="top"><td>CUST_25785_57840</td><td>VIT_18s0001g00980</td><td>GSVIVT01008747001</td><td>-</td><td>&#x02212;0.61</td><td>7.21E-03</td><td>Calcium-dependent protein kinase 9 CPK9</td></tr><tr valign="top"><td>CUST_45042_37341</td><td>VIT_15s0021g01150</td><td>GSVIVT01018316001</td><td>AT1G28710</td><td>&#x02212;1.59</td><td>4.74E-07</td><td>Calcium-dependent protein kinase-related</td></tr><tr valign="top"><td>CUST_46046_19308</td><td>VIT_01s0010g02150</td><td>GSVIVT01010291001</td><td>AT1G14590</td><td>1.08</td><td>3.25E-07</td><td>Calcium-dependent protein kinase-related</td></tr><tr valign="top"><td>CUST_25533_22696</td><td>VIT_05s0020g04240</td><td>GSVIVT01018059001</td><td>AT5G57110</td><td>0.76</td><td>7.26E-04</td><td>Calcium ATPase 12 (ACA12)</td></tr><tr valign="top"><td>CUST_38995_37629</td><td>VIT_14s0030g02090</td><td>GSVIVT01021803001</td><td>AT3G63380</td><td>1.41</td><td>2.60E-05</td><td>Calcium ATPase 12 (ACA12)</td></tr><tr valign="top"><td>CUST_40093_46251</td><td>VIT_05s0020g04260</td><td>GSVIVT01018061001</td><td>AT3G22910</td><td>&#x02212;0.63</td><td>9.81E-03</td><td>Calcium ATPase 13 (ACA13)</td></tr><tr valign="top"><td>NG2_7370_1539</td><td>VIT_09s0018g01840</td><td>GSVIVT01016118001</td><td>AT3G13320</td><td>0.99</td><td>1.10E-05</td><td>Cation exchanger (CAX2)</td></tr><tr valign="top"><td>CUST_43832_58554</td><td>VIT_08s0056g01480</td><td>GSVIVT01029961001</td><td>AT5G17860</td><td>0.97</td><td>4.47E-03</td><td>Cation exchanger (CAX7)</td></tr><tr valign="top"><td>NG11_49713_18843</td><td>VIT_14s0068g02190</td><td>GSVIVT01033108001</td><td>AT3G27170</td><td>&#x02212;0.61</td><td>7.55E-05</td><td>Chloride channel B (CLC-b)</td></tr><tr valign="top"><td>NG11_46088_11883</td><td>VIT_19s0015g01850</td><td>GSVIVT01014852001</td><td>AT1G55620</td><td>1.37</td><td>2.85E-34</td><td>Chloride channel F (CLC-f)</td></tr><tr valign="top"><td>NG11_51750_10097</td><td>VIT_06s0004g03520</td><td>GSVIVT01025107001</td><td>AT3G45650</td><td>1.27</td><td>7.60E-19</td><td>Nitrate excretion transporter 1</td></tr><tr valign="top"><td>NG11_44542_25973</td><td>VIT_06s0004g03530</td><td>GSVIVT01025106001</td><td>AT3G45650</td><td>1.61</td><td>1.24E-32</td><td>Nitrate excretion transporter 2</td></tr><tr valign="top"><td>NG11_46422_21127</td><td>VIT_11s0016g05170</td><td>GSVIVT01015522001</td><td>AT2G26690</td><td>&#x02212;1.22</td><td>2.58E-19</td><td>Nitrate transporter 1.4</td></tr><tr valign="top"><td>CUST_37073_22417</td><td>VIT_01s0127g00070</td><td>GSVIVT01013802001</td><td>AT1G12940</td><td>0.63</td><td>6.29E-03</td><td>Nitrate transporter 2.5</td></tr><tr valign="top"><td>CUST_42271_1540</td><td>VIT_14s0066g00850</td><td>GSVIVT01032430001</td><td>AT5G14570</td><td>1.59</td><td>8.82E-06</td><td>Nitrate transporter 2.7</td></tr><tr valign="top"><td>NG2_12101_30038</td><td>VIT_03s0097g00510</td><td>GSVIVT01038513001</td><td>AT5G64410</td><td>0.87</td><td>1.65E-06</td><td>Oligopeptide transporter OPT4</td></tr><tr valign="top"><td>NG12_21396_16431</td><td>VIT_12s0035g01820</td><td>GSVIVT01023146001</td><td>AT1G59740</td><td>0.52</td><td>2.47E-05</td><td>Proton-dependent oligopeptide transport (POT) family protein</td></tr><tr valign="top"><td>NG11_4749_12704</td><td>VIT_17s0000g05550</td><td>GSVIVT01008072001</td><td>AT3G47960</td><td>0.54</td><td>1.88E-04</td><td>Glucosinolate transporter 1 (GTR1)</td></tr><tr valign="top"><td>NG11_7897_10153</td><td>VIT_14s0066g02020</td><td>GSVIVT01032550001</td><td>AT5G14940</td><td>0.64</td><td>3.31E-07</td><td>Proton-dependent oligopeptide transport (POT) family protein</td></tr><tr valign="top"><td>NG11_35177_1429</td><td>VIT_18s0041g00670</td><td>GSVIVT01026058001</td><td>AT1G72140</td><td>0.89</td><td>7.91E-14</td><td>Proton-dependent oligopeptide transport (POT) family protein</td></tr><tr valign="top"><td>NG11_25530_14040</td><td>VIT_04s0008g03580</td><td>GSVIVT01035643001</td><td>AT1G22550</td><td>1.11</td><td>2.21E-24</td><td>Nitrate transporter 1.11</td></tr><tr valign="top"><td>NG11_31776_20297</td><td>VIT_16s0050g01860</td><td>GSVIVT01028789001</td><td>AT5G24030</td><td>0.54</td><td>4.61E-05</td><td>SLAH3 (SLAC1 Homologue 3)</td></tr><tr valign="top"><td>CUST_21950_56777</td><td>VIT_07s0191g00070</td><td>GSVIVT01003419001</td><td>AT4G40010</td><td>&#x02212;1.01</td><td>1.28E-04</td><td>SNF1-related protein kinase 2.7 (SnRK2.7)</td></tr><tr valign="top"><td>CUST_41758_42394</td><td>VIT_00s0710g00020</td><td>GSVIVT01002389001</td><td>AT4G33950</td><td>&#x02212;0.56</td><td>1.54E-02</td><td>SNF1-related protein kinase 2.6 (SnRK2.6)</td></tr><tr valign="top"><td>CUST_27252_1533</td><td>VIT_01s0011g06550</td><td>GSVIVT01011573001</td><td>AT2G01980</td><td>&#x02212;2.30</td><td>1.53E-05</td><td>SOS1 (Na+/H&#x02009;+&#x02009;antiporter)</td></tr><tr valign="top"><td>CUST_15165_41173</td><td>VIT_06s0004g07830</td><td>GSVIVT01024587001</td><td>AT5G58380</td><td>&#x02212;0.73</td><td>1.72E-06</td><td>SOS2 (salt overly sensitive 2)</td></tr><tr valign="top"><td>CUST_27642_7432</td><td>VIT_16s0098g01870</td><td>GSVIVT01038549001</td><td>AT5G24270</td><td>&#x02212;0.67</td><td>8.65E-03</td><td>SOS3 (salt overly sensitive 3)</td></tr></tbody></table><table-wrap-foot><p>List of significantly differentially expressed genes (<italic>P</italic> &#x0003c;0.05, &#x02265; &#x000b1;1.41 fold) between the contrasting grapevine rootstocks 140 Ruggeri and K51-40 in the absence of Cl^&#x02212; treatment that have putative roles in ion homeostasis. Positive log(2) FC values&#x02009;=&#x02009;higher in 140 Ruggeri.</p></table-wrap-foot></table-wrap><table-wrap id="Tab2"><label>Table 2</label><caption><p> <bold>Highly significantly differentially expressed genes between contrasting rootstocks under control conditions</bold> </p></caption><table frame="hsides" rules="groups"><thead><tr><th> <bold>Probe ID</bold> </th><th> <bold>12xV1 blast hit</bold> </th><th> <bold>12xV0 gene ID</bold> </th><th> <bold>Arabidopsis homolog</bold> </th><th> <bold>Log2 FC 140 R - K51-40</bold> </th><th> <bold>p-value</bold> </th><th> <bold>B</bold> </th><th> <bold>Functional annotation</bold> </th></tr></thead><tbody><tr><td>NG11_47168_24630</td><td>VIT_09s0002g02430</td><td>GSVIVT01016879001</td><td>AT3G21250</td><td>&#x02212;1.90</td><td>1.69E-42</td><td>89.62</td><td>ABC transporter C member 12</td></tr><tr valign="top"><td>NG11_46088_11883</td><td>VIT_19s0015g01850</td><td>GSVIVT01014852001</td><td>AT1G55620</td><td>1.37</td><td>2.85E-34</td><td>70.44</td><td>CLCf (chloride channel F)</td></tr><tr valign="top"><td>NG11_44542_25973</td><td>VIT_06s0004g03530</td><td>GSVIVT01025106001</td><td>AT3G45650</td><td>1.61</td><td>1.24E-32</td><td>66.55</td><td>Nitrate excretion transporter 2</td></tr><tr valign="top"><td>NG2_21308_18913</td><td>VIT_11s0016g02570</td><td>GSVIVT01015240001</td><td>AT2G19690</td><td>3.01</td><td>1.05E-26</td><td>56.42</td><td>Phospholipase A2 precursor</td></tr><tr valign="top"><td>NG2_12381_40127</td><td>VIT_06s0004g06340</td><td>GSVIVT01024768001</td><td>AT5G58800</td><td>3.35</td><td>5.46E-26</td><td>54.67</td><td>Flavodoxin-like quinone reductase 1</td></tr><tr valign="top"><td>NG2_21123_37199</td><td>VIT_12s0028g02740</td><td>GSVIVT01020642001</td><td>-</td><td>&#x02212;4.06</td><td>1.36E-24</td><td>51.28</td><td>Isoflavone methyltransferase/Orcinol O-methyltransferase 1 oomt1</td></tr><tr valign="top"><td>NG11_25530_14040</td><td>VIT_04s0008g03580</td><td>GSVIVT01035643001</td><td>-</td><td>1.11</td><td>2.21E-24</td><td>47.44</td><td>Nitrate transporter 1.11</td></tr><tr valign="top"><td>NG2_12165_35517</td><td>VIT_13s0073g00250</td><td>GSVIVT01034634001</td><td>AT2G26230</td><td>&#x02212;5.07</td><td>9.34E-23</td><td>46.84</td><td>Urate oxidase</td></tr><tr valign="top"><td>NG2_48691_28703</td><td>VIT_15s0046g01950</td><td>GSVIVT01026987001</td><td>-</td><td>2.97</td><td>4.01E-22</td><td>45.28</td><td>Anthocyanidine rhamnosyl-transferase</td></tr><tr valign="top"><td>NG2_28672_23579</td><td>VIT_10s0003g03780</td><td>GSVIVT01021513001</td><td>AT1G30130</td><td>2.18</td><td>7.23E-22</td><td>44.65</td><td>Unknown protein</td></tr><tr valign="top"><td>NG2_35994_23405</td><td>VIT_18s0001g13850</td><td>GSVIVT01009855001</td><td>AT4G31500</td><td>&#x02212;3.20</td><td>1.93E-21</td><td>43.62</td><td>Cytochrome P450, family 83, subfamily B, polypeptide 1</td></tr><tr valign="top"><td>NG2_48494_21157</td><td>VIT_18s0001g13820</td><td>GSVIVT01009854001</td><td>AT4G31500</td><td>&#x02212;3.32</td><td>1.78E-19</td><td>38.90</td><td>Cytochrome P450, family 83, subfamily B, polypeptide 1</td></tr><tr valign="top"><td>NG2_5431_23220</td><td>VIT_00s0153g00040</td><td>GSVIVT01001251001</td><td>-</td><td>&#x02212;2.73</td><td>1.90E-19</td><td>38.83</td><td>S-locus receptor kinase</td></tr><tr valign="top"><td>NG2_48249_3223</td><td>VIT_03s0038g01760</td><td>GSVIVT01024088001</td><td>-</td><td>3.48</td><td>2.76E-19</td><td>38.44</td><td>Disease resistance protein (CC-NBS class)</td></tr><tr valign="top"><td>NG2_33320_2332</td><td>VIT_08s0007g01590</td><td>GSVIVT01034034001</td><td>-</td><td>&#x02212;1.80</td><td>5.70E-19</td><td>37.65</td><td>Fructose 1,6-bisphosphatase</td></tr><tr valign="top"><td>NG2_21199_29690</td><td>VIT_06s0004g00730</td><td>GSVIVT01025431001</td><td>AT3G13550</td><td>2.00</td><td>8.19E-19</td><td>37.26</td><td>Ubiquitin-conjugating enzyme E2 D/E</td></tr><tr valign="top"><td>NG2_11819_5360</td><td>VIT_05s0094g00120</td><td>GSVIVT01038099001</td><td>AT3G59600</td><td>2.61</td><td>3.10E-18</td><td>35.84</td><td>DNA-directed RNA polymerase II subunit H</td></tr><tr valign="top"><td>NG11_46422_21127</td><td>VIT_11s0016g05170</td><td>GSVIVT01015522001</td><td>AT2G26690</td><td>&#x02212;1.22</td><td>2.58E-19</td><td>35.59</td><td>Nitrate transporter 1.4</td></tr><tr valign="top"><td>NG2_12023_10427</td><td>VIT_18s0001g05430</td><td>GSVIVT01036371001</td><td>-</td><td>3.07</td><td>4.99E-18</td><td>35.33</td><td>(+)-delta-cadinene synthase isozyme XC14</td></tr><tr valign="top"><td>NG2_45557_29139</td><td>VIT_10s0042g01130</td><td>GSVIVT01026257001</td><td>AT4G19670</td><td>3.69</td><td>7.83E-18</td><td>34.85</td><td>Zinc finger (C3HC4-type ring finger)</td></tr><tr valign="top"><td>NG2_40716_2133</td><td>VIT_06s0061g00120</td><td>GSVIVT01031543001</td><td>-</td><td>3.17</td><td>7.99E-18</td><td>34.82</td><td>Beta-1,3-glucanase [<italic>Vitis riparia</italic>]</td></tr><tr valign="top"><td>NG11_51750_10097</td><td>VIT_06s0004g03520</td><td>GSVIVT01025107001</td><td>AT3G45650</td><td>1.27</td><td>7.60E-19</td><td>34.49</td><td>Nitrate excretion transporter 1</td></tr><tr valign="top"><td>NG2_45497_36221</td><td>VIT_12s0028g02810</td><td>GSVIVT01020636001</td><td>-</td><td>&#x02212;1.58</td><td>2.64E-17</td><td>33.55</td><td>Isoflavone methyltransferase/Orcinol O-methyltransferase 1 oomt1</td></tr><tr valign="top"><td>NG2_6989_23958</td><td>VIT_06s0004g05440</td><td>GSVIVT01024878001</td><td>AT2G29260</td><td>&#x02212;1.23</td><td>3.46E-17</td><td>33.27</td><td>Tropinone reductase</td></tr><tr valign="top"><td>NG2_5127_34182</td><td>VIT_03s0097g00620</td><td>GSVIVT01038529001</td><td>AT5G64440</td><td>1.71</td><td>4.59E-17</td><td>32.98</td><td>N-acylethanolamine amidohydrolase</td></tr><tr valign="top"><td>NG2_7581_45053</td><td>VIT_06s0080g00800</td><td>GSVIVT01036089001</td><td>AT5G22360</td><td>2.01</td><td>7.43E-17</td><td>32.47</td><td>Vesicle-associated membrane protein 714</td></tr><tr valign="top"><td>NG2_12628_32903</td><td>VIT_10s0071g00440</td><td>GSVIVT01034406001</td><td>AT4G11900</td><td>&#x02212;3.24</td><td>7.90E-17</td><td>32.40</td><td>Serine/threonine-protein kinase receptor ARK3</td></tr><tr valign="top"><td>NG2_48742_21119</td><td>VIT_08s0007g09030</td><td>GSVIVT01033230001</td><td>-</td><td>&#x02212;1.40</td><td>2.03E-16</td><td>31.39</td><td>DnaJ homolog, subfamily A, member 5</td></tr><tr valign="top"><td>NG2_575_20076</td><td>VIT_16s0098g01670</td><td>GSVIVT01038570001</td><td>AT5G53070</td><td>1.57</td><td>3.23E-16</td><td>30.90</td><td>Ribosomal protein L9</td></tr><tr valign="top"><td>NG2_5167_10137</td><td>VIT_05s0029g00770</td><td>GSVIVT01020981001</td><td>-</td><td>&#x02212;1.67</td><td>3.40E-16</td><td>30.84</td><td>Nematode resistance-like protein</td></tr><tr valign="top"><td>NG2_12777_24696</td><td>VIT_18s0117g00080</td><td>GSVIVT01012796001</td><td>AT5G36930</td><td>3.34</td><td>3.95E-16</td><td>30.68</td><td>R protein L6</td></tr><tr valign="top"><td>NG2_5559_11115</td><td>VIT_02s0025g00930</td><td>GSVIVT01019469001</td><td>AT3G59140</td><td>&#x02212;1.63</td><td>5.03E-16</td><td>30.43</td><td>Multidrug resistance-associated protein 14</td></tr><tr valign="top"><td>NG2_36555_51297</td><td>VIT_03s0088g00390</td><td>GSVIVT01037045001</td><td>AT5G23590</td><td>1.34</td><td>6.02E-16</td><td>30.24</td><td>DnaJ homolog, subfamily C, member 17</td></tr></tbody></table><table-wrap-foot><p>Highly significantly differentially expressed unique genes (<italic>P</italic> &#x0003c;0.05, &#x02265; &#x000b1;1.41 fold, B &#x0003e;30) between 140 Ruggeri and K51-40 root tissue under control conditions identified using the B-statistic. Positive log(2) FC values&#x02009;=&#x02009;higher in 140 Ruggeri.</p></table-wrap-foot></table-wrap></p></sec><sec id="Sec18"><title><bold><italic>NRT</italic></bold>/<bold><italic>POT</italic></bold></title><p>The NRT or proton dependent oligopeptide (POT) gene family is involved in the acquisition and whole plant homeostasis of nitrogen; different family members transport NO₃^&#x02212;, amino acids and various peptides [<xref ref-type="bibr" rid="CR55">55</xref>]. In our study, 8 <italic>NRT1</italic> genes were expressed differently between rootstocks (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Grapevine <italic>NRT1</italic> gene family members were poorly annotated in functional databases. To assign putative functions, we produced a phylogeny of the grapevine <italic>NRT</italic>s uncovered in our microarray screen using Arabidopsis <italic>NRT1</italic>s. Homologs of <italic>AtNRT1.4</italic>, <italic>AtNRT1.11</italic>, <italic>nitrate excretion transporter 1 (AtNAXT1), AtNAXT2</italic> and <italic>glucosinolate transporter 1</italic> (<italic>AtGTR1)</italic> were identified, as well as three other <italic>Vitis NRT</italic>s with uncharacterised Arabidopsis homologs (Figure&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>). Two grapevine <italic>NRT</italic>s homologous to Arabidopsis <italic>AtNRT2.5</italic> and <italic>AtNRT2.7</italic>, as well as a homolog of Arabidopsis oligopeptide transporter 4 (OPT4) were more abundantly expressed in 140 Ruggeri (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Differential expression of <italic>VvNAXT1</italic>, <italic>VvNAXT2</italic>, <italic>VvNRT1.11</italic> (all higher in 140 Ruggeri) and <italic>VvNRT1.4</italic> (higher in K51-40) was also highly significant (Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>).<fig id="Fig4"><label>Figure 4</label><caption><p> <bold>Phylogenetic relationship between Arabidopsis and grapevine</bold> <bold><italic>NRT/POT</italic></bold> <bold>gene family members.</bold> Unrooted neighbour-joining tree of Arabidopsis and grapevine (bold) <italic>NRT/POT</italic> family members with bootstrap values from 1000 iterations. Scale&#x02009;=&#x02009;substitutions per site. Gene identifiers for the protein sequences used are shown in Additional file <xref rid="MOESM2" ref-type="media">2</xref>, while the multiple sequence alignment is shown in Additional file <xref rid="MOESM3" ref-type="media">3</xref>.</p></caption><graphic xlink:href="12870_2014_273_Fig4_HTML" id="MO4"/></fig></p><p>In Arabidopsis roots, <italic>AtNRT1.8</italic> is induced and <italic>AtNRT1.5</italic> repressed by salt and cadmium stress [<xref ref-type="bibr" rid="CR56">56</xref>]. AtNRT1.5 is the only NRT1 isoform with a confirmed role in root xylem loading of NO₃^&#x02212; [<xref ref-type="bibr" rid="CR57">57</xref>], and mutants of <italic>atnrt1.5</italic> grow better under NaCl stress than wildtype [<xref ref-type="bibr" rid="CR58">58</xref>]. Conversely, AtNAXT1 effluxes NO₃^&#x02212; under acid load, and is regulated at the post-transcriptional level [<xref ref-type="bibr" rid="CR59">59</xref>]. We further investigated expression patterns of <italic>Vitis</italic> orthologs of these genes. <italic>VvNRT1.8</italic> and <italic>VvNRT1.5</italic> were identified phylogenetically (Figure&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>). They were oppositely regulated by salt stress in Cabernet Sauvignon and 140 Ruggeri, but not K51-40, although the expression changes were small (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>A and B). <italic>VvNAXT1</italic> was unresponsive to salt in all three genotypes (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>C), which is consistent with the response of its homolog in Arabidopsis [<xref ref-type="bibr" rid="CR59">59</xref>]. Interestingly, <italic>VvNRT1.4</italic> was strongly repressed (3 fold) by salt stress in K51-40 (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>D). In spite of these differences in salt response, the largest transcriptional differences in grapevine <italic>NRT1</italic> mRNAs were observed between genotypes under control conditions, especially between the contrasting rootstocks 140 Ruggeri and K51-40 (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>E &#x02013; H). This suggests of a role of some of these genes in Cl^&#x02212; exclusion in the absence of stress (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>). Arabidopsis <italic>AtNRT1.5</italic> is considered important for plant salt tolerance [<xref ref-type="bibr" rid="CR58">58</xref>], possibly due a role in anion loading to the xylem [<xref ref-type="bibr" rid="CR57">57</xref>]. In grapevine, <italic>VvNRT1.5</italic> was not preferentially expressed in the root stele under salt stress (Additional file <xref rid="MOESM13" ref-type="media">13</xref>), which contrasts with <italic>AtNRT1.5</italic> [<xref ref-type="bibr" rid="CR57">57</xref>]. Furthermore, <italic>VvNRT1.5</italic> was more abundant in 140 Ruggeri than K51-40 (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>F; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>B). These data reduce the likelihood of VvNRT1.5 having a role in xylem loading of Cl^&#x02212; in grapevine. Based on transcriptional data, we suggest that <italic>VvNRT1.4</italic> is the best <italic>NRT1</italic> candidate for xylem loading of Cl^&#x02212; due to a much greater abundance in K51-40 roots under control conditions (Figure&#x000a0;<xref rid="Fig5" ref-type="fig">5</xref>D; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>C).<fig id="Fig5"><label>Figure 5</label><caption><p> <bold>mRNA expression changes of four</bold> <bold><italic>Vitis NRT1</italic></bold> <bold>family members in three grapevine genotypes under salt stress and control conditions. (A &#x02013; D)</bold> Log2 mRNA fold changes of <italic>VvNRT1.8</italic> <bold>(A)</bold> <italic>VvNRT1.5</italic> <bold>(B)</bold> <italic>VvNAXT1</italic> <bold>(C)</bold> <italic>VvNRT1.4</italic> <bold>(D)</bold> in response to 50&#x000a0;mM Cl^&#x02212; treatment as determined by qRT-PCR (filled symbols) and microarray hybridisation (open symbols). <bold>(E &#x02013; H)</bold> Log2 mRNA fold differences of <italic>VvNRT1.8</italic> <bold>(E)</bold> <italic>VvNRT1.5</italic> <bold>(F)</bold> <italic>VvNAXT1</italic> <bold>(G)</bold> <italic>VvNRT1.4</italic> <bold>(H)</bold> between grapevine genotypes under control conditions as determined by qRT-PCR (filled symbols) and microarray hybridisation (open symbols). For qRT-PCR data points, the bars represent the mean&#x02009;&#x000b1;&#x02009;SEM of four biological replicates. CS&#x02009;=&#x02009;Cabernet Sauvignon, 140 R = 140 Ruggeri. The E-value of <italic>VvNRT1.5</italic> probe is above the threshold used for all other probes analysed in this study.</p></caption><graphic xlink:href="12870_2014_273_Fig5_HTML" id="MO5"/></fig><fig id="Fig6"><label>Figure 6</label><caption><p> <bold>Relative transcript abundances of membrane proteins in roots of grapevine genotypes under control conditions measured by qRT-PCR, and a model indicating possible molecular mechanisms for reduced net xylem loading of Cl</bold> ^{<bold>&#x02212;</bold>} <bold>in 140 Ruggeri. (A &#x02013; B)</bold> relative expression levels of <italic>VvNAXT1</italic> <bold>(A)</bold> and <italic>VvNRT1.5</italic> <bold>(B)</bold> measured by qRT-PCR, which represent possible avenues for cortical or epidermal efflux of Cl^&#x02212; out of roots. <bold>(C &#x02013; E)</bold> relative expression levels of <italic>VvNRT1.4</italic> <bold>(C)</bold>, <italic>VvALMT1</italic> <bold>(D)</bold> and <italic>VvSLAH3</italic> <bold>(E)</bold> measured by qRT-PCR, which represent possible avenues for xylem loading of Cl^&#x02212;. Bars represent the mean of four biological replicates&#x02009;&#x000b1;&#x02009;SEM. Transcript abundance is relative to the Cabernet Sauvignon biological replicate with the lowest cycle threshold (Ct) value, which was set to 1. Statistical differences were determined using one way ANOVA with Holm-Sidak's multiple comparisons test to compare the means. <bold>(F - G)</bold> proposed model for reduced net xylem loading of Cl^&#x02212; in 140 Ruggeri relative to K51-40. <bold>(F)</bold> In 140 Ruggeri, anion efflux from cortical or epidermal root cells could be mediated through putative anion channels VvNRT1.5 and VvNAXT1 which are transcriptionally more abundant in the Cl^&#x02212; excluder. Xylem loading of Cl^&#x02212; could be restricted through reduced VvNRT1.4 abundance, or inhibition of VvSLAH3 and VvALMT by higher [Ca²⁺]_cyt mediated by VvCAX3 (directly, or in partnership with Ca²⁺ dependent protein kinases). <bold>(G)</bold> In K51-40, anion efflux to the xylem apoplast could be enhanced through increased abundance of VvALMT1 and VvNRT1.4, and activation of VvALMT1 and VvSLAH3 by SnRK2 kinases.</p></caption><graphic xlink:href="12870_2014_273_Fig6_HTML" id="MO6"/></fig></p></sec><sec id="Sec19"><title>ALMT</title><p>Chelation of toxic aluminium in the rhizosphere by the efflux of organic acids from roots is facilitated by plasma membrane aluminium-activated malate transporters (ALMT) [<xref ref-type="bibr" rid="CR60">60</xref>]. ALMTs are a large multigene family with multiple roles; despite their name most ALMTs are not activated by aluminium and they allow the permeation of various anions. For example, ALMTs function in anion homeostasis and mineral nutrition, (ZmALMT1) [<xref ref-type="bibr" rid="CR61">61</xref>], or Cl^&#x02212; transport across the tonoplast (AtALMT9) [<xref ref-type="bibr" rid="CR62">62</xref>]. Root ALMTs might therefore have a role in Cl^&#x02212; exclusion. Three <italic>ALMT1</italic> homologs were differentially expressed between rootstocks (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Whether these proteins mediate Cl^&#x02212; fluxes, and the directionality of such fluxes, remains unresolved, but Cl^&#x02212; exclusion could arise through efflux of Cl^&#x02212; to the rhizosphere (higher expression in 140 Ruggeri, <italic>VIT_06s0009g00450</italic>, <italic>VIT_08s0105g00250</italic>) (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) or reduced Cl^&#x02212; entry in the cortex and restricted xylem loading of Cl^&#x02212; (lower expression in 140 Ruggeri, <italic>VIT_06s0080g00170</italic>) (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>D and G).</p></sec><sec id="Sec20"><title>Calcium transporters (CAX and ACA)</title><p>Calcium exchangers (CAX) mediate Ca²⁺/cation antiport activity across the tonoplast. Roles of CAXs include cell specific storage of Ca²⁺ by CAX1 [<xref ref-type="bibr" rid="CR63">63</xref>], while Arabidopsis <italic>cax3</italic> mutants are sensitive to NaCl, LiCl and acidic pH, suggesting a possible role in salt tolerance [<xref ref-type="bibr" rid="CR64">64</xref>]. Three grapevine <italic>CAX</italic> transcripts were more abundant in roots of 140 Ruggeri compared to K51-40 (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). In addition to CAX, the plant plasma and vacuolar membranes harbour auto-inhibited Ca²⁺-ATPases (ACA), of which Arabidopsis <italic>ACA4</italic> can improve salt tolerance of yeast [<xref ref-type="bibr" rid="CR65">65</xref>]. Six <italic>ACA</italic>s were differentially expressed between 140 Ruggeri and K51-40. These data indicate that genes regulating cytosolic free calcium ([Ca²⁺]_cyt) in roots could be important for grapevine Cl^&#x02212; exclusion.</p></sec><sec id="Sec21"><title>CLC</title><p>Two CLCs showed differential expression between rootstocks under control conditions. A gene homologous to Arabidopsis <italic>AtCLCb</italic> (<italic>VIT_14s0068g02190</italic>) was less abundant in 140 Ruggeri (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Another <italic>CLC</italic> with homology to <italic>AtCLCf</italic> (<italic>VIT_19s0015g01850</italic>) was less abundant in K51-40 (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Differential expression of <italic>VvCLCf</italic> was also identified as highly statistically significant (Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>).</p></sec><sec id="Sec22"><title>SLAH3 and ABA signalling</title><p>Homologs of the Arabidopsis SLAC1 anion channel (AtSLAH1 and AtSLAH3) are plasma membrane localized, expressed in the root vasculature, and functionally complement guard cell anion efflux in the <italic>slac1</italic> mutant [<xref ref-type="bibr" rid="CR23">23</xref>]. This indicates that SLAHs might be involved in anion homeostasis [<xref ref-type="bibr" rid="CR23">23</xref>] and loading to the xylem sap [<xref ref-type="bibr" rid="CR54">54</xref>]. <italic>VvSLAH3</italic> was more abundant in the Cl^&#x02212; excluder 140 Ruggeri compared to K51-40 under control conditions (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>E). This contrasts with <italic>Citrus</italic>, where <italic>CcSLAH1</italic> was up-regulated by 90&#x000a0;mM salt stress in a Cl^&#x02212; accumulating rootstock [<xref ref-type="bibr" rid="CR22">22</xref>]. Reconstitution in <italic>X. laevis</italic> oocytes has demonstrated that plant SLAC/SLAH activity is tightly regulated by kinase/phosphatase activity following an ABA signal [<xref ref-type="bibr" rid="CR66">66</xref>]. Homologs of the Arabidopsis ABA signalling machinery were differentially expressed between rootstocks. The ABA receptor <italic>VvPYL1/RCAR11</italic> was more highly expressed in 140 Ruggeri (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Two SNF1-related protein kinase 2 <italic>(SnRK2)</italic> family members including the <italic>Vitis</italic> ortholog of <italic>SnRK2.6</italic> (open stomata 1 (OST1)) were repressed in 140 Ruggeri, and multiple calcium dependent protein kinases (<italic>CPK</italic>) were differentially expressed between rootstocks (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). Homologs of these genes in other plants have proven roles in ABA induced activation of SLAC1 in guard cells [<xref ref-type="bibr" rid="CR67">67</xref>] and might be involved in SLAH3 regulation in <italic>Vitis</italic> roots.</p></sec><sec id="Sec23"><title>Other candidates</title><p>Two ABC transporters were significantly differentially expressed between rootstocks; a C-type (<italic>ABCC 12</italic>) (<italic>VIT_09s0002g02430</italic>) (higher in 140 Ruggeri) and multidrug resistance 12-type (<italic>VIT_02s0025g00930</italic>) (higher in K51-40) (Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>). A role in Cl^&#x02212; transport has not been identified for ABC transporters in plants, although reports suggest roles in arsenic tolerance [<xref ref-type="bibr" rid="CR68">68</xref>] and salt tolerance [<xref ref-type="bibr" rid="CR69">69</xref>]. A C3HC4-type ring finger protein was more abundant in the tolerant variety 140 Ruggeri (Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>). A C3HC4 protein was potentially crucial for abiotic stress tolerance in rice roots [<xref ref-type="bibr" rid="CR70">70</xref>]. A phospholipase A2 precursor (<italic>VIT_11s0016g02570</italic>) was also expressed alternatively between rootstocks (Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>). The product of a phospholipase A2 activates a tonoplast H⁺/Na⁺ antiporter in cultured cells of California poppy [<xref ref-type="bibr" rid="CR71">71</xref>].</p></sec></sec><sec id="Sec24" sec-type="discussion"><title>Discussion</title><p>Shoot chloride exclusion is one of several traits that underpins salt tolerance. However, the root-localised anion transport proteins (or their regulators) thought to be crucial for salt tolerance remain unidentified [<xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR15">15</xref>,<xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR22">22</xref>]. We therefore analysed the genome wide transcriptional response of grapevine roots to Cl^&#x02212; stress. Cabernet Sauvignon repressed transcripts encoding respiratory proteins, probably to reduce ROS production under salt stress. Although ROS may act as signalling molecules in eukaryotes [<xref ref-type="bibr" rid="CR72">72</xref>,<xref ref-type="bibr" rid="CR73">73</xref>], it has previously been reported that ROS production in grapevine cells that contrast in salt-tolerance represents a manifestation of cellular damage rather than an adaptive response [<xref ref-type="bibr" rid="CR74">74</xref>]. We therefore propose a hierarchy exists in the magnitude of transcriptional responses to Cl^&#x02212; stress (K5140&#x0003e;&#x02009;&#x0003e;&#x02009;Cabernet Sauvignon&#x0003e;&#x02009;&#x0003e;&#x02009;140 Ruggeri) that correlates with the amount of damage in the laminae. However, these differences in varietal responses to stress do not explain differential Cl^&#x02212; exclusion, which was statistically significant before salt stress (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>).</p><p>Studies indicate that there is natural variation in the ability to tolerate salt stress in various plant species including <italic>Citrus</italic> [<xref ref-type="bibr" rid="CR22">22</xref>], rice [<xref ref-type="bibr" rid="CR70">70</xref>], barley [<xref ref-type="bibr" rid="CR75">75</xref>,<xref ref-type="bibr" rid="CR76">76</xref>], and Arabidopsis [<xref ref-type="bibr" rid="CR77">77</xref>]. Our data support the hypothesis that Cl^&#x02212; exclusion in grapevine is mediated by anion transporters or channels that are differentially expressed between non-stressed 140 Ruggeri and K51-40. To this end, we have proposed a testable model for Cl^&#x02212; exclusion based on the expression levels of candidate genes identified in our study (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>). These candidate genes for Cl^&#x02212; exclusion are subsequently discussed in the context of this model and existing literature.</p><p>In plant roots, anion movement across the plasma membrane of xylem parenchyma cells for loading to xylem vessels occurs through unidentified anion channels with fast and slow activation kinetics (X-QUAC and X-SLAC respectively) [<xref ref-type="bibr" rid="CR17">17</xref>]. Anion conductances in Arabidopsis guard cells with homologous activation kinetics have been characterised, and the channels eliciting these currents identified. The slowly activating anion conductance in guard cells has been attributed to AtSLAC1 and AtSLAH3 channels [<xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR78">78</xref>], while guard cell QUAC is mediated by AtALMT12 [<xref ref-type="bibr" rid="CR79">79</xref>]. It is therefore feasible that X-SLAC and X-QUAC arise from SLAH and ALMT channels in root cells. We identified <italic>VvSLAH3</italic> and three <italic>VvALMT1</italic> transcripts that were expressed differently between rootstocks. Thermodynamics predicts that the loading of Cl^&#x02212; into the xylem under low apoplastic [Cl^&#x02212;] occurs by passive transport [<xref ref-type="bibr" rid="CR80">80</xref>]. Therefore, transcripts that encode putative anion transport proteins with a high abundance in K51-40 are good candidates for controlling xylem loading of Cl^&#x02212;. Our results suggest that VvALMT1 may be involved in xylem loading of Cl^&#x02212; (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>D and G). <italic>VvSLAH3</italic> transcript was more abundant in 140 Ruggeri (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>E). For it to be involved in xylem loading of Cl^&#x02212; there are two alternatives. Arabidopsis SLAH3 has been shown to be much more permeable for NO₃^&#x02212; than Cl^&#x02212; [<xref ref-type="bibr" rid="CR78">78</xref>]. If this is the case in grapevine, the pathway for anion transport in 140 Ruggeri could be more NO₃^&#x02212; selective than in K51-40, thus resulting in greater discrimination against Cl^&#x02212; loading of the xylem in 140 Ruggeri. Alternatively, SLAH3 could be permeable to Cl^&#x02212; but the extent of post-translational control differs between varieties, as elaborated below.</p><p>Cellular anion conductance must be tightly regulated to avoid uncontrolled electrolyte efflux [<xref ref-type="bibr" rid="CR54">54</xref>], and for this reason complex signalling networks exist in plants. Upon an ABA induced rise in [Ca²⁺]_cyt, guard cell SLAC may be activated by calcium dependent protein kinases CPK23 and CPK21 [<xref ref-type="bibr" rid="CR67">67</xref>]. Alternatively, the Ca²⁺ independent kinase SnRK2.6 (OST1) can activate both guard cell SLAC and QUAC in response to ABA [<xref ref-type="bibr" rid="CR81">81</xref>]. In contrast, opposite regulation by ABA and [Ca²⁺]_cyt occurs in root cells, with X-QUAC being inhibited by ABA and by high [Ca²⁺]_cyt [<xref ref-type="bibr" rid="CR17">17</xref>,<xref ref-type="bibr" rid="CR19">19</xref>]; whether kinases are involved in this regulation have not yet been determined. A transcript encoding the <italic>VvPYL1/RCAR11</italic> ABA receptor was significantly more abundant in roots of 140 Ruggeri compared to K51-40. The Cl^&#x02212; excluder might therefore be more sensitive to ABA, or may be primed for any slight increase in ABA concentration. Differential expression of vacuolar <italic>CAXs</italic> and <italic>ACAs</italic> between rootstocks might function to maintain [Ca²⁺]_cyt signals in root cells of 140 Ruggeri, thus participating in the Ca²⁺ dependent down-regulation of X-QUAC and X-SLAC (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>F). AtCPK20 interacts with AtSLAH3 in Arabidopsis pollen tubes [<xref ref-type="bibr" rid="CR82">82</xref>]. Differential expression of <italic>VvCPK20</italic>, among other <italic>CPKs</italic>, between rootstocks might indicate an involvement of these kinases in <italic>VvSLAH3</italic> regulation. In addition, differential expression of <italic>VvSnRK2.6</italic> and <italic>VvSnRK2.7</italic> between rootstocks implicates both the Ca²⁺ dependent and independent ABA signalling machinery in grapevine roots as possible mediators of Cl^&#x02212; exclusion (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>G). The sheer number of genes potentially involved in X-QUAC and X-SLAC mediated pathways, possible kinase redundancy or multiple kinase targets, could explain the observations that Cl^&#x02212; exclusion in grapevine is polygenic [<xref ref-type="bibr" rid="CR15">15</xref>,<xref ref-type="bibr" rid="CR83">83</xref>].</p><p>Arabidopsis has 53 <italic>NRT1</italic> genes and rice has 80 <italic>NRT1</italic> members. This has led to the question [<xref ref-type="bibr" rid="CR55">55</xref>]: are there unidentified anionic substrates for NRTs beyond just nitrate or peptides to account for such large gene families? The large number of <italic>NRT1</italic> genes identified in our screen suggests they might play some key role in Cl^&#x02212; homeostasis. Plasma membrane localisation of plant NRTs heightens the possibility for roles in cellular Cl^&#x02212; fluxes. However, the anion selectivities of plant NRTs have been rarely examined [<xref ref-type="bibr" rid="CR9">9</xref>]. AtNAXT1 was shown not to transport Cl^&#x02212; [<xref ref-type="bibr" rid="CR59">59</xref>], but characterisation of the remaining 6 Arabidopsis NAXTs is yet to be reported. If permeable to Cl^&#x02212;, greater abundance of <italic>VvNAXT1</italic> and <italic>VvNAXT2</italic> in 140 Ruggeri compared to K51-40 (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>; Table&#x000a0;<xref rid="Tab2" ref-type="table">2</xref>; Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>A) could allow the Cl^&#x02212; excluding rootstock to excrete Cl^&#x02212; back to the external medium instead of transporting it to the shoot (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>F). This function might be enhanced under salinity stress if cytosolic pH is reduced, as <italic>AtNAXT1</italic> actively excretes anions under acid load, possibly to balance proton extrusion by H⁺-ATPases [<xref ref-type="bibr" rid="CR59">59</xref>]. This cannot work as the sole mechanism of Cl^&#x02212; exclusion, as 140 Ruggeri still retains more Cl^&#x02212; in the roots compared to Cabernet Sauvignon and K51-40 (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>A). Other stress responsive plant <italic>NRT1</italic>s (<italic>VvNRT1.8, VvNRT1.5</italic>) showed similar expression profiles to orthologous genes in Arabidopsis. However, <italic>VvNRT1.5</italic> was less abundant in the root stele compared to the cortex (Additional file <xref rid="MOESM13" ref-type="media">13</xref>), indicating that this gene is more likely to be involved in cortical efflux of Cl^&#x02212; rather than Cl^&#x02212; loading to the xylem (Figure&#x000a0;<xref rid="Fig6" ref-type="fig">6</xref>F and G). Excessive Cl^&#x02212; in the root zone or cytoplasm could inhibit NO₃^&#x02212; transport (both uptake and efflux) due to the well-documented antagonism between these anions [<xref ref-type="bibr" rid="CR84">84</xref>,<xref ref-type="bibr" rid="CR85">85</xref>]. Therefore, differences in <italic>NRT1</italic> expression in salt stressed grapevine roots also could be a compensatory mechanism to overcome this ionic antagonism.</p><p>Multiple studies have linked plant CLCs to salt tolerance [<xref ref-type="bibr" rid="CR86">86</xref>-<xref ref-type="bibr" rid="CR89">89</xref>]. <italic>VvCLCb</italic> and <italic>VvCLCf</italic> were expressed differently between rootstocks under control conditions. In Arabidopsis, <italic>AtCLCb</italic> is a vacuolar NO₃^&#x02212;/H⁺ exchanger [<xref ref-type="bibr" rid="CR90">90</xref>], as is <italic>AtCLCa</italic> [<xref ref-type="bibr" rid="CR91">91</xref>]. A single missense mutation in <italic>AtCLCa</italic> changes the selectivity from NO₃^&#x02212; to Cl^&#x02212; [<xref ref-type="bibr" rid="CR92">92</xref>,<xref ref-type="bibr" rid="CR93">93</xref>]. It is therefore possible that <italic>VvCLCb</italic> in <italic>Vitis</italic> roots participates in Cl^&#x02212; sequestration in cell vacuoles, although greater expression in K51-40 does not fully support this. AtCLCf is associated with the trans-Golgi network [<xref ref-type="bibr" rid="CR94">94</xref>], so a role in salt tolerance is less likely. Further study into <italic>Vitis</italic> CLCs is therefore needed before concluding a role in grapevine salt tolerance.</p><p>Candidate genes for plant Cl^&#x02212; exclusion identified by Brumos <italic>et al.</italic> [<xref ref-type="bibr" rid="CR22">22</xref>] were not highlighted in our study. Our array design had two probes for the putative Cl^&#x02212; conductance regulator <italic>VvICln</italic> (<italic>VIT_16s0022g01560</italic>) and neither were salt responsive, consistent with short-term stress response in <italic>Citrus</italic> but contrasting with the long-term results [<xref ref-type="bibr" rid="CR22">22</xref>]. On the other hand, one probe showed statistically significant differential expression between varieties under control conditions but was greater in the Cl^&#x02212; excluding rootstock (data not shown). This means if <italic>VvICln</italic> contributes to Cl^&#x02212; exclusion in 140 Ruggeri, it must act as a negative regulator of Cl^&#x02212; conductance. This seems unlikely given data in animals and plants [<xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR26">26</xref>]. <italic>VvCCC</italic> and <italic>VvSLAH1</italic> were also not differentially expressed, and so if they are involved may be regulated at the post-translational level, which cannot be highlighted by microarray technology. Indeed the activity of many plant anion channels is modified by phosphorylation events such as AtNRT1.1 [<xref ref-type="bibr" rid="CR95">95</xref>], AtSLAC1 [<xref ref-type="bibr" rid="CR96">96</xref>] and AtCLCa [<xref ref-type="bibr" rid="CR97">97</xref>]. Differences in expression of SLAC/SLAH regulators <italic>SnRK2</italic> and <italic>CPK</italic> between rootstocks ensures <italic>VvSLAH1</italic> remains a candidate for Cl^&#x02212; homeostasis in <italic>Vitis</italic>. In future studies, it would be valuable to identify interacting partners of the protein kinases identified as differentially expressed in this study, and any functional changes induced by such interactions.</p></sec><sec id="Sec25" sec-type="conclusion"><title>Conclusions</title><p>Using a whole root transcriptome approach, a detailed analysis of root mRNA profiles of contrasting grapevine genotypes is presented. This provides a complement to earlier physiological studies of the same varieties that have demonstrated the mechanism of shoot Cl^&#x02212; exclusion as the restriction of its net xylem loading at the root [<xref ref-type="bibr" rid="CR15">15</xref>,<xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR98">98</xref>]. A valuable list of candidate genes likely to mediate shoot Cl^&#x02212; exclusion has been identified. Future functional characterisation of these genes, including the elucidation of protein-protein interactions, may enable their use in grapevine rootstock breeding efforts. More broadly the further study of these genes and their homologs in other species will aid our understanding of long distance Cl^&#x02212; transport in plants.</p><sec id="Sec26"><title>Availability of supporting data</title><p>Data supporting the results of this article are available in the Gene Expression Omnibus repository (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>) under accession number GSE57770. An ArrayQualityMetrics report is available at <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.6070/H4CZ354H">http://dx.doi.org/10.6070/H4CZ354H</ext-link>.</p></sec></sec>

The Rare Sprengel Deformity: Our Experience with Three Cases

<p>Sprengel shoulder is a rare congenital deformity of one or both scapulae that is usually detected at birth. It occurs due to failure of the scapula to descend during intrauterine development and its cause is still unknown. Although the deformity appears randomly most of the time, familial cases have been reported. Sprengel shoulder is often associated with Klippel&#x02013;Feil syndrome and other congenital skeletal deformities. Anteroposterior X-ray imaging can accurately diagnose Sprengel deformity. However, computed tomography and magnetic resonance scans with three-dimensional reconstruction are nowadays used in everyday practice in order to diagnose concomitant abnormalities, study in detail the anatomy of the affected shoulder(s), and plan appropriate management. We present here our imaging experience from three pediatric cases with Sprengel shoulder and take the opportunity to discuss this rare entity, which is, nevertheless, the commonest congenital defect of the scapula.</p>

<contrib contrib-type="author"><name><surname>Bindoudi</surname><given-names>Antonia</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kariki</surname><given-names>Eleni P</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Vasiliadis</surname><given-names>Konstantinos</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Tsitouridis</surname><given-names>Ioannis</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-55-g001.jpg" position="float"/></p><p>Sprengel deformity, also known as congenital high scapula, undescended scapula, or scapula elevata, is a rare congenital deformity of one or both scapulae that appears at birth,[<xref rid="ref1" ref-type="bibr">1</xref>] although Pellegrin <italic>et al</italic>., have published a single case in which the patient presented with Sprengel shoulder reported as appearing after trauma.[<xref rid="ref2" ref-type="bibr">2</xref>] Undescended scapula results from failed migration of the scapula during early embryonic life,[<xref rid="ref3" ref-type="bibr">3</xref>] leading to a scapula that protrudes in the neck of the patient. However, the deformity is not simply an aesthetic concern, but more importantly it causes restricted mobility of the shoulder and the cervical spine.[<xref rid="ref4" ref-type="bibr">4</xref>] Sprengel deformity appears either as a single defect or in association with other abnormalities, most commonly Klippel&#x02013;Feil syndrome, scoliosis and rib defects.[<xref rid="ref5" ref-type="bibr">5</xref>] Conventional anteroposterior radiography of the chest including both shoulders is a simple and effective means of diagnosing Sprengel deformity, particularly when combined with appropriate clinical information. Computed tomography (CT) with three-dimensional (3-D) reconstruction and magnetic resonance imaging (MRI) are necessary nowadays for the diagnosis of coexisting abnormalities and treatment planning. Although the management of Sprengel deformity depends on the severity of the abnormality and the degree of motion restriction, the most common therapeutic choice for patients and orthopedic surgeons is surgical intervention for cosmetic and functional recovery of the shoulder.[<xref rid="ref5" ref-type="bibr">5</xref>]</p></sec><sec id="sec1-2"><title>CASE REPORTS</title><sec id="sec2-1"><title>Case 1</title><p>A 6-year-old boy presented with a short neck and bilateral lumps on both sides of his neck. Both scapulae were elevated, although the left one was at a higher level compared to the right one. On physical examination, the patient had limited range of movement of the cervical spine, 60&#x000b0; abduction on his right shoulder, and 90&#x000b0; adduction on his left shoulder, without neurovascular compromise. The little boy was first examined with an anteroposterior X-ray of the thorax and anteroposterior and lateral X-rays of the cervical spine [<xref ref-type="fig" rid="F1">Figure 1</xref>], which demonstrated asymmetrically high-positioned scapulae, in conjunction with cervical vertebrae synostosis and thoracic cage deformities. Surgery was decided and a CT scan was performed to delineate the anatomy in detail, assess the degree of asymmetry, and provide the details for morphometric analysis and surgery planning [<xref ref-type="fig" rid="F2">Figure 2</xref>]. The Sprengel deformity was initially corrected on the left side (higher grade) and 5 months later on the contralateral side.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Case 1. 6-year-old boy who presented with a short neck and bilateral lumps on his neck was diagnosed with bilateral Sprengel deformity. a) An AP X-ray of the thorax shows asymmetrical position of the scapulae and high position of scapular bases (arrows); compare the level of the glenoid cavity (asterisk) and infraglenoid tubercle (arrowhead) on each side. b) An AP X-ray of the cervical&#x02013;upper thoracic spine demonstrates thoracic spine scoliosis (arrows) and fused ribs (asterisks). c) Lateral X-ray of the cervical&#x02013;upper thoracic spine shows disturbance of the normal thoracic spine kyphosis (arrows).</p></caption><graphic xlink:href="JCIS-4-55-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Case 1. 6-year-old boy who presented with a short neck and bilateral lumps on his neck was diagnosed with bilateral Sprengel deformity. Axial CT images a) At the C7 level shows where the right scapula appears (double arrow), the upper third of the left scapula is already visible (arrow). b) At the T1 level where the right acromion lies (closed arrowhead), the scan reveals the left scapular body just inferior to the scapular spine (open arrowhead). c) At the T2, the scan shows the level of the upper part of the body of the right scapula (asterisk), only the lower third of the left scapular body (double asterisk), as is expected since the right scapula is at a lower level compared with the left one.</p></caption><graphic xlink:href="JCIS-4-55-g003"/></fig></sec><sec id="sec2-2"><title>Case 2</title><p>A 7-year-old girl was referred to our department for imaging studies prior to surgical repair of her left undescended scapula. She had been diagnosed with Klippel&#x02013;Feil syndrome and Sprengel deformity of the left scapula when she was 6-months old. Since then, she had been undergoing physiotherapy and it was now decided to treat her surgically. Anteroposterior X-ray scans of the shoulders were performed, followed by a CT and an MRI scan, in order to evaluate the omovertebral association and demonstrate the spectrum of abnormalities that was required for thorough planning of surgery [Figures <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Case 2. 7-year-old girl with Klippel&#x02013;Feil syndrome and left Sprengel deformity referred for imaging studies before operative intervention. a) A synthesis AP X-ray of the right and left shoulder shows the undescended and medially rotated left scapula (arrows). There is also a right cervical rib (asterisk). b) Axial CT images at the level of lower cervical vertebrae show the left scapula (white arrowheads) and the left glenohumeral joint (double asterisk) lying at a higher level compared to the contralateral side. There are also cervical unfused vertebral arches (empty asterisks).</p></caption><graphic xlink:href="JCIS-4-55-g004"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Case 2. 7-year-old girl with Klippel&#x02013;Feil syndrome and left Sprengel deformity. 3D-CT reconstruction images show the difference between the position of the scalpulae: (a and b) giving the superior displacement of the affected scapula relative to the normal one. The A/B ratio measures the rotational deformity of the affected scapula. Lines A and B are drawn between the inferior scapular angle (yellow arrowhead) and a line perpendicular to the axis along the spinous processes (asterisks). Lines A and B connect the center of the glenoid cavity (green dots) to the medial border of the scapular spine (blue dots).</p></caption><graphic xlink:href="JCIS-4-55-g005"/></fig></sec><sec id="sec2-3"><title>Case 3</title><p>A 14-year-old boy visited our department for investigation of an undetermined deformity on the right side of his neck. CT and MRI scans were performed. These revealed a mild Sprengel deformity and provided the details of the bony associations and anatomy of extra-osseous structures as needed for preparation of the intervention [<xref ref-type="fig" rid="F5">Figure 5</xref>].</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>Case 3. 14-year-old boy with a right-sided nuchal deformity, shown with CT and MRI to be a Sprengel scapula. (a and b) Axial CT-scan images demonstrate the elevated position of the right scapula (white arrows). The shoulder joints (asterisks) are asymmetrical. (c) Coronal MRI reveals mildly hypoplastic musculature of the right shoulder (double arrow). (d) 3D-CT reconstruction images show that the right scapula (black arrowhead) is higher than the left (yellow arrowhead). Note the associated deformity of the rib cage on the right side (yellow arrow) and the asymmetry of the shoulder joints (double asterisks).</p></caption><graphic xlink:href="JCIS-4-55-g006"/></fig></sec></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Otto Sprengel published four clinical cases of upward dislocation of the scapula in 1891.[<xref rid="ref6" ref-type="bibr">6</xref>] However, Michael Eulenberg, a German surgeon, was the first to describe the anatomy of one case of an undescended scapula in 1863, and claimed that it occurred due to traumatic dislocation of the scapula.[<xref rid="ref7" ref-type="bibr">7</xref>] Alfred Willet and William Walsham described another case in 1883, in which the high scapula was associated with the formation of an osteochondral bridge with the lower cervical vertebrae.[<xref rid="ref7" ref-type="bibr">7</xref>] After Sprengel, Vittorio Putti, an Italian radiologist and orthopedic surgeon, further studied and described the anatomical abnormalities of a Sprengel shoulder.</p><p>The embryonic primordium of the scapula appears during the fifth week of intrauterine life and acquires the final morphology by the end of the eighth week (end of embryonic period).[<xref rid="ref3" ref-type="bibr">3</xref>] It initially appears at the level of the fourth to fifth cervical vertebrae.[<xref rid="ref3" ref-type="bibr">3</xref>] During the same period, the brachial plexus gives rise to the peripheral nerves of the upper limb, which operates as a stimulus for development of the upper limb muscles.[<xref rid="ref3" ref-type="bibr">3</xref>] During its growth, the scapula descends over the upper five ribs to reach the correct anatomical position that holds at birth.[<xref rid="ref3" ref-type="bibr">3</xref>] This developmental migration of the scapula reflects a progressive adaptation of the bone, which serves for brachiation and not for bearing weight.[<xref rid="ref3" ref-type="bibr">3</xref>] The increased demands for greater range of movement and flexibility in humans have altered the morphology and dynamics, both of the shoulder joint and the scapula. The scapula acquired a spine, enlarged in size, especially in the part beneath its spine, widened (high breadth to length ratio), and moved backward, standing at an angle of approximately 45&#x000b0; relative to the midline[<xref rid="ref3" ref-type="bibr">3</xref>] [<xref ref-type="fig" rid="F6">Figure 6</xref>]. Failure of scapula to descend leads to Sprengel deformity, in which case the bone sits 2&#x02013;10 cm higher than expected. The maintenance of high position of the scapula in the process of skeletal development leads to a series of other musculoskeletal defects including hypoplasia, medialization, and adduction[<xref rid="ref8" ref-type="bibr">8</xref>] of the scapula, prominence of its upper angle, distal rotation and lateral angulation of the glenoid cavity, changes in the position of the clavicle, anomalies of the cervicothoracic vertebrae and ribs, and muscular hypoplasia or atrophy of the shoulder musculature.[<xref rid="ref5" ref-type="bibr">5</xref>] A fibrous structure may bridge the cervical spine with the undescended scapula, which when ossified is called the omovertebral bone (omo derives from the Greek word for shoulder &#x0201c;&#x003ce;&#x003bc;o&#x003c2;&#x0201d;). The result in practice is limitation of the abduction at the shoulder joint[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] and &#x0201c;growing out&#x0201d; of the shoulder blade.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Although Sprengel deformity is rare, it is described as the commonest congenital defect of the scapula and nowadays, as a rule, it is recognized either at birth or in early childhood. Its cause is unknown and there have been only very few cases of familial Sprengel deformity described. Most authors agree that the undescended scapula affects women three times more often than men, although Kadavkolan and others report that the entity occurs equally in both sexes.[<xref rid="ref9" ref-type="bibr">9</xref>] It can occur on both sides concurrently but is most often unilateral, with a predilection for the left side. Although patients with bilateral Sprengel deformity might seem less disfigured, their functional limitation is more debilitating, compared to patients with a unilateral defect. Since the affected scapula is not only mis-positioned but also smaller than the bone on the other side, it is often difficult to accurately estimate the grade of the defect, especially in bilateral Sprengel shoulder. In other words, the estimation of the height difference between the healthy-sat and the high-sat scapula using the easily visualized inferior angle of the scapulae on an anteroposterior X-ray of the chest is not always accurate enough for the estimation of the severity of the deformity and for choosing the appropriate treatment.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p>Line drawings show normal anatomy, (a) dorsal and (b) ventral views of the left scapula, and (c) Sprengel deformity. The affected scapula (arrow) sits 2-10 cm higher than normal in medial rotation: 1, the line from the midpoint of the acromioclavicular joint (asterisks) to the midpoint of the sternoclavicular joint (double asterisk); 2, the line connecting the middle of the acromioclavicular joint (asterisks) to the inferior angle of the scapula (arrowheads); and 3, a line along the spinous processes of the vertebrae (red line). SSA: Superior scapular angle, ISA: Inferior scapular angle.</p></caption><graphic xlink:href="JCIS-4-55-g007"/></fig><p>There have been various indices proposed for the study of the morphology of the scapula and the estimation of the degree of scapular deformity. The scapular index is an indicator of the relationship between the breadth and the length of the scapula. It measures the width along the base of the scapula (length of spinous process), the length between its superior and inferior angles, and it is expressed as a percentage (100 &#x000d7; scapular breadth/scapular length). The infraspinatus index describes the width of the scapula relative to the length of the infaspinatus fossa. Both of these indices are estimated radiologically. <xref ref-type="table" rid="T1">Table 1</xref> shows the clinical and radiologic classification of Sprengel deformity.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>]</p><table-wrap id="T1" position="float"><label>Tabel 1</label><caption><p>Cavendish clinical and Rigault radiological classification of Sprengel shoulder</p></caption><graphic xlink:href="JCIS-4-55-g008"/></table-wrap></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>CT with 3-D reconstruction and MRI are particularly useful for the accurate calculation of the degree of Sprengel deformity, determination of associated osseous and/or muscular deformities, and for choosing and planning the treatment. Considering that surgical treatment, when appropriate, is better applied when the child is under 8 years of age, early and accurate diagnosis of the deformity is important. CT 3-D images are a powerful tool for detailed study of the anatomical features of the bony structures in Sprengel deformity. This evaluation is necessary to understand the morphological characteristics, biomechanics, and etiology of this entity.</p></sec>

Ewing's Sarcoma of the Finger

<p>Ewing's sarcoma is a mesenchymal cell tumor usually seen in long bones but very rarely seen in the bones of a finger. Swelling and pain are the most common complaints of the affected finger. In radiological imaging, it may be seen as permeative bone destruction accompanied by a soft tissue component or an expansile bone lesion. A 27-year-old right-hand dominant female patient presented with a swelling on the proximal phalanx of her right 3^rd finger that had existed for 3 years. However, the mass started to gradually increase in size and the pain worsened over a period of 5 weeks. The mass was excised under regional intravenous anesthesia and Ewing's sarcoma was confirmed following a histopathological evaluation. No local recurrence or metastasis was detected 1 year after surgery. Since Ewing's sarcoma is rarely seen in the finger, we present this case with its radiological and clinical findings.</p>

<contrib contrib-type="author"><name><surname>G&#x000f6;kalp</surname><given-names>Mehmet Ata</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kaplano&#x0011f;lu</surname><given-names>Veysel</given-names></name><xref ref-type="aff" rid="aff2">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>&#x000dc;nsal</surname><given-names>Seyyid &#x0015e;erif</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Erten</surname><given-names>Remzi</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-57-g001.jpg" position="float"/></p><p>Ewing's sarcoma (ES) was first described in 1921.[<xref rid="ref1" ref-type="bibr">1</xref>] It is the second most common primary bone tumor of childhood and adolescence.[<xref rid="ref2" ref-type="bibr">2</xref>] It usually affects long bones; however, it is very rarely seen in the hand, especially in the fingers.[<xref rid="ref1" ref-type="bibr">1</xref>] Compared to the typical radiological findings on other localizations, this sarcoma usually shows different radiological features when it appears on the hand or foot.[<xref rid="ref3" ref-type="bibr">3</xref>] In this paper, we present this case of ES that was treated surgically.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 27-year-old right-hand dominant female patient presented with a 3-year history of swelling on the proximal phalanx of her right 3^rd finger. However, the mass started to gradually increase in size and the pain worsened over a period of 5 weeks. There was no recent history of trauma, fever, or weight loss. On physical examination, the mass was immobile and fixed. All the laboratory findings were within the normal range. Conventional radiographs revealed preserved bone contours, surrounding soft tissue swelling, and increase in the opacity of the proximal phalanx of the 3^rd finger [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Magnetic resonance imaging (MRI) revealed a homogeneous soft tissue lesion about 4.7 &#x000d7; 3.3 cm in size on the right 3^rd proximal phalanx, next to the flexor tendon. The lesion showed significant enhancement with focal lobulation on the proximal point. It was hypointense on T1-weighted (T1W) images and hyperintense on T2-weigthed (T2W) images [Figure <xref ref-type="fig" rid="F3">2a</xref>&#x02013;<xref ref-type="fig" rid="F3">c</xref>]. The neighboring bone cortex was natural in appearance and no metastasis was detected in the preoperative computed tomography (CT) of the thorax and in the full-body scintigraphy. The lesion was surgically excised and a microscopic evaluation showed tumor cells that were round in shape with vesicular nuclei, finely dispersed chromatin, narrow cytoplasm, and stratified monotonous cells with indistinct borders. In places, the cytoplasm could be clearly seen due to the glycogen content [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The immunohistochemical study showed widespread and powerful staining of the cells with cluster of differentiation 99 (CD99) antigen. Cytokeratin and epithelial membrane antigen (EMA) showed focal staining. The cells stained positive for vimentin, but did not stain positive for S-100, cytokeratin 7, cytokeratin 19, desmin, actin, CD34, factor 8, and calretinin. The Ki67 proliferation index was 30%. The pathological diagnosis was Ewing's sarcoma. The patient received postoperative chemotherapy consisting of VAC + IE [cyclophosphamide (C), actinomycin (A), vincristine (V), ifosfamide (I), and etoposide (E)]. On follow-up after 1 year, no postoperative local recurrence or metastasis was detected.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>27-year-old right-hand dominant female patient with a swelling on the proximal phalanx of her right 3^rd finger diagnosed as Ewing's sarcoma. X-ray of the right hand AP view shows swelling of the soft tissue and increase in the density of the proximal phalanx of the 3^rd finger (arrow).</p></caption><graphic xlink:href="JCIS-4-57-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>27-year-old right-hand dominant female patient with a swelling on the proximal phalanx of her right 3^rd finger diagnosed as Ewing's sarcoma. (a) T1W coronal, (b) T2W sagittal, (c) T1W axial contrast enhanced images of the right hand 3^rd finger, proximal phalanx palmar side, neighboring the flexor tendon, show a lesion hypointense on T1W (arrow), hyperintense on T2W (arrow), with contrast enhancement (solid arrow).</p></caption><graphic xlink:href="JCIS-4-57-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>27-year-old right-hand dominant female patient with a swelling on the proximal phalanx of her right 3^rd finger diagnosed as Ewing's sarcoma. Excised tissue stained with hematoxylin and eosin (&#x000d7;400) shows Ewing's sarcoma tumor cells in parts with clear appearance due to presence of glycogen (arrow).</p></caption><graphic xlink:href="JCIS-4-57-g004"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Ewing's sarcoma is a mesenchymal cell tumor.[<xref rid="ref1" ref-type="bibr">1</xref>] It is usually seen in the first or second decade of life, generally affecting long tubular bones and the pelvis.[<xref rid="ref2" ref-type="bibr">2</xref>] It is very rarely seen in the bones of the hand. Less than 1% of the cases diagnosed with ES are localized in the hand and the wrist. The most common locations in the hand are the metacarpal and proximal phalanxes.[<xref rid="ref3" ref-type="bibr">3</xref>] The thumb (28%) and the middle finger (28%) are most commonly affected.[<xref rid="ref1" ref-type="bibr">1</xref>] Men (69%) are affected more than the women, with the average age of occurrence being 18.5 years (5 months&#x02013;51 years).[<xref rid="ref1" ref-type="bibr">1</xref>] Pain and swelling are the most common complaints of the affected finger.[<xref rid="ref2" ref-type="bibr">2</xref>] At the beginning of the clinical presentation, the patient's general health condition is good with no fever or weight loss being present.[<xref rid="ref4" ref-type="bibr">4</xref>] In the current case, the patient was in good health, with the only complaints being pain and swelling of the finger.</p><p>Detection of the site and size of a primary tumor is very important for the planning and application of optimal local treatment. Therefore, X-ray and MRI of the primary site, chest CT, and bone scintigraphy should be performed for staging. An X-ray will show typical destructive lesions with indistinct margins, onion-skin&#x02013;type periosteal reactions and soft tissue masses in the diaphysis or, more rarely, in the metaphyseal/diaphyseal region.[<xref rid="ref5" ref-type="bibr">5</xref>] A sclerotic appearance may be seen on smooth bones. MRI is an ideal method for evaluating soft tissues, the intramedullary involvement of the disease, and the involvement of the primary tumor in the surrounding soft tissues. Bone scintigraphy assists in detecting the bone involvement and, to an extent, the amount of intramedullary involvement. Conventional Tc99 bone scintigraphy is ideal for metastatic disease scanning.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>ES is clinically characterized by fever and an increase in the erythrocyte sedimentation rate. This may be confused with tuberculous dactylitis.[<xref rid="ref3" ref-type="bibr">3</xref>] Radiologically, it may be seen as bone destruction and expansile lesion. On X-ray, it may be confused with osteomyelitis due to bone destruction.[<xref rid="ref1" ref-type="bibr">1</xref>] Furthermore, tuberculous dactylitis is characterized by expansile lesion of the hand and absence of periosteal reaction. However, permeating bone destruction, onion-skin&#x02013;type periosteal reaction, large soft tissue swelling, and cystic lesions causing thickening of the bone are only seen in ES.[<xref rid="ref7" ref-type="bibr">7</xref>] Osteosarcoma is more commonly found in the metaphysis, while ES occurs in the diaphysis.[<xref rid="ref8" ref-type="bibr">8</xref>] Furthermore, an increase of alkaline phosphatase (ALP) is seen in osteosarcoma, but not in ES. Differing from ES, in a primitive neuroectodermal tumor, the large soft tissue components reach inside the bone. Askin tumors, however, are only seen on the chest wall.[<xref rid="ref9" ref-type="bibr">9</xref>] The tumor may be clinically confused with infection because it causes pain and local inflammatory symptoms. In addition, metastatic diseases and hematological malignancies should be considered in the differential diagnosis.[<xref rid="ref9" ref-type="bibr">9</xref>]</p><p>Radiological features such as permeating bone destruction and laminated or spiculated periosteal reaction may not be seen in hand lesions.[<xref rid="ref8" ref-type="bibr">8</xref>] Bone expansion, cystic or honeycomb appearance, periosteal reaction, and cortical thickening of the bone are less seen in the ES lesion on the hand or the foot.[<xref rid="ref8" ref-type="bibr">8</xref>] Regardless of the treatment method, the localization of the tumor on the hand is an important prognostic factor. When the distal parts of the extremities are affected, the survival rate is much higher. The best treatment combination is radical excision and postoperative chemotherapy. This treatment approach is successful in tumors of the fingers. Radiotherapy is useful when radical surgical excision is not performed and in partial response to chemotherapy. Currently, the most effective treatment method is a combination of surgery and chemotherapy.[<xref rid="ref1" ref-type="bibr">1</xref>]</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Although fingers are a rare site for localization of Ewing's sarcoma, ES should be considered in the differential diagnosis of lesions in this location.</p></sec>

Pelvic Pheochromocytoma Mimicking as Urinary Bladder Pheochromocytoma: Looking Beyond the Obvious

<p>Pheochromocytomas located outside the adrenal glands are called paragangliomas. A pelvic location is rare, the most common location for a paraganglioma being the retroperitoneal space. Paragangliomas arise from neural crest cells. Pelvic pheochromocytomas may mimic urinary bladder pheochromocytomas on imaging studies. Patients may present with hypertensive crisis during micturition. We present a 26-year-old female who presented to us with accelerated hypertension with episodes of severe headache and palpitation during micturition. Based on imaging studies, she was diagnosed to have a urinary bladder pheochromocytoma. However, on exploration, the patient was found to have an extravesical pheochromocytoma arising from the left posterolateral pelvic wall, which was excised while preserving the bladder. We present this case report as pelvic pheochromocytomas can mimic bladder pheochromocytomas and are difficult to differentiate on radiological imaging and can lead to inadvertent cystectomy.</p>

<contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Santosh</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Parmar</surname><given-names>Kalpesh Mahesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Shivanshu</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Rani</surname><given-names>Jyotsna</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-56-g001.jpg" position="float"/></p><p>Pheochromocytomas are chromaffin cell tumors derived from the adrenal gland and sympathetic nervous system.[<xref rid="ref1" ref-type="bibr">1</xref>] Extra-adrenal pheochromocytomas, also known as paragangliomas, account for 10&#x02013;20% of all pheochromocytomas. More than 85% of these extra-adrenal lesions are found below the diaphragm, especially along the sympathetic chain or the organs of Zuckerkandl. Pelvic organ and urinary bladder involvement is rare.[<xref rid="ref2" ref-type="bibr">2</xref>] The urinary bladder pheochromocytomas may present with paroxysmal hypertension induced by micturition and hematuria.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 26-year-old female patient presented to our institute with a 3-month history of uncontrolled hypertension. She had accelerated hypertension, diabetes mellitus, and eclampsia during pregnancy, which were controlled after delivery with antihypertensive medication. The patient also complained of episodic attacks of headache and palpitation during micturition. No history of hematuria or lower urinary tract symptoms was present. The patient's pulse was 90 bpm and BP was 188/96 mm Hg. General physical examination was within normal limits. Routine biochemical and hematological work-up was normal. In view of uncontrolled hypertension in a young patient, further work-up revealed serum metanephrines to be elevated (660 pg/ml), normal range being 12&#x02013;60 pg/ml. Contrast-enhanced computed tomography (CECT) abdomen revealed a large, lobulated, heterogeneously enhancing soft tissue mass measuring 6 &#x000d7; 4.5 &#x000d7; 5 cm in size, arising from the left posterolateral wall of the urinary bladder, with a few non-enhancing areas suggestive of necrosis [<xref ref-type="fig" rid="F1">Figure 1</xref>]. The soft tissue had predominant endoluminal component projecting into the urinary bladder lumen. Bilateral adenexa were normal. Cystoscopy was done after optimizing the patient, which revealed a large bulge from the left postero-lateral wall of the urinary bladder with normal overlying mucosa, giving the impression of an intramural growth. Positron emission tomography (PET)/computed tomography (CT) using 68 Ga DOTANOC (111In-DOTA-1-Nal3-octreotide) showed intense tracer uptake (SUVmax 9.8) with somatostatin receptor expression in a large heterogeneously hyperenhancing soft tissue lesion 5 &#x000d7; 6 &#x000d7; 5 cm in size, seen arising from the left posterolateral wall of the urinary bladder [<xref ref-type="fig" rid="F2">Figure 2</xref>]. With the diagnosis of bladder pheochromocytoma and in view of the large mass with involvement of trigone and bladder neck, the patient was prepared for radical cystectomy. After pre-operative optimization with alpha and beta blockers, the patient underwent exploratory laparotomy via an infraumbilical midline incision. During mobilization of the urinary bladder from its lateral attachments, there was an accidental rent in the anterior wall of the urinary bladder, which was extended intentionally for better exposure. The mass was seen arising from the left lateral pelvic wall closely abutting the urinary bladder [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The mass was dissected meticulously and completely excised [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Bladder was preserved and closed in two layers with absorbable sutures. Post-operative recovery was uneventful and patient remained normotensive without antihypertensive medications. Histopathological report of the specimen revealed a circumscribed tumor with cells arranged in an organoid pattern separated by thin fibrovascular septae. Tumor cells were monomorphic containing round to oval nuclei with finely dispersed chromatin, inconspicuous nucleoli, and abundant granular eosinophilic cytoplasm. Immunostain for synaptophysin, chromogranin, and CD56 were positive, consistent with paraganglioma [<xref ref-type="fig" rid="F5">Figure 5</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>26-year-old female with accelerated hypertension and elevated serum metanephrines diagnosed with pelvic paraganglioma. CECT abdomen axial cut shows heterogeneously enhancing intraluminal growth (arrow) from the left anterolateral wall of the urinary bladder with predominant intraluminal component.</p></caption><graphic xlink:href="JCIS-4-56-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>26-year-old female with accelerated hypertension and elevated serum metanephrines diagnosed with pelvic paraganglioma. PET/CT image shows intense tracer uptake and somatostatin receptor expressing lesion (arrow) in the left lateral wall of the urinary bladder.</p></caption><graphic xlink:href="JCIS-4-56-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>26-year-old female with accelerated hypertension and elevated serum metanephrines diagnosed with pelvic paraganglioma. Intraoperative image shows tumor arising from the left lateral pelvic wall (arrow) and the bladder appears normal (thin arrow).</p></caption><graphic xlink:href="JCIS-4-56-g004"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>26-year-old female with accelerated hypertension and elevated serum metanephrines diagnosed with pelvic paraganglioma. Photograph of the cut specimen of the excised mass shows areas of necrosis in its inferior aspect (arrow).</p></caption><graphic xlink:href="JCIS-4-56-g005"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>26-year-old female with accelerated hypertension and elevated serum metanephrines diagnosed with pelvic paraganglioma. Photomicrograph of the tissue on immunohistochemical staining (&#x000d7;10) shows positivity for chromogranin marker, suggestive of pheochromocytoma (arrow).</p></caption><graphic xlink:href="JCIS-4-56-g006"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Paragangliomas are extra-adrenal pheochromocytomas.[<xref rid="ref1" ref-type="bibr">1</xref>] These tumors are rare (10% of pheochromocytomas are paragangliomas), slow-growing, hypervascular lesions arising from the neural crest cells. Pelvic location is rare (2%)[<xref rid="ref2" ref-type="bibr">2</xref>] and the most common location is the retroperitoneal space.[<xref rid="ref3" ref-type="bibr">3</xref>] There is an increased incidence in women and in patients living at high altitudes. Paragangliomas occur mainly in adults[<xref rid="ref4" ref-type="bibr">4</xref>] (mean age, 37 years; range, 11&#x02013;70 years) and are usually benign. The signs and symptoms can be related to excess catecholamine secretion and include paroxysmal hypertension accompanied by anxiety, sweating, a throbbing headache, and either facial pallor or flushing during an attack.[<xref rid="ref1" ref-type="bibr">1</xref>] On the other hand, bladder pheochromocytoma is also a rare tumor, accounting for less than 1% of all pheochromocytomas.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>The hypertensive crises result from excessive catecholamine secretion usually accompanying voiding.[<xref rid="ref6" ref-type="bibr">6</xref>] Our index case was diagnosed to have accelerated hypertension and eclampsia, which were under control after delivery on antihypertensive medications. However, she presented with complaints of uncontrolled hypertension after about 1 year of delivery, with episodes of headache and palpitation on micturition. Catecholamine and vanillylmandelic acid levels in the urine or catecholamine levels in the plasma help in the diagnosis of a symptomatic pheochromocytoma. In our case, patient's urinary and metanephrine levels were not markedly raised on evaluation for uncontrolled hypertension. Imaging is essential in evaluating the location, extent, and mutifocality of the disease. CT can detect larger tumors, but its sensitivity is just 82%. In our index case, CT revealed a large, lobulated, heterogeneously enhancing soft tissue mass arising from the left posteroinferior wall of the urinary bladder, with areas of necrosis. With the suspicion of urinary bladder pheochromocytoma, the patient underwent cystoscopy, which showed normal bladder mucosa with a bulge in the left anterolateral wall near the bladder neck with a non-visualized left ureteric orifice suggesting submucosal growth from the urinary bladder wall. The treatment of pelvic pheochromocytoma is primarily surgical, and patient preparation is an essential step, which includes pre-operative treatment with alpha and beta blocking agents. Surgical resection is the treatment of choice.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>The patient was planned for radical cystectomy in view of the large endophytic bladder growth and taken for surgery after optimization of her blood pressure with alpha and beta blockers. Intra-operatively, we found an exophytic growth arising from the left lateral pelvic wall abutting the bladder in the lateral wall. The bladder wall adjacent to the mass was normal. The mass was dissected from the adjacent tissues and excised preserving the bladder. Post-operatively, the patient recovered well and has normal blood pressure, not requiring any antihypertensive medication.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Pelvic pheochromocytomas are a common location of paragangliomas and may mimic urinary bladder pheochromocytomas on imaging studies due to their location and restricted space in the pelvic region. Detailed imaging investigations along with pre-operative evaluation and preparation are important prior to planning intervention to prevent inadvertent cystectomy.</p></sec>

Hermansky-Pudlak Syndrome Complicated by Pulmonary Fibrosis: Radiologic-Pathologic Correlation and Review of Pulmonary Complications

<p>Hermansky&#x02013;Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous hypopigmentation, platelet dysfunction, and in many cases, life-threatening pulmonary fibrosis. We report the clinical course, imaging, and postmortem findings of a 38-year-old female with HPS-related progressive pulmonary fibrosis, highlighting the role of imaging in assessment of disease severity and prognosis.</p>

<contrib contrib-type="author"><name><surname>Kelil</surname><given-names>Tatiana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Shen</surname><given-names>Jeanne</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>O&#x02019;Neill</surname><given-names>Ailbhe C</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Howard</surname><given-names>Stephanie A</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff3">3</xref></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-59-g001.jpg" position="float"/></p><p>A 38-year-old female from Puerto Rico with complaints of hypoxia was transferred to our facility. She had been diagnosed 5 years earlier with Hermansky-Pudlak syndrome (HPS) subtype 1, complicated by pulmonary fibrosis. She had experienced a slow deterioration in pulmonary function, manifesting as worsening dyspnea, cough, and increasing oxygen dependence. In addition to pulmonary fibrosis, she had other manifestations of HPS, including multiple major bleeding episodes requiring blood transfusions, and oculocutaneous albinism with legal blindness. She required 24 h supplemental home oxygen with a baseline flow requirement of 6 l/min via nasal cannulae and was under evaluation for lung transplantation prior to her terminal hospital admission.</p><p>Pulmonary function tests [<xref ref-type="table" rid="T1">Table 1</xref>] showed increasing severe restrictive ventilatory deficit over the 2 years prior to admission, with total lung capacity going from 64% in 2010 to 57% in 2011 and 44% in 2012.</p><table-wrap id="T1" position="float"><label>Tabel 1</label><caption><p>Pulmonary function test results over a course of 2 years revealing progressive worsening of restrictive ventilatory deficit</p></caption><graphic xlink:href="JCIS-4-59-g002"/></table-wrap><p>The patient presented with increasing dyspnea, pleuritic chest pain, and cough. Exercise capacity on admission measured via the 6-min walk test was 24% predicted. She was transferred to the intensive care unit due to hypoxia, with an oxygen saturation of 44% on 6 l nasal cannula. She was started on empiric antibiotics and supported with noninvasive ventilation, but continued to desaturate and was subsequently intubated.</p><p>The patient developed multiorgan failure as evidenced by elevated creatinine, transaminitis, and right heart failure. Blood and urine cultures on admission were negative, and white blood cell count was normal at 10.9 &#x000d7; 10⁹ cells/L. Her oxygen saturation continued to drop with passive movement despite ventilator assistance. Given her poor prognosis, she was terminally extubated.</p><sec id="sec2-1"><title>Radiologic features</title><p>Chest X-ray upon terminal admission demonstrated diffuse reticular interstitial opacities throughout the lungs [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Baseline CT of the chest obtained 16 months [<xref ref-type="fig" rid="F2">Figure 2a</xref>] and 3 months before admission [<xref ref-type="fig" rid="F2">Figure 2b</xref>] demonstrated progressive worsening of pulmonary fibrosis, as evidenced by increasing traction bronchiectasis, interlobular septal thickening, and consolidative opacities. Chest CT upon admission showed multifocal ground glass opacities, subpleural cysts, septal and peribronchovascular thickening, reticulation, and traction bronchiectasis involving greater than two-thirds of the lungs. Also, there was new honeycombing in the bilateral upper lobes suggesting end-stage fibrosis [<xref ref-type="fig" rid="F2">Figure 2c</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>38-year-old female with a known history of Hermansky&#x02013;Pudlak syndrome complicated by pulmonary fibrosis, admitted with hypoxia. Posteroanterior chest radiograph performed on admission demonstrates bilateral, mid to lower lung predominant, diffuse reticular interstitial opacities (arrows). Tubing from oxygen cannulation projects over the image (arrowhead).</p></caption><graphic xlink:href="JCIS-4-59-g003"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>38-year-old female diagnosed with Hermansky&#x02013;Pudlak syndrome complicated by pulmonary fibrosis, as evidenced by worsening dyspnea, cough, and increasing oxygen dependence. Baseline high-resolution CT (HRCT) of the chest: (a) Axial image on lung windows at the level of the carina demonstrates scattered, predominantly peripheral, ground glass opacities with associated fine peripheral reticulation in the lungs bilaterally (curved arrows); (b) high-resolution CT of the chest from the same level obtained 13 months later demonstrates progression of the peripheral opacities that have become more consolidative (curved arrows) with more conspicuous subpleural opacity in the right upper lobe (straight thin arrow); there is new bronchiectasis (arrowhead) and septal thickening along the left main fissure (thick arrow); and (c) axial CT of the chest on lung windows at terminal admission, performed as part of a CT pulmonary angiogram protocol (16 months after initial imaging), demonstrates further progression with increased size of peripheral consolidative opacities and increasing bronchiectasis. There is new honeycombing in the upper lobes bilaterally (curved arrow). Of note, the study did not demonstrate a pulmonary embolus.</p></caption><graphic xlink:href="JCIS-4-59-g004"/></fig></sec><sec id="sec2-2"><title>Pathologic features</title><p>At postmortem examination, both lungs were found to be heavy with diffuse nodular parenchymal fibrosis [Figure <xref ref-type="fig" rid="F3">3a</xref> and <xref ref-type="fig" rid="F3">3b</xref>]. There was end-stage remodeling, characterized by cystic spaces lined by bronchial epithelium and surrounded by well-developed fibrosis on microscopic examination. The cysts, airspaces, and bronchioles were filled with mucus, suppurative exudate, and abundant vacuolated macrophages [Figure <xref ref-type="fig" rid="F4">4a</xref> and <xref ref-type="fig" rid="F4">b</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>38-year-old female with history of Hermansky&#x02013;Pudlak syndrome and end -stage pulmonary fibrosis, admitted with hypoxia and was terminally extubated after sustaining multiorgan failure. Postmortem autopsy was performed. (a and b) Photographs of coronal cut sections of a lung demonstrate diffuse nodular parenchymal fibrosis (arrows) with apparent bronchiolocentric distribution.</p></caption><graphic xlink:href="JCIS-4-59-g005"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>38-year-old female with history of Hermansky&#x02013;Pudlak syndrome and end-stage pulmonary fibrosis, admitted with hypoxia. Photomicrographs of postmortem microscopic examination of lung tissue stained with hematoxylin&#x02013;eosin stain: (a) &#x000d7;200 magnification demonstrates end-stage remodeling, with honeycomb cystic spaces lined by metaplastic bronchial epithelium and surrounded by well-developed fibrosis (arrows); (b) &#x000d7;400 magnification shows alveolar septal thickening associated with prominent diffuse vacuolization of type II pneumocytes (arrow).</p></caption><graphic xlink:href="JCIS-4-59-g006"/></fig></sec></sec><sec sec-type="discussion" id="sec1-2"><title>DISCUSSION</title><p>HPS is a heterogeneous group of autosomal recessive disorders characterized by oculocutaneous hypopigmentation, platelet dysfunction (easy bruising and prolonged bleeding time despite normal platelet count), and lysosomal accumulation of ceroid-lipofuscin in the lungs, which primarily accounts for the associated morbidity.[<xref rid="ref1" ref-type="bibr">1</xref>] In North America, most patients with HPS are from Puerto Rico, where the prevalence is estimated to be 1 in 1800 or occurring in 5 of every 6 albinos.[<xref rid="ref2" ref-type="bibr">2</xref>] Groups of affected individuals have also been identified in Japan and a small village in Switzerland.[<xref rid="ref3" ref-type="bibr">3</xref>] Patients usually present in childhood, often with recurrent epistaxis or prolonged bleeding after dental procedures.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Nine subtypes of HPS are described, most of which are associated with a mutation in the HPS gene on the long arm of chromosome 10. Type 1 is the most common and most severe variant. This is also the most common subtype found in Puerto Rican patients.[<xref rid="ref2" ref-type="bibr">2</xref>] This genotype leads to a high risk of pulmonary disease, hemorrhage, and, in approximately 15% of patients, granulomatous colitis.[<xref rid="ref4" ref-type="bibr">4</xref>] Only Type 4 approaches Type 1 in severity, with the remaining subtypes behaving more mildly clinically and with little risk of restrictive lung disease.[<xref rid="ref2" ref-type="bibr">2</xref>] While most patients with <italic>HPS1</italic> die of pulmonary complications, approximately 13% die of complications from bowel inflammation, with this granulomatous colitis mimicking Crohn's disease and often occurring in adolescence or early adulthood.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>The pathophysiology is believed to be impaired intracellular trafficking of melanosomes, platelet dense bodies, and lysosomes.[<xref rid="ref5" ref-type="bibr">5</xref>] Impaired formation of platelet-dense bodies leads to the bleeding dyscrasia, whereas impaired intracellular trafficking of melanin in the melanosomes of the skin and retina is postulated to be the cause of oculocutaneous albinism. Systemic complications are associated with accumulation of ceroid-lipofuscin, an amorphous lipid&#x02013;protein complex in multiple organs and the reticuloendothelial system, leading to pulmonary fibrosis, granulomatous colitis, cardiomyopathy, and renal failure.[<xref rid="ref6" ref-type="bibr">6</xref>] Ceroid-lipofuscin accumulation in the pulmonary alveolar macrophages is believed to be the primary mechanism for the development of pulmonary fibrosis. Recurrent hemorrhage with resulting hemosiderosis and inflammatory response is also suggested as an alternative mechanism.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Pulmonary fibrosis in HPS is twice as common in women, occurring between the third and fifth decades.[<xref rid="ref8" ref-type="bibr">8</xref>] It initially presents as dyspnea on exertion or restrictive lung disease on pulmonary function tests, and is the most common cause of death in affected patients.</p><p>The radiographic appearance of HPS is nonspecific and chest radiographs may be normal at presentation. Various radiographic abnormalities have been described, including reticular or reticulonodular opacities, pleural thickening, interstitial infiltrates, and perihilar fibrosis.[<xref rid="ref7" ref-type="bibr">7</xref>] These findings may involve both lungs symmetrically or asymmetrically. Chest radiograph in our patient at the time of admission demonstrated symmetric reticular opacities throughout both lungs [<xref ref-type="fig" rid="F1">Figure 1</xref>]. As chest radiographs sometimes underestimate the extent of disease, high-resolution CT is the imaging modality of choice for characterizing the parenchymal abnormalities and evaluating the extent of pulmonary involvement.</p><p>CT findings also vary depending on disease severity. Early stages of the disease are characterized by subtle reticulations, interlobular septal thickening, and peripheral ground glass opacities. In more advanced stages, there are more severe reticulations, peribronchovascular thickening, subpleural cysts, and bronchiectasis involving central airways.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Abnormalities on high-resolution CT tend to be evenly distributed throughout the lungs, with a slight predilection for the middle and lower lungs. Chest CT from our patient demonstrated diffuse septal thickening and ground glass opacities throughout both lungs, while honeycombing was predominantly seen in the bilateral upper lobes [Figure <xref ref-type="fig" rid="F2">2a</xref>&#x02013;<xref ref-type="fig" rid="F2">c</xref>]. HPS is more likely than the usual interstitial pneumonias or collagen diseases to also involve the upper lobes, particularly later in the course of the disease.[<xref rid="ref9" ref-type="bibr">9</xref>]</p><p>Pulmonary hemorrhage, which typically presents with diffuse ground glass opacity on CT, should be considered in these patients given the underlying platelet dysfunction. Other entities that might have overlapping appearance on CT images include idiopathic pulmonary fibrosis (IPF), which features irregular reticulation, fibrosis and honeycombing in the posterior subpleural lung bases, and nonspecific interstitial pneumonitis (NSIP), which has bilateral and symmetric ground glass opacity and less honeycombing. However, IPF most commonly occurs in older patients and NSIP is not a progressive process. HPS has been classified by the American Thoracic Society and the European Respiratory Society as a mimic of IPF and one of the few diseases that has histological features similar, but not identical to usual interstitial pneumonia.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>A prior imaging study examining patients with HPS used a four-point score to grade the severity of CT appearance of disease. Grade 0 showed no changes. Grade 1 showed minimal changes (thickened interlobular septa, reticular disease, subpleural cysts, and areas of ground glass). Grade 2 had moderate disease (traction bronchiectasis, peribronchovascular thickening, and tracheal retraction in one-third or two-thirds of the lungs). Grade 3 had the findings of Grades 1 and 2, but involved more than two-thirds of the lungs. The study found that patients under 20 years of age typically had no CT findings (Grade 0) and those between 20 and 29 years had minimal CT changes (average grade 0.25 &#x000b1; 0.16). Among patients older than 30 years, those with <italic>HPS1</italic> mutations had significantly more severe disease on CT compared with those patients without this mutation (<italic>P</italic> = 0.001). Worsening CT appearance correlated highly with poorer pulmonary function; as CT severity score increased, the forced vital capacity (FVC) fell dramatically (<italic>P</italic> &#x0003c; 0.001). Also, higher CT severity score correlated with time to death, with over half the patients with Grade 3 disease dying within 4 months.[<xref rid="ref6" ref-type="bibr">6</xref>] Our patient had a chest CT 16 months prior to admission, which showed Grade 2 findings [<xref ref-type="fig" rid="F2">Figure 2a</xref>]. FVC at this time was 59% predicted. Subsequent chest CT 3 months before admission showed significant worsening of fibrosis, now involving greater than two-thirds of the lungs and meeting the criteria for Grade 3 disease [<xref ref-type="fig" rid="F2">Figure 2b</xref>]. FVC at this time was 45% predicted. Final chest CT upon admission revealed extensive traction bronchiectasis, peribronchovascular thickening, and honeycombing, consistent with end-stage fibrosis [<xref ref-type="fig" rid="F2">Figure 2c</xref>].</p><p>There is no effective treatment for pulmonary fibrosis due to HPS, besides lung transplantation. High-dose corticosteroids are administered to patients with advanced disease, though their efficacy has not been proven. Cigarette smoke and other pulmonary irritants must be avoided, and the antibiotic pirfenidone may slow the progression of pulmonary fibrosis in patients with significant residual lung. Supplemental oxygen is used in patients with dyspnea to alleviate discomfort.[<xref rid="ref6" ref-type="bibr">6</xref>] Our patient had an objective and symptomatic decline over the last 12 months of her life, which rendered her oxygen dependent with a baseline requirement of 6 l/min and was under evaluation for lung transplantation prior to her terminal admission.</p></sec><sec sec-type="conclusion" id="sec1-3"><title>CONCLUSION</title><p>In summary, HPS is a rare congenital disorder, most often seen in Puerto Ricans, that manifests as easy bruising, epistaxis, prolonged bleeding, granulomatous colitis, and a high risk of pulmonary fibrosis. Patients with mutations in <italic>HPS1</italic> are much more likely to develop more severe disease, typically after 30 years of age. CT correlates well with pulmonary function, and more severe CT changes significantly correlate with time to death. Transplantation remains the only current durable treatment for the disease.</p></sec>

Diagnostic Imaging for Dental Implant Therapy

<p>Dental implant is a device made of alloplastic (foreign) material implanted into the jaw bone beneath the mucosal layer to support a fixed or removable dental prosthesis. Dental implants are gaining immense popularity and wide acceptance because they not only replace lost teeth but also provide permanent restorations that do not interfere with oral function or speech or compromise the self-esteem of a patient. Appropriate treatment planning for replacement of lost teeth is required and imaging plays a pivotal role to ensure a satisfactory outcome. The development of pre-surgical imaging techniques and surgical templates helps the dentist place the implants with relative ease. This article focuses on various types of imaging modalities that have a pivotal role in implant therapy.</p>

<contrib contrib-type="author"><name><surname>Nagarajan</surname><given-names>Aishwarya</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Perumalsamy</surname><given-names>Rajapriya</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Thyagarajan</surname><given-names>Ramakrishnan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Namasivayam</surname><given-names>Ambalavanan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-4-g001.jpg" position="float"/></p><p>Imaging plays an important part in dental implant procedures. The imaging modalities vary from standard projections routinely available in the dental office to more complex radiographic techniques typically available only in radiology centers. Implant imaging provides accurate and reliable diagnostic information of the patient's anatomy at the proposed implant site. Standard projections include intra-oral (periapical, occlusal) and extra-oral (panoramic, lateral cephalometric) radiographs. More complex imaging techniques include conventional X-rays, computed tomography (CT), and cone beam computed tomography (CBCT). Multiple factors influence the selection of radiographic techniques for a particular case including cost, availability, radiation exposure, and patient's anatomy. The dentist aspires to find a balance between these factors with an aim to minimize risk of any complications to the patient.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>The objectives of imaging are to decide if implant treatment is appropriate for the patient, to identify the location of vital anatomical structures such as the inferior alveolar nerve and maxillary sinus, to ascertain bone quantity, height, buccolingual width, and angulation of alveolar process, to detect any possible pathological conditions, and also to decide the length and width of the implant to be placed.</p><p>Imaging modality is useful in three phases of treatment.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Phase 1: Pre-prosthetic implant imaging</p><p>Imaging in this phase determines the quantity, quality, and angulation of bone; relationship of critical structures to prospective implant sites; and the presence or absence of disease at the proposed surgical sites.</p><p>Phase 2: Surgical and interventional implant imaging</p><p>Imaging in this phase evaluates the surgical sites during and immediately after surgery, assists in the optimal positioning and orientation of dental implants, and ascertains the healing and integration phase of implant surgery. It also ensures appropriate abutment positioning and prosthesis fabrication.</p><p>Phase 3: Post-prosthetic implant imaging</p><p>This phase commences just after placement of the prosthesis and continues as long as the implant remains in the jaw. Imaging in this phase evaluates the long-term change, if any, in the implant's fixed position and function, including the crestal bone levels around each implant, and evaluates the status and prognosis of the dental implant. It also helps to routinely assess the bone adjacent to the dental implant to note any changes in mineralization or bone volume.</p><p>The goals of imaging are:</p><p> <list list-type="bullet"><list-item><p>To measure bone height and width (bone dimensions)</p></list-item><list-item><p>To assess bone quality</p></list-item><list-item><p>To determine the long axis of alveolar bone</p></list-item><list-item><p>To identify and localize internal anatomy</p></list-item><list-item><p>To establish jaw boundaries</p></list-item><list-item><p>To detect any underlying pathology.</p></list-item></list> </p></sec><sec id="sec1-2"><title>PLANAR IMAGING MODALITIES</title><sec id="sec2-1"><title>Periapical radiography</title><p>Periapical radiography is used to find the presence of pathosis and location of anatomic structures around the implant site, and evaluate implants postoperatively. It is used to determine vertical height of the edentulous region, architecture, and bone quality. For the edentulous and resorbed jawbone, however, this technique can be difficult as adequate support for positioning the instrument is not available. Attaching a film holder to the tube, while carefully positioning the jaw bone under investigation parallel to the film, may offer a solution. <xref ref-type="fig" rid="F1">Figure 1</xref> shows the postoperative periapical radiograph of an osseointegrated implant.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>45-year-old male patient imaged for the evaluation of the implant. Periapical radiograph of the left mandibular posterior region shows the osseointegrated implant (arrows).</p></caption><graphic xlink:href="JCIS-4-4-g002"/></fig></sec><sec id="sec2-2"><title>Intra-oral imaging using electronic or charge-coupled device imaging techniques</title><p>Charge-coupled device (CCD) detectors consist of a serial chain of signal components, such as phosphors, fiber optics or lenses, image intensifiers, and the CCD, which serve to convert the X-ray energy to light or electron-hole pairs and to record the spatially resolved image. With CCDs, pre-surgical implant assessment of a single site becomes precise. CCD provides accurate measurement of implant sites preoperatively and gives information about osseo-integration postoperatively.</p><p>CCD detectors are readily available and offer high-resolution images with minimal distortion.</p><p>However, CCDs have some disadvantages. Only a small area can be imaged and CCDs produce 2D images that are difficult to reproduce (due to non-uniformities in the phosphor, the optical couplings, the transmission of the fiber optics, and the response of the CCD pixels). Image elongation or foreshortening is also common (due to image intensifiers and optical coupling in the system).</p></sec><sec id="sec2-3"><title>Occlusal radiography</title><p>High-resolution planar images of the mandible or the maxilla are produced by occlusal radiography. Structures like maxillary sinus, nasal cavity, and nasopalatine canal can be assessed through occlusal radiography. Mandibular occlusal radiograph projection is less distorted than the maxillary occlusal radiograph.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>As periapical radiographs are unable to produce any cross-sectional information, occlusal radiographs are sometimes used to determine the facio-lingual dimensions of the mandibular alveolar ridge.</p><p>Occlusal imaging does not provide detailed information of the facial&#x02013;lingual dimension of the alveolar ridge. It has limited reproducibility (replication of the imaging region), and superimposition of images (lack of distinctiveness of the image due to overlapping of the structures) is common.</p></sec><sec id="sec2-4"><title>Cephalometric radiography</title><p>Lateral cephalometric radiography helps in the analysis of the quality of the bony site (ratio of compact to cancellous bone), especially in the anterior region of the mandible. Though it gives limited information about the symphyseal area, the inclination and buccolingual dimensions of the anterior jawbone region can be obtained. These images do not provide useful information when planning placement of implants lateral to the mid-sagittal plane. Overly optimistic bone volume assessments are created due to the presence of genial tubercles.</p><p>The advantage of lateral cephalometric radiographs is that they outline the geometry of the alveolus in the anterior region and the relationship of the lingual plate to the patient's skeletal anatomy.</p><p>The disadvantages of this technique are that it is not useful for demonstrating the bone quality and only demonstrates a cross-sectional image of the alveolus where the central rays of the X-ray device are tangent to the alveolus. Other disadvantages include low power magnification and superimposition of images (lack of distinctiveness of the image due to overlapping of the structures).</p></sec><sec id="sec2-5"><title>Panoramic radiography</title><p>These are narrow beam rotational tomographs, which use two or more centers of rotation with a predefined focal trough, to produce an image of both the upper and lower jaws. Optimal patient positioning is crucial in this procedure because jaw positioning errors in the sagittal plane can occur easily, especially in the edentulous patient. It provides an approximation of bone height, vital structures, and any pathological conditions that may be present.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p><xref ref-type="fig" rid="F2">Figure 2</xref> shows the evaluation of sub-periosteal implant using panoramic radiograph.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>65-year-old female with completely edentulous maxillary and mandibular arches. Panoramic radiograph demonstrates a mandibular subperiosteal implant (arrows).</p></caption><graphic xlink:href="JCIS-4-4-g003"/></fig><p>Advantages of panoramic radiography are that they provide information on opposing landmarks, vertical height of the bone, assess crestal alveolar bone and cortical boundaries, and evaluate gross anatomy of the jaws and related pathologic conditions.</p><p>Disadvantages of this modality are distortion of the visualized structures, low level of reproducibility, 1.1-1.7 times magnification of the structures, difficulty in assessing hard tissue morphology and density of the bone, little information about the buccolingual cross-sectional dimension, inadequate identification of critical structures, and no data of spatial relationships between the structures.</p></sec><sec id="sec2-6"><title>Zonography</title><p>Zonography is a modification of the panoramic X-ray machine and generates cross-sectional image of the jaws. The tomographic layer is around 5 mm. Zonography allows appreciation of spatial relationship between the critical structures and the implant site.</p><p>However, the disadvantages are presence of relatively thick tomographic layers, blurred adjacent structures superimposed on the image, and inability to identify the differences in bone densities or presence of disease pathology at the implant site.</p></sec></sec><sec id="sec1-3"><title>QUASI 3D IMAGING MODALITIES</title><sec id="sec2-7"><title>X-ray tomography</title><p>This is a special X-ray technique that enables visualization of a section of patient's anatomy above and below the section of interest. Tomography is derived from the Greek words &#x0201c;Tomo&#x0201d; (slice) and &#x0201c;Graph&#x0201d; (picture).</p></sec><sec id="sec2-8"><title>Conventional tomography</title><p>Conventional tomography differs from X-ray tomography in its ability to resolve details as small as a few microns in size, even when imaging objects is made of high-density materials. In the last two decades, conventional tomographic machines have been introduced in oral health care. Some extra-oral X-ray equipments may also offer possibilities for cross-sectional imaging. Sharp visualization of structures in the focal plane, while blurring all other structures is achieved by working with different tomographic movements in the linear, circular, spiral, elliptical, and hypocycloidal planes. Complex tomographic movements (e.g. spiral and hypocycloidal) are the most widely used.</p><p>Digital tomographic images offer increased image quality by contrast enhancement, reduction of blurring, and image manipulation. Further image processing may yield precise information on bone volume, (relative) bone density, and help to simulate implant surgery by visualizing the planned implant in relation to the anatomic structures.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>For radiographic visualization of the mandibular canal, cross-sectional imaging provides the best information. When comparing computed to conventional tomography (hypocycloidal and spiral) for measuring the distances to the mandibular canal CT does not seem to be more accurate. Spiral tomography performs better than hypocycloidal tomography as the borders of the canal are better identified with the former technique. The greatest inaccuracy is found when using panoramic images.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Conventional tomography has the advantage in that it can determine the spatial relationship between the critical structures and the implant site and help in quantification of the geometry of the alveolus. The reformatted CT images provide axial, panoramic, and cross-sectional images that are all cross-referenced to one another allowing rapid correlation of different views.</p><p>Teeth, particularly those with metallic restorations adjacent to the area of interest, obscure the tomographic image. Limited resolution caused by use of an intensifying screen cassette makes the identification of anatomical structures and assessment of bone topography more difficult. Conventional tomography cannot assess the bone quality and does not identify bone and dental disease.</p></sec><sec id="sec2-9"><title>Computed tomography</title><p>CT was invented by Sir Godfrey Hounsfield and was introduced in 1972.[<xref rid="ref5" ref-type="bibr">5</xref>] This modality gives rise to high-density resolution images, and allows soft tissues to be visualized. The reformatted CT image generates axial, panoramic, and cross-sectional images that allow rapid correlation of the different views.</p><p>CT scan provides tangential and cross-sectional tomographic images of the implant site. <xref ref-type="fig" rid="F3">Figure 3</xref> explains the various sections of a CT scan. Reformatted images are characterized by in-plane resolution of 1 pixel by scan spacing (0.5-1.5 mm) and a section thickness of 1 pixel (0.25 mm) resulting in a geometric resolution similar to that of planar imaging. The density of structures produced in the image is absolute and quantitative, and can be used to differentiate tissues in the region and characterize bone quality.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>39-year-old male with mandibular edentulous posterior region. CT of the edentulous area of the mandibular posterior region for the evaluation of bone for implant placement. a) Various sections of CT (arrow); b) shows the inferior alveolar nerve (arrow); and c) shows the arbitrary implant placement (arrow).</p></caption><graphic xlink:href="JCIS-4-4-g004"/></fig><p>Disadvantages include limited availability of CT scanners, high cost of machines, image artifacts caused by metal, technique errors, and the need for special training in image interpretation.</p></sec><sec id="sec2-10"><title>Interactive computed tomography</title><p>Interactive CT (ICT) allows the transfer of images to the clinician as a computer file. It helps the clinician measure the length and the width of the alveolus and also bone quality. An important aspect of ICT is that the clinician and radiologist can together perform &#x0201c;electronic surgery&#x0201d; (ES).</p></sec><sec id="sec2-11"><title>Cone-beam computerized tomography</title><p>CBCT scanners are designed specifically for diagnosis and treatment planning in implant therapy. Multiple pictures of the region of interest are generated in a single scan. This enables the dentist to perform minimally invasive surgery without raising a flap, thereby reducing surgery time, postoperative pain and swelling, and faster recovery time.[<xref rid="ref6" ref-type="bibr">6</xref>] A master cast can be fabricated pre-surgically using the information that is stored in the surgical plate, and a provisional restoration can be placed immediately after surgery.</p><p>Radiographic markers can be inserted at the time of the scan and these identify the precise location of the proposed implants.[<xref rid="ref7" ref-type="bibr">7</xref>] Stents provide radiographic landmarks that can be used to correlate proposed clinical location and angulation of implants with the available alveolar bone.</p><p>With the help of Digital Imaging and Communications in Medicine (DICOM) data, computer-generated surgical guides (stereolithographic models) can be fabricated from the CBCT data. The guide helps the surgeon place implants in their optimal and exact position. <xref ref-type="fig" rid="F4">Figure 4</xref> demonstrates the stereolithographic model that was fabricated using the data obtained from CBCT.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>62-year-old female with completely edentulous maxillary arch. Image shows the stereolithographic model of the maxillary region.</p></caption><graphic xlink:href="JCIS-4-4-g005"/></fig><p>A stereotactic surgical template is fabricated from a model that replicates the patient's alveolar structure. The template determines the position and orientation of the guide cylinders and this accurately identifies the position and orientation of the proposed implants. <xref ref-type="fig" rid="F5">Figure 5</xref> shows the implant site assessment that is carried out using CBCT.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>30-year-old female patient with edentulous mandibular anterior region. CBCT shows implant placement site in the mandibular anterior region.</p></caption><graphic xlink:href="JCIS-4-4-g006"/></fig><p>Advantages of CBCT are that it generates a 3D dataset (digitally reconstructed image with 670 projections), has the potential for generating all 2D images (e.g. orthopantomogram, lateral cephalogram), and allows vertical scanning with the patient in a seated position. <xref ref-type="fig" rid="F6">Figure 6</xref> demonstrates the various sections of CBCT.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p>40-year-old male with mandibular posterior region. CBCT of mandibular posterior region for the evaluation of implant placement (a) shows the implant size (arrow) using the available bone and (b) points out the cross section of the inferior alveolar nerve (arrow).</p></caption><graphic xlink:href="JCIS-4-4-g007"/></fig><p>CBCT generates high-resolution images of anatomical structures, bone trabeculae, periodontal ligament (PDL), and root formation. The other advantages include rapid scanning procedure, lower radiation dose, reduced disturbance from metal artifacts, lower cost, easy accessibility, easy handling, and DICOM compatibility.</p><p>CBCT has some disadvantages. It has a low contrast range, restricted field of view (FOV), reduced scanned volume caused by limited detector size, gives little information about the inner soft tissue, and increased noise from scatter radiation and artifacts.</p></sec><sec id="sec2-12"><title>Magnetic resonance imaging</title><p>Magnetic resonance imaging (MRI) was first discovered by Lauterbur. The presence of ferromagnetic (high magnetic susceptibility) metals can distort the magnetic field and compromise the images. Non-ferromagnetic alloys do not produce image deformities, whereas non-precious ferromagnetic alloys (cobalt&#x02013;chromium) produce large image deformations. Devge <italic>et al</italic>., found that MRI images are not affected by implants of the Branemark system.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p>MRI differentiates the inferior alveolar canal and neurovascular bundle from adjacent trabecular bone, and visualizes the fat in the trabecular bone.[<xref rid="ref9" ref-type="bibr">9</xref>] MRI avoids radiation hazards associated with CT.</p><p>MRI is contraindicated for patients with ferromagnetic metallic implants in their bodies. Unlike multi-plane reformatted CT images, cross-sectional MRI images are obtained using conventional reformatting due to the lack of software support for MRI data. Thus, MRI is not useful in characterizing bone mineralization.</p></sec></sec><sec id="sec1-4"><title>DOSIMETRY</title><p>Appropriate selection criteria must be applied to choose the correct imaging modality.[<xref rid="ref10" ref-type="bibr">10</xref>] Dentist must consider potential risks versus perceived benefits of each imaging procedure [<xref ref-type="table" rid="T1">Table 1</xref>].[<xref rid="ref1" ref-type="bibr">1</xref>] When the dentist prescribes a radiographic examination, the ALARA principle (as low as reasonably achievable) of radioactive materials or radiation-producing devices should be kept in mind. Radiographs should only be prescribed when the information required cannot be obtained by any lesser invasive modalities.</p><table-wrap id="T1" position="float"><label>Tabel 1</label><caption><p>Effective doses and risk from implant imaging</p></caption><graphic xlink:href="JCIS-4-4-g008"/></table-wrap></sec><sec id="sec1-5"><title>RECENT ADVANCES IN IMAGING</title><p>Using the platform of the SCANORA (Sordex Orion Corporation, Helsinki, Finland), a limited-volume CBCT system 3DX Accuitomo was developed. PSR9000N [<xref ref-type="fig" rid="F7">Figure 7</xref>] is also a limited-volume CBCT system, which is an inherited technology from another dento-maxillary multimodal tomographic system, the AZ3000 (Asahi Roentgen, Japan).[<xref rid="ref14" ref-type="bibr">14</xref>]</p><fig id="F7" position="float"><label>Figure 7</label><caption><p>New Tom CBCT system.</p></caption><graphic xlink:href="JCIS-4-4-g009"/></fig><p>Cross-sectional images of a small, defined area in the jaws and dental arches are produced by these dental tomographic platforms. Minimization of the radiation dose is a remarkable advantage of limited-volume CBCT.</p><p>The main disadvantage of low-dose CBCT is the difficulty in the acquisition of reliable CT values, which leads to poor soft tissue resolution. But this helps to reduce metal and beam hardening artifacts, which are inherent in CT imaging. In limited-volume CBCT imaging, the size of FOV is small compared to the head and the intensity of transparent X-radiation fluctuates during the 360&#x000b0; scan. Metal artifacts in conventional fan-beam CT images are common. Metals producing artifacts are usually detectable. It is difficult to detect the appearance of halation artifacts in CBCT because their cause is outside the imaged area. Further studies are necessary to identify the characteristics of these presumed halation artifacts.</p></sec><sec sec-type="discussion" id="sec1-6"><title>DISCUSSION</title><p>Medical images can be assessed for their technical quality and diagnostic value. The clinician has to carefully weigh the pros and cons of each modality and choose a particular technique accordingly.</p><p>Literature suggests that when evaluating the alveolar ridge, the radiologist should also determine the angle that the ridge makes with the vertical axis. This is important because the occlusal force vector that acts upon the fixture should be parallel to the vertical axis through the alveolar ridge. If excess angulation exists between the vertical axis through the fixture than that through the alveolar ridge, the resultant force vector may fall in an area which is unable to withstand the occlusal forces and breakdown of the surrounding bone may occur.</p><p>The various radiographic methods detailed here have their unique characteristics and each of these applied judiciously, where required, will help the diagnostician as well as the clinician to accurately plan, execute, and evaluate implant treatment.</p><p>Periapical radiographs produce a high-resolution planar image of a limited region of the jaws and do not provide any cross-sectional information of the jaws. Occlusal radiography produces high-resolution planar images of the body of the mandible or the maxilla. Cephalometric radiographs are a useful tool for the development of an implant treatment plan, especially for the completely edentulous patient, or for placement of implants near the midline for overdentures.</p><p>MRI is used in implant imaging as a secondary imaging technique where primary imaging techniques such as CT or ICT have failed. MRI visualizes the fat in the trabecular bone and differentiates the inferior alveolar canal and neurovascular bundle from the adjacent trabecular bone.</p><p>Since the introduction of CBCT scanners in the late 1990s, there has been great interest in these devices in the field of oral and maxillofacial surgery, orthodontics, and dentistry. Swennen and Schutyser stated that with CBCT, the image value of a voxel of an organ depends on the position in the image volume.[<xref rid="ref15" ref-type="bibr">15</xref>] Several studies have described the value of computer-guided implant bed preparation for dental implantology. Gert Wittwer <italic>et al</italic>., evaluated a novel approach in the placement of interforaminal mandibular dental implants with computer-assisted navigation and without conventional elevation and reflection of mucoperiosteal flaps or mucosal punching at the surgical site.[<xref rid="ref16" ref-type="bibr">16</xref>] Patel <italic>et al</italic>., stated that perhaps the most clinically useful aspect of CBCT imaging is the highly sophisticated software that allows the huge volume of data collected to be broken down and processed or reconstructed into a format that closely resembles that produced by other imaging modalities.[<xref rid="ref17" ref-type="bibr">17</xref>] Vannier stated that when new developments in the synthesis and optimization of CBCT reconstruction algorithms allow the full exploitation of the potential of area detectors in CBCT, it will provide important benefits for craniofacial imaging.[<xref rid="ref18" ref-type="bibr">18</xref>] It is expected that improvements in cone-beam reconstruction algorithms and post processing will solve or reduce this problem.</p><p>Flapless transmucosal implant placement is also an ideal method to treat cases of medically compromised patients. One major limitation of this new technique is the cost of CT images and the navigation technology. However, the benefits outweigh its cost.</p></sec><sec sec-type="conclusion" id="sec1-7"><title>CONCLUSION</title><p>With the excellent imaging modalities that exist today, one can enhance the success of implant placement. Selection of an appropriate imaging modality should be made based on the type and number of implants, location of the implant, and surrounding anatomy. As in the case of all imaging techniques, appropriate selection criteria must be applied before selecting one which is most suitable for each patient.</p></sec>

MDCT Venography Evaluation of a Rare Collateral Vein Draining from the Left Subclavian Vein to the Great Cardiac Vein

<p>Congenital vascular anomalies of the venous drainage in the chest affect both cardiac and non-cardiac structures. Collateral venous drainage from the left subclavian vein to the great cardiac vein is a rare venous drainage pattern. These anomalies present a diagnostic challenge. Multi-detector computed tomography (MDCT) is useful in the diagnosis and treatment planning of these clinically complex disorders. We present a case report of an 18-year-old Caucasian male who came to our institute for evaluation of venous drainage patterns to the heart. We describe the contrast technique of bilateral dual injection MDCT venography and the imaging features of the venous drainage patterns to the heart.</p>

<contrib contrib-type="author"><name><surname>Abchee</surname><given-names>Antoine</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Saade</surname><given-names>Charbel</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Al-Mohiy</surname><given-names>Hussain</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>El-Merhi</surname><given-names>Fadi</given-names></name><xref ref-type="aff" rid="aff2">1</xref><xref ref-type="corresp" rid="cor1"/></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-58-g001.jpg" position="float"/></p><p>Multi-detector computed tomography (MDCT) venography is an effective tool in demonstrating the complex venous vasculature present in congenital heart disease, especially extra-cardiac pathology.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] In most syndromes, patients often have unusual arteriovenous connections. MDCT venography allows accurate identification of the cardiac and extra-cardiac arteries and veins and their relationships to both atria and ventricles. The enhanced preoperative understanding of congenital heart disease provided by MDCT venography allows intensive surgical planning with the potential to reduce post-surgical complications.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>An 18-year-old Caucasian man with a complex congenital heart disease had attacks of ventricular tachycardia and oxygen saturation levels that showed discrepancy between the upper and the lower extremities. The patient was referred to our institution for evaluation of the complex angioarchitecture of venous blood draining into the heart, especially the superior vena cava. Bilateral MDCT venography of the thoracic venous system performed demonstrated significant collateral venous blood supply from the left subclavian vein to the coronary sinus via the left superior intercostal vein and unusual collateral left vein that drained into the great cardiac vein, and markedly dilated superior vena cava, inferior vena cava, and azygous vein [Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>18-year-old male with complex congenial heart disease had attacks of ventricular tachycardia and oxygen saturation levels that showed discrepancy between the upper and the lower extremities, later diagnosed with a rare collateral vein draining from the left subclavian vein to the great cardiac vein. Three-dimensional MDCT of the heart and thorax demonstrates extraordinary dilated azygous vein (small arrowhead) draining into the superior vena cava (large arrow head), and the inferior vena cava (large arrow) draining directly into the right atrium (small arrow).</p></caption><graphic xlink:href="JCIS-4-58-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>18-year-old male with complex congenial heart disease had attacks of ventricular tachycardia and oxygen saturation levels that showed discrepancy between the upper and the lower extremities, later diagnosed with a rare collateral vein draining from the left subclavian vein to the great cardiac vein. Three-dimensional MDCT of the heart and thorax demonstrates the collateral vein (large arrowhead) from the left subclavian vein (small arrowhead) draining into the great cardiac vein (arrow).</p></caption><graphic xlink:href="JCIS-4-58-g003"/></fig><sec id="sec2-1"><title>MDCT venography technique</title><p>Bilateral dual injection MDCT venography was performed using a 256-MDCT scanner (Philips iCT; Philips Healthcare, Clevland Ohio, USA) with the patient positioned supine with arms by his side. Anterior-posterior and lateral scout scans were performed, with a scan range from the apex of the chest to the costophrenic angle. The scan parameters were: Detector width 256 &#x000d7; 0.625 mm; pitch 0.9; rotation time 0.4 s; exposure factors 100 kVp, 200 mA, with z-axis modulation; and scanning time of 2.6 s. A caudocranial scan direction was employed.</p></sec><sec id="sec2-2"><title>Contrast bolus geometry</title><p>Bolus geometry is the pattern of enhancement, after intravascular injection of contrast material, measured in a region of interest (ROI), plotted on a time (s)/attenuation Hounsfield units (HU) curve. We employed the test bolus technique where one ROI was plotted inside the superior vena cava and another in the pulmonary trunk. A small amount of contrast material was (10 ml) injected at the same rate as the main bolus using a power injector, where the initial rate was 3 ml/s which was then decreased exponentially to 2.2 ml/s over the duration of the injection using bolus shaping software. This ROI assessed the time to peak (TTP) and determined the arteriovenous circulation time for thoracic vasculature [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>18-year-old male with complex congenial heart disease had attacks of ventricular tachycardia and oxygen saturation levels that showed discrepancy between the upper and the lower extremities, later diagnosed with a rare collateral vein draining from the left subclavian vein to the great cardiac vein. Determination of contrast bolus transit time using test bolus injection shows that the region of interest is placed inside the superior vena cava (circle A) and in the pulmonary trunk (circle B). Resulting enhancement curves display the time needed to reach the peak of maximum contrast enhancement for test bolus.</p></caption><graphic xlink:href="JCIS-4-58-g004"/></fig></sec><sec id="sec2-3"><title>Contrast medium administration</title><p>Contrast material was injected with an automated dual barrel power injector (Optivantage; Covidien, Cincinnati, OH, USA) via a 20-gauge venous catheter in each arm (right and left sides). Bilateral antecubital venous access was used in this study because it provides uniform contrast distribution into the heart for the contrast material to pass through the venous system with the least amount of dilution, promoting good image quality during computed tomography angiography (CTA) at reduced contrast volumes.[<xref rid="ref3" ref-type="bibr">3</xref>] Bilateral simultaneous injection occurred at 3 ml/s, employing a 20:80 contrast to saline ratio mix in each syringe. The volume of contrast used totaled to 40 ml (this included 20 ml of contrast and 80 ml of saline in each syringe).</p></sec></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Identification of the components of the cardiac congenital anomalies is vital for devising a management plan. Echocardiography and angiography are the current modalities of choice for evaluating congenital heart diseases. However, there has been an increased role of MDCT as a superior imaging modality used prior to surgical intervention for providing detailed anatomical and functional information of venous anomalies with 2D-reformatted and 3D-reconstructed images.[<xref rid="ref4" ref-type="bibr">4</xref>] This patient required an assessment of the systemic venous return to the heart, especially to the left superior intercostal vein, collateral vein draining into the coronary sinus, and the great cardiac vein. Optimal and diagnostic images are vital in understanding patients&#x02019; anatomy and cardiovascular hemodynamics. Additionally, it is necessary to take into consideration the contrast medium administration and parameters that affect bolus geometry as it needs to be carefully configured to match the venous enhancement pattern of complex angioarchitecture during MDCT venography.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>]</p><p>There are limited studies on CT direct venography for evaluating thoracic venous return. However, one study conducted by Kim et al., using CT venography in the diagnosis of benign thoracic central venous obstruction, employed bilateral upper extremity injection with a total volume of 200 ml of contrast medium and scan delay of 20-40 s.[<xref rid="ref10" ref-type="bibr">10</xref>] However, in our case, we employed a reduced amount of contrast medium (40 ml in total) by using the test bolus technique at the level of the superior vena cava for optimal enhancement.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Evaluation of complex venous angioarchitecture in patients with vascular anomalies has considerably improved with the use of MDCT direct venography when injecting contrast media bilaterally and simultaneously via the upper extremity.</p></sec>

Unusual Unilateral Fracture of the Condylar and Coronoid Processes of the Mandible

<p>The incidence of condylar fracture is very high and most are not caused by direct trauma. On the other hand, fracture of the coronoid process is reported less often than fracture of other parts of the mandible. We report a case of right subcondylar and coronoid fractures without any evidence of direct trauma to the zygomatic area or an indirect trauma to the mandibular corpus or sypmheseal region. The possible cause was identified as acute reflex contraction of the temporalis muscles leading to coronoid and condylar stress fractures.</p>

<contrib contrib-type="author"><name><surname>Baykul</surname><given-names>Timu&#x000e7;in</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ayd&#x00131;n</surname><given-names>M As&#x00131;m</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Aksoy</surname><given-names>M&#x000fc;ge &#x000c7;&#x00131;na</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>F&#x00131;nd&#x00131;k</surname><given-names>Yavuz</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib>

Journal of Clinical Imaging Science

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><graphic xlink:href="JCIS-4-3-g001.jpg" position="float"/></p><p>Mandible, because of its position and prominence, is the second most commonly fractured part of the maxillofacial skeleton after the nasal bone. The proportion of condylar fractures among all mandibular fractures is between 17.5% and 52%.[<xref rid="ref1" ref-type="bibr">1</xref>] Most are not caused by direct trauma, but follow indirect forces transmitted to the condyle from a blow elsewhere. Mandibular and condylar fractures are relatively common injuries, however, fractures of the coronoid process are very uncommon and account for only 1% of all mandible fractures.[<xref rid="ref2" ref-type="bibr">2</xref>] Isolated coronoid fractures due to direct trauma are very uncommon because the coronoid process is anatomically protected by the zygomaticomalar complex and its associated muscles.[<xref rid="ref3" ref-type="bibr">3</xref>] Most coronoid fractures are due to indirect blunt or penetrating trauma. There are various reports of iatrogenic fractures of the coronoid process, occurring during the extractions of maxillary and mandibular third molars, sagittal split osteotomy, and cystectomies.[<xref rid="ref4" ref-type="bibr">4</xref>] In the literature, reports of unilateral fractures of both condylar and coronoid process without any evidence of trauma to the mandible are very rare.[<xref rid="ref5" ref-type="bibr">5</xref>] We report a very rare unilateral fracture of condylar and coronoid processes due to a sudden contraction of the temporalis muscle.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 16-year-old male patient was referred to our institution with the chief complaint of pain in the right temporomandibular joint (TMJ) region and difficulty in mastication and mouth opening. He had sustained a trauma to the right temporal region because of a traffic accident. Patient did not present an initial loss of conscience or anterograde amnesia. Clinical examination showed little swelling over the right TMJ region with an inadequate mouth opening. No facial palsy was observed. Intraoral examination showed abnormal occlusion, restricted mouth opening, deviation from the midline to the right side, protrusion, absence of lateral movement to the left side and on palpitation, tenderness and pain over right ascending ramus and right temporal region. A computed tomography (CT) scan showed unilateral fracture of the right coronoid and condylar processes [<xref ref-type="fig" rid="F1">Figure 1</xref>]. There was no evidence of direct trauma or fracture to the zygomatic region or mandibular symphyseal and corpus area. Radiological examination revealed a well-defined radiolucent line at the base of both condylar and coronoid processes [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Under nasal intubation, arch bar was fixed in the maxillary and mandibular dental arches and the inter-maxillary fixation was done with elastic bands. The fractures were corrected using the help of the patients&#x02019; occlusion as a guide. Pre-accident occlusion was achieved. Local analgesic with epinephrine (adrenaline) was infiltrated into the submandibular incision site. A mini submandibular incision was made 2 cm below the angle and inferior border of the mandible. Sharp dissection was performed down through the platysma, and then blunt dissection was begun to minimize risk to the marginal mandibular branch of the facial nerve. Periosteum and attachments for the masseter were completely stripped off the lateral cortex of the vertical ramus up to the level of the sigmoid notch. The subcondylar and coronoid region was exposed and fracture lines were seen. Because of insufficient vision and inability to fix the screws to the plaque in correct directions, a small incision was made to the skin and a trocar was placed for fixing the screws. A silk suture was used at this stage in the lower part of the plates for positioning of the plates in the correct position [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Fractured fragments were fixed with mini plates. To avoid hematoma formation, a small penrose drain was placed in the operation area. The patient was followed-up postoperatively without any esthetic or functional problems [<xref ref-type="fig" rid="F4">Figure 4</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>16-year-old male patient with the chief complaint of pain in the right mandibleramus region, diffuculty in mastication, and mouth opening diagnosed with fracture of the condylar and coronoid processes. CBCT image shows the fractures of both condylar and coronoid processes of the mandible. (black arrows point to the fracture lines).</p></caption><graphic xlink:href="JCIS-4-3-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>16-year-old male patient with the chief complaint of pain in the right mandibleramus region, diffuculty in mastication, and mouth opening diagnosed with fracture of the condylar and coronoid processes. Panaromic X-ray image shows the fractures of condylar and coronoid part of the mandible. (blue arrrows point to the fracture lines).</p></caption><graphic xlink:href="JCIS-4-3-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>16-year-old male patient with the chief complaint of pain in the right mandibleramus region, diffuculty in mastication, and mouth opening diagnosed with fracture of the condylar and coronoid processes. Photograph shows surgical intervention being performed. A silk suture is used at this stage for positioning of the plates in the correct directions.</p></caption><graphic xlink:href="JCIS-4-3-g004"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>16-year-old male patient with the chief complaint of pain in the right mandibleramus region, diffuculty in mastication, and mouth opening diagnosed with fracture of the condylar and coronoid processes. Panoramic X-ray image of the patient's jaw after the operation, shows coronoid and condylar processs of the mandible fixed with two miniplates (arrows).</p></caption><graphic xlink:href="JCIS-4-3-g005"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>The coronoid process is considered a relatively weak part of the mandible. Coronoid fractures due to direct trauma are very uncommon because of their protected position under the zygomaticomalar complex. On the other hand, incidence of condylar fracture is very high and most are not caused by direct trauma.[<xref rid="ref6" ref-type="bibr">6</xref>] The fracture of the coronoid process mostly result from direct, penetrating trauma or if there occurs concomitant sudden and violent contraction of the temporalis muscle at the time of impact.[<xref rid="ref7" ref-type="bibr">7</xref>] Coronoid process has no relation with cranial bones so indirect trauma cannot create fractures. In the present case, fractures occurred due to the sudden contraction of the temporalis muscle because there was no evidence of trauma to the facial bones and there were no trauma-related facial skin injuries [<xref ref-type="fig" rid="F5">Figure 5</xref>]. The literature shows that unilateral fracture of both condylar and coronoid processes without any direct or indirect trauma, like in our case, is very rare.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>16-year-old male patient with the chief complaint of pain in the right mandibleramus region, diffuculty in mastication, and mouth opening diagnosed with fracture of the condylar and coronoid processes. Line diagram explains the probable mechanism. Arrows show the possible cause of the fractures.</p></caption><graphic xlink:href="JCIS-4-3-g006"/></fig><p>The coronoid region can be considered as part of the ramus, the angle region encompasses both the ramus and body, and the symphysis is the anterior part of the body. Each of these areas present unique characteristics, and the decision to reconstruct or repair certain areas is dependent on the goals to be achieved. The coronoid process is considered part of the mandible, and its loss can be considered to be critical. As in other parts of the mandible, the relationship of the coronoid process to the surrounding bones is critical.</p><p>Malpositioning of the coronoid process can impede opening of the jaw owing to interferences with the zygoma and zygomatic arch. Also, trismus may also occur secondary to fibrous or fibro-osseous ankylosis of the coronoid to the zygomatic arch. On the other hand, significantly displaced coronoid process can be pulled in a cephalad direction, occasionally into the infratemporal fossa, by the temporalis muscle.[<xref rid="ref8" ref-type="bibr">8</xref>] Coronoid fracture may manifest as a swelling below the zygomatic arch and soft tissue swelling and ecchymosis in the retromolar trigone region.</p><p>Treatment depends on the degree of the displacement of the fractured coronoid and condylar process and the severity of the symptoms. Fractures with minimal displacement can be managed with a soft diet and mouth opening exercises to avoid osseous adhesions to the surrounding tissues. Fractures with significant displacement that limits mandibular movements can be treated with internal fixation and surgery. If the coronoid fracture edges are properly reduced like in the present case, mini plaques are good treatment choices for fixing the fragments and restoring unrestricted motion of the mandible. Although coronoid fractures are traditionally managed conservatively, surgery may be indicated like in the present case, where the patient was not a good candidate for prolonged intermaxillary fixation. A variety of surgical approaches to treat the fractured condyle have been suggested, including intraoral, submandibular, retromandibular, preauricular, and, more recently, endoscopic. The most important factor in determining the approach used is the level at which the fracture has occurred. Modifying factors such as the degree of displacement or dislocation and the planned method of fixation may also have a bearing on the approach selected. Because of the well-arranged fracture edges and the need to avoid any facial nerve damage, submandibular incision was selected for our case. Submandibular incision is enough for exposing both coronoid and condylar processes. On the other hand, for fixing the screws to the plates, this incision is not enough. We encountered this difficulty in our case, so a small incision was made into the skin for trocar placement. With trocar fixation, screws were easily fixed to the plates. In this step, facial nerve must be protected to avoid any facial nerve damage. Complications of trauma to the TMJ are far-reaching in their effects and not always immediately apparent. Ankylosis, however, is infrequent and has been estimated to occur in only 0.2-0.4% of condylar fractures.[<xref rid="ref9" ref-type="bibr">9</xref>] Therefore, one should apply early mobilization treatment at the expense of a stable occlusion.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>In conclusion, both coronoid and condyle fractures may occur due to reflex contractions of the muscles, and surgical treatment, we present in this article, is a successful choice for management of these types of fractures.</p></sec>

Cytomorphology of columnar cell variant of papillary carcinoma thyroid: A case report and review of the literature

<p>A 58 years old lady reported with history of progressively increasing lump in the neck. Patient had earlier undergone sub-total thyroidectomy (details not available) in a private institute one year back. Fine needle aspiration cytology (FNAC) of the present lump revealed features of papillary carcinoma thyroid. Patient subsequently underwent total thyroidectomy along with excision of a tumor nodule in the larynx. Gross examination of the specimen revealed a tumor nodule in the right lobe of the thyroid. Microscopic examination of the tumor nodule in the thyroid and larynx revealed a columnar cell variant of papillary carcinoma thyroid. Very few reports describing the cytomorphologic features of this variant of papillary carcinoma are available in the published literature. These reports highlight the absence or paucity of nuclear grooves and intranuclear inclusions in this variant. We describe a case of columnar cell variant of papillary carcinoma where nuclear grooves were prominently seen. In addition, we report the occurrence of rosette-like structures which were brought out better on FNA smears. These rosette-like structures have not been emphasized earlier in the published literature. The cytomorphologic features of this rare variant are also reviewed in this report.</p>

<contrib contrib-type="author"><name><surname>Sen</surname><given-names>Arijit</given-names></name><degrees>MD</degrees><email xlink:href="aseniaf@rediffmail.com">aseniaf@rediffmail.com</email><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Nalwa</surname><given-names>Aasma</given-names></name><degrees>MD</degrees><email xlink:href="aasmanalwa@gmail.com">aasmanalwa@gmail.com</email><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mathur</surname><given-names>Sandeep R.</given-names></name><degrees>MD</degrees><email xlink:href="mathuraiims@gmail.com">mathuraiims@gmail.com</email><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Deepali</given-names></name><degrees>MD</degrees><email xlink:href="deepalijain76@gmail.com">deepalijain76@gmail.com</email><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Iyer</surname><given-names>Venkateswaran K.</given-names></name><degrees>MD</degrees><email xlink:href="iyer_venkat@hotmail.com">iyer_venkat@hotmail.com</email><xref ref-type="aff" rid="aff1"/></contrib>

CytoJournal

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Columnar cell variant of papillary carcinoma thyroid is a very rare neoplasm. The entity was first described by Evans in 1986.[<xref rid="ref1" ref-type="bibr">1</xref>] It is an aggressive tumor associated with widespread dissemination and a fatal outcome. The cytology of the neoplasm is quite elusive since the various classical nuclear features of papillary carcinoma like ground glass nuclei, nuclear grooves and intranuclear inclusions have not been found to be consistently present in the published reports on this neoplasm.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] Biological behavior and histomorphology of this neoplasm is quite dissimilar to the conventional papillary carcinoma thyroid and hence it demands attention. The neoplasm consists of papillary fronds and follicles lined by columnar cells with pseudostratification, hyperchromasia of the nuclei and clear or vacuolated cytoplasm.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] This variant of papillary carcinoma usually shows aggressive behavior with invasion of the surrounding soft tissue and even distant metastasis.[<xref rid="ref8" ref-type="bibr">8</xref>] Herein, we report the fine-needle aspiration (FNA) features of a case of columnar cell variant of papillary carcinoma thyroid, which had cytological features that have not been observed in the earlier published cases.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 58-year-old female patient presented with a history of progressively increasing lump in the neck. Review of past medical documents revealed that she had undergone sub-total thyroidectomy 1 year back in a private institute. Slides and blocks were not available for review. FNA was performed on the present lump. Papanicolaou and May Gr&#x000fc;nwald Giemsa stained smears were available. A cytological diagnosis of papillary carcinoma of thyroid was rendered based upon architectural characteristics and nuclear features. Patient underwent total thyroidectomy. The tumor was found to invade the larynx. Following the histopathological diagnosis of columnar cell variant of papillary carcinoma thyroid, a retrospective review of cytomorphology was performed.</p><sec id="sec2-1"><title>Cytopathologic findings</title><p>The smears were cellular and showed tumor cells in aggregates and present singly. The tumor cells were arranged in papillary fronds, syncytial sheets and at places in a rosette-like pattern. The papillary fronds, periphery of syncytial fragments as well as the rosette-like clusters of tumor cells showed prominent pseudostratification [<xref ref-type="fig" rid="F1">Figure 1a</xref>] of the nuclei. Many singly lying cells were also seen having a columnar configuration and wispy cytoplasm [<xref ref-type="fig" rid="F1">Figure 1b</xref>, inset]. There was evidence of nucleomegaly, moderate degree of anisonucleosis, stippled chromatin, and the presence of occasional inconspicuous nucleoli. However, nuclear grooves [<xref ref-type="fig" rid="F1">Figure 1c</xref>] were frequently encountered in the tumor cells. Intranuclear cytoplasmic inclusions were conspicuous by their absence. Mitotic figures were seen [<xref ref-type="fig" rid="F1">Figure 1c</xref>, inset]. Rosette like clusters of the tumor cells were also observed having peripherally aligned nuclei and columnar cytoplasmic processes merging toward the center [<xref ref-type="fig" rid="F1">Figure 1b</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) A papillary cluster of tumor cells displaying prominent pseudostratification of nuclei toward the periphery (Papanicolaou, &#x000d7;400). (b) Rosette-like clusters of tumor cells with tall cytoplasm merging toward the lumen. Inset shows singly lying cells with columnar configuration and wispy cytoplasm (Papanicolaou, &#x000d7;400). (c) Prominent nuclear grooves (black arrows) were seen in the fine needle aspiration smears. Inset shows presence of mitotic figures (Papanicolaou, &#x000d7;400). (d) Histologic section of tumor showing trabecular arrangement with presence of pseudostratification and rosette-like structures (black arrow). Inset highlights the same rosette-like structures (H and E, &#x000d7;200)</p></caption><graphic xlink:href="CJ-11-27-g001"/></fig></sec><sec id="sec2-2"><title>Histopathologic findings</title><p>The gross examination of the specimen revealed a thyroid gland 30 g in weight. Right lobe had a nodule 3.5 cm &#x000d7; 3.0 cm &#x000d7; 2.5 cm. The cut surface of the nodule in the thyroid was solid and homogenous. Microscopic examination revealed an encapsulated tumor mass with invasion of the capsule. There were areas of necrosis. The tumor cells had mostly trabecular arrangement with pseudostratification of nuclei [<xref ref-type="fig" rid="F1">Figure 1d</xref>]. Cells were columnar and had clear to pale pink cytoplasm. Rosette-like structures were also noted in the broad trabeculae of columnar cells [<xref ref-type="fig" rid="F1">Figure 1d</xref>, black arrow and inset]. The nuclei showed mild to moderate anisokaryosis with speckled nuclear chromatin. Single conspicuous nucleoli were visible in few cells with many nuclei showing presence of nuclear grooves. However, no intranuclear cytoplasmic inclusions or psammoma bodies were identified. The tumor showed frequent mitotic figures averaging 4-5/10 hpf. The nodule excised from the larynx showed a tumor mass with similar histomorphology. Immunohistochemistry performed showed diffuse positivity for thyroglobulin and carcinoembryonic antigen. The cells were negative for CK19, chromogranin, and synaptophysin. The case was diagnosed as columnar cell variant of papillary carcinoma thyroid.</p></sec></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Fine-needle aspiration of the thyroid did not gain acceptance till early 1980s when Scandinavian investigators firmly reported its diagnostic accuracy.[<xref rid="ref10" ref-type="bibr">10</xref>] Columnar cell variant of papillary carcinoma thyroid is one of the rarest morphological subtypes.[<xref rid="ref11" ref-type="bibr">11</xref>] Earlier this subtype of papillary carcinoma was thought to be always aggressive; however, an encapsulated tumor appears to have a favorable outcome after complete excision.[<xref rid="ref12" ref-type="bibr">12</xref>] There have been very few published reports on the cytomorphologic description of this rare variant of papillary carcinoma. Six case reports of FNA findings are available in the cytological literature.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref13" ref-type="bibr">13</xref>] The details of these isolated cases have been highlighted in <xref ref-type="table" rid="T1">Table 1</xref>. The features of columnar cell variant of papillary carcinoma thyroid are elusive and hence the diagnosis is difficult solely based on FNA. All the five earlier published cases were diagnosed primarily on thyroid FNA, and the present case could not be diagnosed as a case of columnar cell variant of papillary carcinoma thyroid on FNA.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>]</p><table-wrap id="T1" position="float"><label>Tabel 1</label><caption><p>Cytological features and summary of previously reported cases of columnar cell variant of papillary carcinoma thyroid</p></caption><graphic xlink:href="CJ-11-27-g002"/></table-wrap><p>Pseudostratifcation of nuclei is a prominent feature of this neoplasm, and its presence should alert a cytopathologist regarding this variant of papillary carcinoma. Pseudostratification was present in all the published cases and also seen in our case. Tall cell variant of papillary carcinoma, which is the main differential diagnosis, lacks the feature of pseudostratification though it also has tall columnar cells. The tall cell variant has a single layer of columnar cells with deeply eosinophilic cytoplasm, which line the papillary fronds. The nuclear characteristics in tall cell variant are those of papillary carcinoma thyroid.[<xref rid="ref14" ref-type="bibr">14</xref>] FNA findings in two cases of combined columnar and tall cell variant of papillary carcinoma have also been reported.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] Nuclear grooves were present frequently in our case and one case of mixed columnar cell and tall cell features in contrast to their absence in three of the earlier reported cases and their occasional presence in the other two published cases.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref15" ref-type="bibr">15</xref>] Intranuclear cytoplasmic inclusions were absent in all the published cases except in two cases reported by Kini <italic>et al</italic>. and Tranchida <italic>et al</italic>.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref15" ref-type="bibr">15</xref>] Finely stippled chromatin and inconspicuous nucleoli in our case were in agreement with the finding in most of the published cases.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] The present case also had cells scattered singly and in small groups of 2-3 cells with columnar morphology and a wispy cytoplasm. Presence of this feature in the case of papillary carcinoma may also give a clue about the possibility of this variant. Our case in addition also displayed a rosette-like arrangement of columnar tumor cells. These structures were highlighted better in the FNA smears as compared to the histologic sections. To the best of our knowledge, the presence of these rosette-like structures formed by the merging of tall columnar cytoplasm, have not been emphasized earlier in histologic sections or FNA cytology (FNAC) of this tumor.</p><p>Our patient had recurrence within 1 year, and the recurrent tumor showed invasion of the capsule and extension into the larynx. Poorer prognosis and aggressive behavior of this variant of papillary carcinoma warrants an early recognition of the tumor. FNAC being the first line of investigation in the diagnosis of thyroid swellings, recognition of the cytologic characteristics of this aggressive subtype may be of utility in the management of these patients.</p></sec><sec id="sec1-4"><title>COMPETING INTERESTS</title><p>The authors declare that they have no competing interests.</p></sec><sec id="sec1-5"><title>AUTHORSHIP STATEMENT BY ALL AUTHORS</title><p>Each author acknowledges that this final version was read and approved. All authors qualify for authorship as defined by ICMJE <ext-link ext-link-type="uri" xlink:href="http://www.icmje.org/#author">http://www.icmje.org/#author</ext-link>. Each author participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article.</p></sec><sec id="sec1-6"><title>ETHICS STATEMENT BY ALL AUTHORS</title><p>As this is a case report without identifiers, our institution does not require approval from Review Ethics Board (REB).</p></sec><sec id="sec1-7"><title>EDITORIAL/PEER-REVIEW STATEMENT</title><p>To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double blind model (authors are blinded for reviewers and vice versa) through automatic online system.</p></sec>

Is fine needle aspiration cytology a useful diagnostic tool for granular cell tumors? A cytohistological review with emphasis on pitfalls

<sec id="st1"><title>Background:</title><p>Granular cell tumors (GCT) formerly known as Abrikossoff tumor or granular cell myoblastoma, are rare neoplasms encountered in the fine needle aspiration (FNA) service. Named because of their highly granular cytoplasm which is invariably positive for the S-100 antibody, the classic GCT is thought to be of neural origin. The cytomorphological features range from highly cellular to scanty cellular smears with dispersed polygonal tumor cells. The cells have abundant eosinophilic granular cytoplasm, eccentric round to oval vesicular nuclei with small inconspicuous nucleoli. The fragility of the cells can result in many stripped nuclei in a granular background. The differential diagnosis occasionally can range from a benign or reactive process to features that are suspicious for malignancy. Some of the concerning cytologic features include necrosis, mitoses and nuclear pleomorphism.</p></sec><sec id="st2"><title>Methods:</title><p>We identified 6 cases of suspected GCT on cytology within the last 10 years and compared them to their final histologic diagnoses.</p></sec><sec id="st3"><title>Results:</title><p>Four had histologic correlation of GCT including one case that was suspicious for GCT on cytology and called atypical with features concerning for a malignant neoplasm. Of the other two cases where GCT was suspected, one showed breast tissue with fibrocystic changes, and the other was a Hurthle cell adenoma of the thyroid.</p></sec><sec id="st4"><title>Conclusions:</title><p>These results imply that FNA has utility in the diagnosis of GCT, and should be included in the differential diagnoses when cells with abundant granular cytoplasm are seen on cytology. Careful attention to cytologic atypia, signs of reactive changes, use of immunohistochemistry, and clinical correlation are helpful in arriving at a definite diagnosis on FNA cytology.</p></sec>

<contrib contrib-type="author"><name><surname>Koshy</surname><given-names>Jason</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Schnadig</surname><given-names>Vicki</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Nawgiri</surname><given-names>Ranjana</given-names></name><degrees>MD</degrees><email xlink:href="ranawgir@utmb.edu">ranawgir@utmb.edu</email><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref></contrib>

CytoJournal

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Granular cell tumors (GCT) are rare neoplasms thought to be neuroectodermal in origin. However on fine needle aspiration (FNA), granular cell phenotype may be seen from nonneural entities such as oncocytic neoplasms of the breast and thyroid as well as smooth muscle or fibrohistioctyic tumors. They may arise anywhere in the body with a predilection for the skin, subcutaneous tissue and oral cavity[<xref rid="ref1" ref-type="bibr">1</xref>] with the tongue being the single most common anatomic site involved. Histologic sections of GCTs show relatively consistent morphology while the cytologic appearance of GCT on needle aspiration is variable. The granular cytoplasm reflects the presence of secondary lysosomes and stains strongly for immunohistochemical markers S-100 and neuron specific enolase, as well as other melanocytic markers, CD68, inhibin, and calretinin. They are negative for keratin and muscle markers. Malignant GCTs are much rarer accounting for no more than 2-3% of all GCT. They present with size &#x0003e;4 cm, and show rapid growth and infiltrative borders. Suggested criteria for malignant GCTs include mitoses, necrosis, cellular pleomorphism, increased nuclear/cytoplasm ratios, vesicular nuclei with large nucleoli and high Ki-67 expression.[<xref rid="ref2" ref-type="bibr">2</xref>] Perineural invasion can be seen in both benign and malignant tumors.[<xref rid="ref3" ref-type="bibr">3</xref>] Because of the marked variability seen on cytologic smears, the differential diagnoses of GCT are many and include infection, inflammation and neoplasms, both benign and malignant.</p><p>This paper discusses the clinical features and origin of GCT and highlights the variable cytology seen on FNA preparations. Emphasis is placed on avoiding potential pitfalls by keeping in mind the vast differential diagnosis of an oncocytic lesion on FNA, paying close attention to the cytomorphological characteristics of GCT, as well as the use of special stains and immunohistochemistry on cell block preparations.</p></sec><sec sec-type="methods" id="sec1-2"><title>METHODS</title><p>This study was approved by the Institutional Review Board. An electronic search of institutional cytopathology archives was conducted via the laboratory information system in order to identify cases of GCT. Six cases of suspected GCT within the previous 10 years were identified. The medical records of the patients were then reviewed for final diagnoses as well as histopathologic and clinical findings.</p><p>In all cases, the FNA of the palpable lesions was performed by a cytopathologist using a Cameco syringe holder and 22-23 gauge needles. The smears included air-dried preparations stained with a rapid Romanowski method for on-site evaluation and ethanol-fixed smears stained by the Papanicolaou method. The cytomorphology, histopathology and immunohistochemical stains (if any) were studied.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>Of six cases, four were confirmed as GCT by histology. Of the other two cases that were suspected GCT bycytology, one was a breast mass discovered by routine mammography on a 78-year-old woman. Concomitant and subsequent biopsies showed benign breast tissue with fibrocystic changes and apocrine metaplasia. The other was a nontender, palpable thyroid nodule in a 44-year-old woman with systemic lupus erythematosus. Histologic evaluation of the thyroidectomy specimen rendered a diagnosis of Hurthle cell adenoma.</p><p>Of the cases that were proven to be GCT, one was a 65-year-old woman who presented with an enlarging, tender left posterior neck mass. FNA showed paucicellular smears with rare intact cells and bare, round nuclei within a pale, faintly granular background [Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>]. The nuclei demonstrated mild pleomorphism very prominent macronucleoli, but mitotic figures and necrosis were absent. The excisional biopsy showed a S-100 positive tumor [Figures <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref>]. Another case was a 29-year-old man presenting with a tender scalp mass. FNA showed abundant bare nuclei with mild nuclear enlargement and relatively prominent nucleoli. There were very distinct granules were noted in the cytoplasm and background. The results summary is given in <xref ref-type="table" rid="T1">Table 1</xref>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Diff-quik (&#x000d7;20) stains highlight intact cells with abundant granular cytoplasm and bare nuclei in a granular background</p></caption><graphic xlink:href="CJ-11-28-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Papanicolaou stain (&#x000d7;40) shows few intact cells with abundant granular cytoplasm and bland nuclei</p></caption><graphic xlink:href="CJ-11-28-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>H and E, (&#x000d7;40) histologic section show infiltrative tumor with abundant granular cytoplasm and bland nuclei with small prominent nucleoli</p></caption><graphic xlink:href="CJ-11-28-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>S-100 (&#x000d7;40) shows strong nuclear and cytoplasmic immuno reactivity</p></caption><graphic xlink:href="CJ-11-28-g004"/></fig><table-wrap id="T1" position="float"><label>Tabel 1</label><caption><p>Results summary</p></caption><graphic xlink:href="CJ-11-28-g005"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Granular cell tumor is a rare neoplasm encountered on FNA preparations that can closely mimic other conditions. These include benign and malignant neoplasms as well as infections. Most GCTs are benign, measuring &#x0003c;3 cm, often arise in the head and neck region including the oral mucosa, but can arise anywhere in the body and can be multifocal. Pseudoepitheliomatous hyperplasia of the overlying epidermis or mucosa is common. Females and African-Americans are more affected than males and Caucasians respectively.</p><p>Granular cell tumor cytology show smears of naked nuclei within a coarsely granular background derived from the fragile cytoplasm of tumor cells. Specimen cellularity is variable ranging from scant to cellular preparations depending on the fibrosis associated with the lesion. When smears are paucicellular as was our case of the 65-year-old female with an enlarging left posterior neck mass, careful attention to the finely granular background is especially warranted as it may be the only clue pointing toward a GCT. Intact polygonal tumor cells are uniform and can occur as both syncytial groups and isolated cells. The cells show small round-to-oval nuclei with a minimal pleomorphism, ill-defined cell borders, and small but conspicuous nucleoli with abundant fine to coarse granular cytoplasm. The granules are periodic acid Schiff (PAS)-positive and diastase-resistant, and are believed to be lysosomes. Reported histologic Pustolo-ovoid bodies of Milan, which are aggregates of lysosomal granules, are rarely seen in cytology smears.[<xref rid="ref4" ref-type="bibr">4</xref>] Cell blocks are of value as immunohistochemical stains for S-100 and CD 68 support the diagnosis of GCT. The CD 68 stains the lysosomes, and is not a specific histiocytic marker, as positivity may also seen in fibroblasts, dendritic cells, basophils, osteoclasts and tumors of these cells. GCTs are usually positive for vimentin, neuron-specific enolase (NSE), and negative for smooth muscle actin, desmin, actin, epithelial membrane antigen, cytokeratins, chromogranin, and HMB45. Electron microscopy supports a schwannian derivation.</p><p>The polygonal cells and fragile granular cytoplasm can often be mistaken for histiocytes and necrosis. Therefore, atypical infections, granulomatous inflammation, and histiocytic infiltrates must be considered in the differential diagnosis. Histiocytes however have smaller irregular or bean-shaped nuclei compared to the round to oval nuclei of GCT. Granulomatous inflammation consists of other inflammatory cells as opposed to pure population of neoplastic cells seen in GCT. Other benign nonneoplastic entities such as fat necrosis can cause similar findings. Neoplastic lesions in the differential diagnosis of GCT include rhabdomyomas, fibroxanthomas, dermatofibromas, oncocytic neoplasms, alveolar soft part sarcoma (ASPS) and metastatic renal cell carcinoma.[<xref rid="ref5" ref-type="bibr">5</xref>] This is primarily due to the granular nature of their cytoplasm. Frequent cross-striations, &#x0201c;spider cells&#x0201d;, glycogen and negative S-100 immunostaining favors rhabdomyoma versus GCT. ASPS typically has frequent bi-and multinucleation, and pseudoacinar or pseudoalveolar architecture. Also, molecular studies of ASPS have demonstrated the chromosome rearrangement der (17) t (X; 17) (p11;q25),[<xref rid="ref6" ref-type="bibr">6</xref>] while GCT has typically has no cytogenetic abnormalities. Renal cell carcinoma is S-100 negative and consists of scattered tumor cells with clear cytoplasm in contrast to GCT cells which contain uniformly granular cytoplasm.</p><p>A primary GCT of the thyroid gland is extremely uncommon with six reports seen in the literature.[<xref rid="ref7" ref-type="bibr">7</xref>] Similar to GCT in general, these thyroid lesions are predominantly benign though metastasis has been reported.[<xref rid="ref8" ref-type="bibr">8</xref>] Often these lesions are located in neck structures around or adjacent to the thyroid gland and are misdiagnosed as a primary thyroid neoplasm. Kintanar <italic>et al</italic>. reported a case of a GCT of the trachea that was initially interpreted as a thyroid follicular neoplasm with Hurthle cell change.[<xref rid="ref9" ref-type="bibr">9</xref>] Histology showed similar features seen on cytology including uniform cells with abundant granular amphophilic cytoplasm. Immunohistochemical stains for S-100, NSE, and vimentin were positive rendering the diagnosis of GCT. A similar misdiagnosis of a GCT as a thyroid nodule has been seen in the esophagus.[<xref rid="ref10" ref-type="bibr">10</xref>] Despite its rarity, GCT of the thyroid must be in the differential of a granular cell lesion and can be easily mistaken for a Hurthle cell neoplasm due to the inherent oncocytic nature of their cytoplasm. In our case, FNA smears showed findings seen in both GCT and a Hurthle cell neoplasm including polygonal cells with conspicuous nucleoli and granular cytoplasm. Borderline cases like ours illustrate the necessity of ancillary studies such as PAS stains and immunohistochemistry. Obvious cytoplasmic membranes and larger nuclei with more prominent nucleoli tend to point toward a Hurthle cell lesion.[<xref rid="ref11" ref-type="bibr">11</xref>] A rare differential of GCT in the thyroid is black thyroid syndrome secondary to long-term minocycline therapy. Like GCT, cells of black thyroid syndrome have ill-defined borders and contain abundant granular cytoplasm. However, classic black thyroid findings include blue-gray cytoplasm with dark-brown granules and colloid can often be seen in the background.[<xref rid="ref12" ref-type="bibr">12</xref>] Finally, because of the increased surveillance of thyroid nodules, a histiocytic reaction to a previous FNA should be considered. Histiocytes with foamy granular cytoplasm may mimic the cells of GCT, however the nuclei tend to be reniform as opposed to round and oval and the cytoplasm more foamy than granular. Increased inflammation, scarring, and hemosiderin pigment found in FNA biopsy sites help to distinguish them from GCT.</p><p>Similar pitfalls can be seen in breast masses. Generally, GCT of the breast is a rare lesion with approximately 225 cases reported.[<xref rid="ref13" ref-type="bibr">13</xref>] Almost all reported cases of GCT have been located in the upper quadrants of the breast[<xref rid="ref14" ref-type="bibr">14</xref>] and corresponds to the area of innervation of the supraclavicular nerve.[<xref rid="ref15" ref-type="bibr">15</xref>] This correlates with the theory that GCT is derived from neural cells. These lesions are typically benign but can show clinical signs of malignancy such as an infiltrating spiculated lesion seen on breast imaging.[<xref rid="ref16" ref-type="bibr">16</xref>] Their cytologic features can also mimic carcinoma.[<xref rid="ref17" ref-type="bibr">17</xref>] The fragile granular cytoplasm of GCT can lead to a &#x0201c;dirty&#x0201d; background synonymous with necrosis that can be seen in breast carcinoma and other malignancies.[<xref rid="ref18" ref-type="bibr">18</xref>] In the cases where GCT shows a degree of cellular and nuclear atypia, apocrine cancer and metastatic lesions such as renal cell carcinoma and melanoma must be ruled out.[<xref rid="ref16" ref-type="bibr">16</xref>] This is accomplished with immunohistochemical stains such as S-100, vimentin, and CEA. Apocrine metaplasia, part of the fibrocystic changes spectrum seen in the breast, consists of benign epithelial cells with abundant eosinophilic granular cytoplasm and can be easily mistaken for GCT. The well-defined cellular borders of the apocrine-like breast epithelial cells versus the ill-defined borders seen in GCT is a clue to proper diagnosis. Also, the granularity of apocrine metaplasia is finer and pink on Romonowski stains compared to GCT, which is typically coarser and blue. Fat necrosis too can have granular background, however the cells have a foamy cytoplasm and multinucleated giant cells, and inflammatory cells are frequently seen.</p><p>Granular cell tumor is an uncommon neoplasm that can mimic many different diseases. The ability of GCT on FNA to appear as everything from benign histiocytes to malignant epithelial and mesenchymal cells can cause a diagnostic dilemma for practicing cytopathologist who may not have GCT on their differential. While FNA remains to be a critical, minimally-invasive diagnostic tool, our results underscores the necessity of ancillary studies such as immunohistochemistry and special stains. Awareness of this entity and its variable cytology, its differential diagnoses and careful clinical correlation are critical in accurate interpretation of findings.</p></sec><sec id="sec1-5"><title>COMPETING INTERESTS STATEMENT BY ALL AUTHORS</title><p>All authors declare no competing interests.</p></sec><sec id="sec1-6"><title>AUTHORSHIP STATEMENT BY ALL AUTHORS</title><p>All authors of this article declare that they qualify for authorship as defined by the ICMJE. All authors are responsible for the conception of this study, have participated in its design and coordination, and helped to draft the manuscript. All authors have read and approved the final manuscript.</p></sec><sec id="sec1-7"><title>ETHICS STATEMENT BY ALL AUTHORS</title><p>This study was conducted with approval from the Institutional Review Board (IRB) of the institution. Authors take responsibility to maintain relevant documentation in this report.</p></sec><sec id="sec1-8"><title>EDITORIAL/PEER-REVIEW STATEMENT</title><p>To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double blind model (authors are blinded for reviewers and vice versa) through automatic online system.</p></sec>

Carotid sinus syndrome: Progress in understanding and management

<p>Carotid sinus syndrome (CSS) is a disease of the autonomic nervous system presenting with syncope, especially in older males who often have cardiovascular disease. The aetiology is unknown and epidemiological data is limited. Forty new patients/million population have been estimated to require pacing for CSS and these patients represent &#x0223c;9% of those presenting syncope to a specialist facility. CSS is defined as a response to carotid sinus massage (CSM) that includes reproduction of spontaneous symptoms. Cardioinhibitory CSS shows 3s asystole on CSM and vasodepressor CSS shows &#x0003e;50&#x000a0;mmHg fall in blood pressure (BP), there are mixed forms. The methodology of CSM requires correct massage in the supine and upright with continuous ECG and BP. Assessment of the vasodepressor component implies the &#x02018;method of symptoms&#x02019; using atropine to prevent asystole. Carotid sinus hypersensitivity (CSH) is a related condition where CSM is positive in an asymptomatic patient. CSH cannot be assumed to respond to pacing. CSS patients present syncope with little or no warning. If no cause is revealed by the initial evaluation, CSM should be considered in all patients &#x0003e;40 years. CSM carries a small risk of thromboembolism. Therapy for cardioinhibitory CSS is dual chamber pacing, which is most effective in patients with a negative tilt test. Syncope recurrence is &#x0223c;20% in 5 years in paced patients. Therapy for the vasodepressor component of CSS, as pure vasodepression or mixed, where tilt testing will likely be positive, is often unrewarding: alternative therapeutic measures may be needed including discontinuation/reduction of hypotensive drugs.</p>

<contrib contrib-type="author"><name><surname>Sutton</surname><given-names>Richard</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Definition</title><p>Carotid sinus syndrome is defined, by the new Guidelines of the European Society of Cardiology (ESC)<xref rid="bib1" ref-type="bibr"> ¹ </xref> as syncope with reproduction of symptoms during carotid sinus massage (CSM) of 10s duration. It is cardioinhibitory (CI) when CSM generates &#x0003e;3s asystole (<xref ref-type="fig" rid="fig1">Figure 1</xref>). It is always vasodepressor but the degree of vasodepression varies and when it exists alone, the vasodepressor (VD) form, the systolic blood pressure falls &#x0003e;50&#x000a0;mmHg.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Intermediate or Mixed forms show both features.</p><p>For purposes of clarity, carotid sinus hypersensitivity (CSH) is not carotid sinus syndrome (CSS). CSH is present when a patient has cardioinhibitory, mixed or vasodepressor findings on CSM, with or without symptoms but is asymptomatic otherwise.</p><p>The most recent European Society of Cardiology Guidelines on Pacing<xref rid="bib1" ref-type="bibr"> ¹ </xref> considered altering the previously used 3s duration of asystole to 6s (a new cut-off) for decisions concerning selection of pacing therapy as 3s is too short. The duration of asystole, which causes symptoms in CI and Mixed forms is generally much longer than the historical 3 s cut-off value. On average the duration of asystole to cause symptoms is 7.6&#x000a0;&#x000b1;&#x000a0;2.2 s and the fall in blood pressure is 63&#x000a0;&#x000b1;&#x000a0;24&#x000a0;mmHg.<xref rid="bib2" ref-type="bibr"> ² </xref> CSS is a provocative test and like tilt testing it is difficult to be sure that the provocation reproduces what happens spontaneously.<xref rid="bib3" ref-type="bibr"> ³ </xref> </p></sec><sec><title>Aetiology</title><p>The aetiology of CSS is unknown. It presents in older persons, with a mean age &#x0223c;75 years, and has a strong male dominance &#x0003e;2:1. CSS is an autonomic nervous system disease involving a pathological reflex, the pathophysiology of which has features similar to vasovagal syncope (VVS) with two main elements of its reflex involving cardioinhibition via the vagus nerve and vasodepression, which is thought to be due to sympathetic withdrawal. The abnormal reflex has been attributed to disturbance of baroreceptor function<xref rid="bib4" ref-type="bibr"> ⁴ </xref> and also to degeneration of the medulla.<xref rid="bib5" ref-type="bibr"> ⁵ </xref> CSS has overlap with VVS (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Both may exist in the same patient, but they appear to be independent of each other.<xref rid="bib7" ref-type="bibr"> ⁷ </xref> Carotid sinus syndrome has its name because its initial discovery was by mechanical stimulation of the Carotid sinus as described by Roskam<xref rid="bib8" ref-type="bibr"> ⁸ </xref> and Weiss and Baker.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> Their patients had the so-called spontaneous form of the disease. Tight collars and neck movements<xref rid="bib8" ref-type="bibr"> ^8,9 </xref> have a particular tendency to trigger the reflex and occasionally neck tumours, neck surgery or irradiation may also act as triggers.<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref> Most patients present syncope without any local trigger but the diagnosis is nevertheless made by addressing the carotid sinus by massage, CSM, as described above.</p></sec><sec><title>Epidemiology</title><p>Comprehension of the epidemiology of carotid sinus syndrome is adversely affected by confusion over its definition. The only fairly precise estimates of incidence of CSS were made in the 1980s from Lavagna in Italy<xref rid="bib11" ref-type="bibr"> ¹¹ </xref> and from Worthing, Sussex in the United Kingdom<xref rid="bib12" ref-type="bibr"> ¹² </xref> which gave that of cardioinhibitory CSS as 35&#x02013;40 new patients per million population per year. The reason for the restriction to cardioinhibition reflects selection of patients for treatment by pacing. To my knowledge there have been no good estimates of population incidence that include the vasodepressor form of CSS. The prevalence of CSS has been estimated to be &#x0003c;&#x000a0;4% in patients &#x0003c;&#x000a0;40 years and 41% in those &#x0003e;80 years attending a specialized syncope facility.<xref rid="bib13" ref-type="bibr"> ^13,14 </xref> Estimates of the incidence amongst patients presenting with syncope are better than population data with the latest figures from Lavagna, Italy being 8.8% having CSS in a population of 1855 patients with unexplained syncope by initial evaluation.<xref rid="bib2" ref-type="bibr"> ² </xref> Of these 164 patients 81% had asystole with CSM and 19% had vasodepression. CSS is more common at 8.8% of presenting patients than cardiac syncope of all types, as this represents 10% of patients unexplained by the initial evaluation but only 5% of those after the final diagnosis.<xref rid="bib2" ref-type="bibr"> ² </xref> </p></sec><sec><title>Clinical features</title><p>Patients present with syncope that has little or no prodrome. They are mostly males and often have evidence of cardiovascular disease. With respect to rhythm disturbances there is an association with sinus node disease ranging from 21&#x02013;56% and with atrioventricular block (21&#x02013;37%). Syncope recurrence is common and is reported to be 50% in 2 years.<xref rid="bib15" ref-type="bibr"> ^15,16 </xref> There is also a high mortality, which is considered to be related to co-morbidities and age rather than CSS itself.<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref> When monitored by a special delayed hysteresis pacemaker or by an implantable loop recorder<xref rid="bib15" ref-type="bibr"> ^15,16 </xref> in cardioinhibitory patients the detected arrhythmia is sinus arrest without escape rhythm in 72%. The overlap between CSS and VVS raises difficulties in determination of which is the attributable cause of syncope.<xref rid="bib6" ref-type="bibr"> ^6,17 </xref> </p></sec><sec><title>Methodology of CSM</title><p>Carotid sinus massage should be considered in all patients with syncope over the age of 40 years when the cause of syncope is not detected by the initial evaluation of the patient using clinical history from patient and witness, physical examination, 12-lead ECG and estimation of blood pressure on standing for 3 minutes.<xref rid="bib18" ref-type="bibr"> ¹⁸ </xref> Below the age of 40, CSS is sufficiently rare to allow omission of CSM.</p><p>Carotid sinus massage is conducted in a hospital facility. When the patient undergoes the test, the possible outcome should be explained beforehand. In some countries written consent may be required. It must be regarded as a provocative test that carries a small risk of cerebral embolism almost always associated with complete recovery.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> Nowadays, the test is often performed in a tilt test laboratory as there, supine and erect massage of the two carotids sequentially can be undertaken in a controlled and safe manner. The added diagnostic value of repeating CSM in the upright position has been well documented by Kenny's group.<xref rid="bib20" ref-type="bibr"> ²⁰ </xref> During the test, the ECG, together with beat-to-beat blood pressure, usually non-invasively, is continuously recorded (<xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><p>The carotid artery sinus lies at the anterior margin of the sternocleidomastoid muscle at the level of the cricoid cartilage. Usually the right artery is massaged first for no reason more than right structures are examined first with the physician approaching from the patient's right. Massage of the artery can be performed by the thumb or by the index, middle and ring fingers according to personal preference. The essence of massage is that it is <italic>massage</italic> and not extreme pressure, and certainly not occlusive pressure. If necessary, this can be monitored by a finger of the other hand on the ipsilateral temporal artery. There is some lateralisation of positive responses, with sinus arrest being the more common response to right artery massage, and atrioventricular block being seen occasionally on left massage.<xref rid="bib12" ref-type="bibr"> ¹² </xref> Massage is conducted for 10s. After the right artery, the left is massaged. The tilt table is then raised to 60&#x02013;80 degrees and the right and left massages are repeated. The question of whether this test should be performed before or after a formal tilt test is not, at present, answered. Our own practice is to perform it after the tilt test; Brignole prefers to perform it before tilt, as the effect of tilt on CSM findings is unknown (personal communication). The above given definitions of CSS and CSH are what constitute positive results. The results of CSM are repeatable phenomena but there is potential for fatigue. It is, therefore, recommended that only the minimum number of massages be performed (4 or 6 if the method of symptoms is employed).</p><sec><title>Method of symptoms</title><p>The &#x02018;Method of symptoms&#x02019; was first proposed by Thomas in 1969,<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref> but clinically applied by Brignole.<xref rid="bib21" ref-type="bibr"> ²¹ </xref> It is clear that an asystolic response will have a major effect on blood pressure. So, in order to assess the possible contribution of vasodepression in an asystolic patient atropine can be given intravenously (1&#x000a0;mg or 0.02&#x000a0;mg/kg body weight) and the massage repeated. Asystole is then prevented by the drug and the fall in blood pressure can be measured. Atropine has side-effects and these need to be discussed with the patient at the outset of the test and included in the formal consent, if one is used. Further to the above given definitions, a mixed response to CSM is one where there is an asystolic period of &#x0003e;3s and a fall in blood pressure of &#x0003e;50&#x000a0;mmHg. This can only be assessed using active prevention of the asystole by atropine or possibly by temporary pacing, which is considered too invasive, except in very unusual cases. This describes the &#x02018;Method of symptoms&#x02019; where if there is asystole in the first massage with reproduction of symptoms and symptoms are abolished by atropine in the second massage of the same artery it is revealed that the period of asystole was responsible for the symptoms, see <xref ref-type="table" rid="tbl1">Table 1</xref> &#x02018;Classification of CSS&#x02019;.</p></sec><sec><title>Contraindications to CSM</title><p>Currently, it is accepted that a carotid bruit is a contraindication to CSM but it is known that carotid bruits do not correlate well with degrees of carotid stenosis. A Carotid bruit's elevation of the risk of massage has never been put to the test. However, in small series, patients with quite severe carotid stenoses have safely undergone CSM.<xref rid="bib22" ref-type="bibr"> ²² </xref> There is less controversy about recent (within 3 months) transient ischaemic attacks, strokes and myocardial infarctions providing contraindications to CSM, but the nature of the contraindication should be more considered to the autonomic changes wrought by these conditions altering the results, than the dangers of the CSM at this time.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> CSM occasionally precipitates atrial fibrillation, which quickly reverts to sinus rhythm.</p></sec></sec><sec><title>Carotid sinus hypersensitivity</title><p>As has been stated, carotid sinus hypersensitivity is a positive response to carotid sinus massage in an asymptomatic patient. It could, therefore, be construed that CSH is a precursor of CSS. While this may be true, no data exist to confirm this possibility. However, CSH has been taken to indicate the existence of an abnormal reflex, which may have importance in unexplained falls, where it is necessary to take into account that there may have been syncope but the history of syncope is unavailable due to the relatively common amnesia for the event.<xref rid="bib23" ref-type="bibr"> ²³ </xref> Several studies have been performed to investigate the role of the abnormal reflex in unexplained falls and its possible treatment by pacing to prevent the expected bradycardia and thereby prevent at least some falls.<xref rid="bib24" ref-type="bibr"> ^{24&#x02013;27} </xref> The first trial, SAFE PACE,<xref rid="bib24" ref-type="bibr"> ²⁴ </xref> showed promise that there may be a favourable influence of pacing but this has not been substantiated in the subsequent studies.<xref rid="bib25" ref-type="bibr"> ^{25&#x02013;27} </xref> One of the reasons for these disappointing results may be the lack of equivalence of CSH to CSS in fallers.</p></sec><sec><title>Treatment of cardioinhibitory CSS</title><p>Pacing was introduced for this indication in the 1970s<xref rid="bib28" ref-type="bibr"> ²⁸ </xref> and was developed in the early 1980s when dual chamber pacing became widely available.<xref rid="bib29" ref-type="bibr"> ^29,30 </xref> The results were encouraging to enthusiasts, but neither the diagnosis nor the therapy became accepted practice for an extended period. CSM was not performed in many centres and was considered dangerous until the relatively benign complications incidence was reported.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> Syncope recurrence was also considered to be a problem. That it was higher than in atrioventricular block should have been anticipated, as CSS is a form of reflex syncope involving &#x02013; in every case &#x02013; some degree of vasodepression. Results in the late 1980s, showing a 9% recurrence with dual chamber pacing and 18% with ventricular pacing in 5 years gave a realistic picture at the time.<xref rid="bib31" ref-type="bibr"> ³¹ </xref> This was corroborated by Lopes et al in 2011,<xref rid="bib32" ref-type="bibr"> ³² </xref> using dominantly dual chamber pacing with 10.9% recurrence also in 5 years, but Brignole and Menozzi found 20% in their series over 5 years of follow-up.<xref rid="bib33" ref-type="bibr"> ³³ </xref> Furthermore, in relation to current data, these figures are relatively acceptable with &#x0223c;20% in sinus node disease in 5.5 years<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> and 25% in 2 years in older vasovagal patients.<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> One randomized trial (RCT) was performed on 60 patients in 1992, with a highly significant benefit of pacing being shown (<italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.002),<xref rid="bib36" ref-type="bibr"> ³⁶ </xref> with a second RCT by another group in 2007 serving to confirm the earlier results.<xref rid="bib37" ref-type="bibr"> ³⁷ </xref> </p><p>CSS became fully accepted as having a strong indication for pacing and is classified as a Class 1 indication, level of evidence B in the current ESC Guidelines for Pacing.<xref rid="bib1" ref-type="bibr"> ¹ </xref> The mode of pacing is advised to be dual chamber for all those in sinus rhythm, reserving VVI pacing for those in permanent atrial fibrillation.<xref rid="bib1" ref-type="bibr"> ¹ </xref> </p><p>Recent earlier evidence suggests that, in both CSS and VVS, a positive tilt test implies a less positive outcome, in terms of recurrent syncope (<xref ref-type="fig" rid="fig3">Figure 3</xref>), from pacing,<xref rid="bib2" ref-type="bibr"> ^2,38,39 </xref> which may be explained by the presence of a more potent vasodepressor component of the reflex.<xref rid="bib40" ref-type="bibr"> ⁴⁰ </xref> It is in this regard that consideration of overlap between CSS and VVS is most important.</p><p>There is a case to be made for use of tilt-testing in CSS to risk stratify cardioinhibitory or mixed patients in order to anticipate the likely recurrence of syncope and, perhaps, to go a step further and attempt prophylactic treatment of the vasodepressor component. However, since this treatment is less than satisfactory it may be prudent to await a first recurrence before embarking on therapy.</p></sec><sec><title>Therapy of vasodepressor CSS</title><p>Management of vasodepressor CSS has received relatively little attention.<xref rid="bib41" ref-type="bibr"> ⁴¹ </xref> Patients are advised to take increased volumes of fluid (2+litres/day) and, if safe, to increase salt consumption toward 6g/day. The principle difficulty in management is the frequent coincidence of hypertension. This coincidence becomes even more important when drug therapy of recurrent syncope has to be considered because those drugs, which may offer benefit in supporting low blood pressure during symptoms are likely to raise blood pressure to unacceptable levels at all other times. The first step should be to attempt reduction or cessation of hypotensive medication.<xref rid="bib2" ref-type="bibr"> ² </xref> Following this, if unsatisfactory, fludrocortisone and midodrine are the drugs typically considered, but both are contraindicated in hypertensive patients. Little trial evidence is available to guide the physician. One trial, a RCT, included some vasodepressor CSS patients with a measure of success, but the trial was very small in size and without prolonged follow-up.<xref rid="bib42" ref-type="bibr"> ⁴² </xref> Recent discussions of the aggressiveness of blood pressure control are relevant to these patients<xref rid="bib43" ref-type="bibr"> ⁴³ </xref> and it is reasonable to allow higher than recommended levels of blood pressure to gain benefit at times of hypotension. A newly-available drug, droxidopa, may have some value in these patients but no evidence is yet available in CSS management. The most frequently considered drug is midodrine but one of its common side effects in males is urinary retention, making it untenable in many who might benefit.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Carotid sinus syndrome is a relatively common cause of syncope in patients &#x0003e;40 years old but it is, even today, too infrequently sought as a possible cause. It occurs dominantly in males of advanced age and is diagnosed by CSM with reproduction of spontaneous symptoms.</p><p>Carotid sinus hypersensitivity is a positive result of massage in the absence of clinical symptoms. Thus, these two conditions must be held as distinct entities. CSS is present in 8.8% of patients with syncope unexplained after initial evaluation, cardiac syncope is present in 10% of all patients presenting syncope and in 5% of those unexplained after initial evaluation, thus CSS is more frequent than all types of cardiac syncope combined.</p><p>Cardioinhibitory CSS is treated with acceptable success by pacing dual chamber in all those showing sinus rhythm. Greatest success may be expected in those patients who are tilt-test negative. Treatment of vasodepressor CSS is difficult and often unsatisfactory. Reduction of hypotensive medication and fluid intake increase should be the first step in management if the patient has been taking anti-hypertensive therapy. If recurrent syncope cannot be controlled, careful use of midodrine together with acceptance of higher than usually accepted blood pressure levels is recommended. Tilt-test positive patients may have recurrence of syncope despite adequate pacing. They may benefit from measures to control the vasodepressor component of the reflex.</p></sec>

Molecular candidates for cardiac stretch-activated ion channels

<p>The heart is a mechanically-active organ that dynamically senses its own mechanical environment. This environment is constantly changing, on a beat-by-beat basis, with additional modulation by respiratory activity and changes in posture or physical activity, and further overlaid with more slowly occurring physiological (e.g. pregnancy, endurance training) or pathological challenges (e.g. pressure or volume overload). Far from being a simple pump, the heart detects changes in mechanical demand and adjusts its performance accordingly, both via heart rate and stroke volume alteration. Many of the underlying regulatory processes are encoded intracardially, and are thus maintained even in heart transplant recipients. Over the last three decades, molecular substrates of cardiac mechanosensitivity have gained increasing recognition in the scientific and clinical communities. Nonetheless, the processes underlying this phenomenon are still poorly understood. Stretch-activated ion channels (SAC) have been identified as one contributor to mechanosensitive autoregulation of the heartbeat. They also appear to play important roles in the development of cardiac pathologies &#x02013; most notably stretch-induced arrhythmias. As recently discovered, some established cardiac drugs act, in part at least, via mechanotransduction pathways suggesting SAC as potential therapeutic targets. Clearly, identification of the molecular substrate of cardiac SAC is of clinical importance and a number of candidate proteins have been identified. At the same time, experimental studies have revealed variable&#x02013;and at times contrasting&#x02013;results regarding their function. Further complication arises from the fact that many ion channels that are not classically defined as SAC, including voltage and ligand-gated ion channels, can respond to mechanical stimulation. Here, we summarise what is known about the molecular substrate of the main candidates for cardiac SAC, before identifying potential further developments in this area of translational research.</p>

<contrib contrib-type="author"><name><surname>Reed</surname><given-names>Alistair</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib><contrib contrib-type="author"><name><surname>Kohl</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="aff2 aff3 aff4"> ^2,3,4 </xref></contrib><contrib contrib-type="author"><name><surname>Peyronnet</surname><given-names>R&#x000e9;mi</given-names></name><xref ref-type="aff" rid="aff2 aff3"> ^2,3 </xref><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>Mechanosensitivity is an intrinsic property of cardiac functional autoregulation (<xref ref-type="fig" rid="fig1">Figure 1</xref>), affecting mechanical activity (e.g. via the Frank-Starling effect<xref rid="bib1" ref-type="bibr"> ^1,2 </xref>: an acute stretch-induced increase in cardiac contractility in the absence of raised intracellular calcium) and electrical behaviour (e.g. through the Bainbridge effect<xref rid="bib3" ref-type="bibr"> ³ </xref>: the stretch-induced increase in spontaneous pacemaker rate) of the heart. These mechano-electric feedback (MEF) responses are sustained in denervated (e.g. isolated<xref rid="bib4" ref-type="bibr"> ^4&#x02013;6 </xref> or transplanted<xref rid="bib7" ref-type="bibr"> ^7&#x02013;9 </xref>) hearts, in isolated tissue<xref rid="bib10" ref-type="bibr"> ^10,11 </xref> and even single cells &#x02013; in both cardiac myocytes<xref rid="bib12" ref-type="bibr"> ^{12&#x02013;16} </xref> and non-myocytes.<xref rid="bib17" ref-type="bibr"> ^{17&#x02013;19} </xref> </p><p>Adaptation to a highly dynamic mechanical environment is a crucial feature of normal cardiac function. It is involved in the regulation of beat-by-beat physiology,<xref rid="bib4" ref-type="bibr"> ^4,20,21 </xref> and implicated in the progression of cardiac diseases, including rhythm disturbances.<xref rid="bib22" ref-type="bibr"> ^{22&#x02013;25} </xref> For a compendium of current insight into cardiac mechano-electric coupling and arrhythmias, from pipette to patient, see<xref rid="bib26" ref-type="bibr"> ²⁶ </xref>. Although the mechanisms underlying cardiac mechanotransduction are not completely understood, key players are thought to include mechanosensitive ion channels (MSC). MSC are defined in the broadest sense by their ability to change ion channel open probability in response to mechanical stimuli, thereby converting mechanical energy into the modification of an electrochemical signal.<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> MSC have been demonstrated to act as functional mechanotransducers in a number of different tissues, including the heart, and their block is capable of preventing or terminating certain mechanically-induced arrhythmias.<xref rid="bib28" ref-type="bibr"> ^28,29 </xref> </p><p>MSC can be subcategorised by the type of mechanical stimulation required for channel activation. Although these boundaries are far from clear-cut, it is useful to make this conceptual distinction. In this review we shall focus on <italic>stretch</italic>-activated ion channels (SAC), which are those MSC whose switching from &#x02018;closed&#x02019; to &#x02018;open&#x02019; state can be driven over their full dynamic range by stretch alone, for example through direct mechanical membrane deformation (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>Another subcategory of MSC, <italic>cell volume</italic>-activated ion channels (VAC), are also considered to respond to mechanical stimuli,<xref rid="bib30" ref-type="bibr"> ³⁰ </xref> but these differ in their micro-mechanical deformation properties (cell swelling increases cell diameter with less dominant effects on length, while axial stretch increases cell length and reduces diameter), time course of response (VAC usually show a lag-time of tens of seconds to minutes between the onset of cell swelling and channel opening, while SAC activate instantaneously), and pathophysiological context (it is assumed that &#x02013; in contrast to ischaemia or hypertrophy &#x02013; the normal cycle of contraction and relaxation is not associated with cell volume changes). For these reasons, VAC are unlikely to be main contributors to acute and/or beat-by-beat responses of the heart to mechanical stimuli, and they will not be considered in detail here (for a review on VAC, see<xref rid="bib30" ref-type="bibr"> ³⁰ </xref>).</p><p>SAC were discovered in 1984 in embryonic chick skeletal myocytes by Guharay and Sachs.<xref rid="bib31" ref-type="bibr"> ³¹ </xref> In subsequent years, SAC have been identified in many other cell types<xref rid="bib32" ref-type="bibr"> ^32,33 </xref> including cardiomyocytes.<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> Cardiac SAC can be either cation non-selective (SAC_NS)<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> or potassium-selective (SAC_K)<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> (<xref ref-type="fig" rid="fig3">Figure 3</xref>). The development of the patch clamp technique was vital for the study of cardiac SAC, and it revealed, in addition to stretch-activated whole-cell currents, evidence of single-channel activity in atrial myocytes,<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> foetal<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> and (for SAC_K at least) adult ventricular myocytes,<xref rid="bib36" ref-type="bibr"> ³⁶ </xref> as well as cardiac non-myocytes.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> That said, formation of membrane patches is associated with significant alterations in local mechanical and structural properties, especially in complex and densely &#x02018;crowded&#x02019; cells such as cardiac myocytes. This leaves the potential for false-positive (e.g. channels that would normally be protected from opening, such as by cytoskeletal interaction) and false-negative observations (channels that are constitutively activated by patch formation may not be identified as mechano-sensitive upon additional patch deformation). This highlights the importance of multi-level investigations, combining a range of electrophysiological recording techniques, from lipid bilayers to sub-cellular and cellular studies in expression systems and native cells, to cultures, tissue slices, native tissue and organs, right through to whole animal or patient research. As pointed out elsewhere, much of this hinges on the availability of improved pharmacological agents, and it requires quantitative structure-based integration, such as by computational modelling.</p><p>First insights into the structure and possible mechanisms of operation of these channels were provided by the cloning and crystallization of two bacterial SAC.<xref rid="bib37" ref-type="bibr"> ^37,38 </xref> However, even after an exhaustive search, no sequence homologues of these particular ion channels were found in mammals. The first cloned mammalian SAC was the TREK channel (a &#x02018;tandem of two-pore K⁺ domains in a weak inwardly rectifying K⁺ channel&#x02019; =&#x000a0;TWIK-related potassium channel).<xref rid="bib39" ref-type="bibr"> ³⁹ </xref> Despite these significant steps, the molecular identities of mammalian <italic>cardiac</italic> SAC have yet to be determined.</p><p>In spite of a lack of firm molecular identification, there are several prominent candidates for mammalian cardiac SAC, and these will be reviewed here. For SAC_NS these include transient receptor potential (TRP) channels, and Piezo1. SAC_K candidates are TREK-1, the large-conductance calcium-activated K⁺ channel (BK_Ca; a member of the &#x02018;Big K⁺&#x02019; channel family), and the ATP-sensitive potassium channel (K_ATP); see <xref ref-type="table" rid="tbl1">Table 1</xref>.</p><p>In the following, we evaluate the available evidence for presence and contributions of these main cardiac SAC candidates, including their sensitivity to pharmacological interventions, highlight some of the present experimental challenges, and conclude with a consideration of anticipated further developments in this exciting and dynamic field of translational heart research. We will not discuss alternative mechano-sensors, detailed signalling pathways, or protein-protein interactions, all of which form deserving topics for separate reviews.</p></sec><sec><title>Sac_ns </title><p>Whole-cell currents with a linear current-voltage relationship attributed to SAC_NS (I_SAC,NS), were first identified in cardiac cells by Craelius et al.,<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> using whole-cell patch clamp recordings from neonatal rat ventricular myocytes. By applying a voltage clamp, Zeng et al.<xref rid="bib40" ref-type="bibr"> ⁴⁰ </xref> later described the properties of this current further, including a lack of inactivation and a pronounced sensitivity to block by gadolinium ions (Gd³⁺). The channel's reversal potential is positive to the resting potential of working cardiomyocytes, so that activation of SAC_NS will depolarise resting cells.<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> In contrast to SAC_K, SAC_NS are distinctly sensitive to a peptide, isolated by Sachs et al. from Chilean tarantula venom: GsMTx-4 (Grammostola spatulata Mechano-Toxin 4<xref rid="bib41" ref-type="bibr"> ⁴¹ </xref>). The use of GsMTx-4 has allowed researchers to extend the evidence on whole-cell I_SAC,NS towards identification of SAC_NS effects at the tissue and whole organ levels. At the same time, no SAC_NS single-channel recordings from freshly-isolated adult ventricular cardiomyocytes have been reported. This has led to the suggestion that SAC_NS may be localised in membrane regions that are difficult to access in patch clamp studies, such as transverse tubules (T-tubules<xref rid="bib42" ref-type="bibr"> ⁴² </xref>), caveolae (which, themselves, form a mechanosensitive structural domain that may be integrated into the surface sarcolemma by excess stretch<xref rid="bib43" ref-type="bibr"> ⁴³ </xref>), or at intercalated discs.<xref rid="bib44" ref-type="bibr"> ⁴⁴ </xref> The main molecular candidates for cardiac SAC_NS, TRP channels<xref rid="bib45" ref-type="bibr"> ⁴⁵ </xref> and the recently discovered Piezo1 protein,<xref rid="bib46" ref-type="bibr"> ⁴⁶ </xref> will be discussed in more detail.</p></sec><sec><title>TRP channels</title><p>TRP proteins form a family of widely expressed cation channels, responsible for a variety of cellular functions. Polymodal regulation is a distinct feature of TRP (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/724608">http://www.ncbi.nlm.nih.gov/gene/724608</ext-link>). Known activators of TRP channels include chemical stimuli, temperature elevation, and mechanical interventions ranging from local patch deformation to membrane stretch and shear strain.<xref rid="bib47" ref-type="bibr"> ⁴⁷ </xref> In particular, the so-called &#x02018;canonical&#x02019; TRP channels TRPC1 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/7220">http://www.ncbi.nlm.nih.gov/gene/7220</ext-link>) and TRPC6 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/7225">http://www.ncbi.nlm.nih.gov/gene/7225</ext-link>) have been implicated as candidates for cardiac SAC_NS.</p><p> <italic><bold>TRPC1:</bold></italic> Analysis of mRNA expression suggested that TRPC1 is present in the human heart.<xref rid="bib48" ref-type="bibr"> ⁴⁸ </xref> Using immuno-histochemical labelling and confocal imaging, TRPC1 protein was found to colocalise with phalloidin stain in rat ventricular myocytes.<xref rid="bib42" ref-type="bibr"> ⁴² </xref> This suggests that TRPC1 may be located in T-tubules and is consistent with the hypothesised spatial distribution of endogenous SAC_NS in adult ventricular cardiomyocytes.</p><p>Mechanosensitivity of TRPC1 was first noted by Maroto et al.<xref rid="bib49" ref-type="bibr"> ⁴⁹ </xref> in Xenopus oocytes. In their experiments, I_SAC,NS was measured after membrane protein fractionation and reconstitution of individual proteins in liposomes. A particularly mechanosensitive fraction was found to contain an 80-kDa protein which was immunoreactive to TRPC1 antibody, indicating the presence of a TRPC1 homologue. Further expression of the human TRPC1 (hTRPC1) isoform in Xenopus oocytes and Chinese hamster ovary (CHO) K1 cells increased I_SAC,NS tenfold, whereas microinjection of antisense hTRPC1 RNA greatly reduced I_SAC,NS in both cell types.</p><p>Since publication, these findings have been challenged by several studies, including one by some of the authors of the original report. They found that transfection of hTRPC1 into COS cells (a fibroblast-related cell line, originally derived from kidney tissue of monkey) had no discernible effect, while transfection of a different putative SAC (the SAC_K TREK-1; see below), induced an increase by three orders of magnitude in mechanosensitive whole-cell currents. This result puts into question the significance of the less pronounced (ten-fold) increase seen in the earlier experiments.<xref rid="bib50" ref-type="bibr"> ⁵⁰ </xref> The authors of the later study found limited ion channel expression at the sarcolemma, which is in agreement with a more recent report showing predominantly intracellular expression of transfected TRPC1 in a skeletal muscle cell line, unless co-expressed with Cav3 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/859">http://www.ncbi.nlm.nih.gov/gene/859</ext-link>).<xref rid="bib51" ref-type="bibr"> ⁵¹ </xref> Thus, even if TRPC1 is successfully transfected, it may require associated molecular machinery for a correct subcellular localization and/or proper function. In addition, TRPC1 may require other TRPC isoforms to form a functional heteromeric channel.<xref rid="bib52" ref-type="bibr"> ⁵² </xref> </p><p>The conflicting results reported above highlight problems that can be associated with the use of heterologous expression systems to study cardiac ion channels. Clearly, the intracellular environment of stable cell lines differs significantly from that of cardiomyocytes, while additional transfection with exogenous ion channels can alter the structure and function of recipient cells.<xref rid="bib50" ref-type="bibr"> ⁵⁰ </xref> Given the dependence of SAC gating properties on micro-mechanical and structural properties of a cell, it is difficult to establish suitable control protocols,<xref rid="bib50" ref-type="bibr"> ⁵⁰ </xref> or to arrive at definitive conclusions from these experiments.</p><p>Interestingly, mice in whom TRPC1 has been knocked out (TRPC1^{&#x02212;&#x000a0;/&#x000a0;&#x02212;&#x000a0;}) exhibit no significant difference in ventricular slow force response (SFR, initially characterised in cat papillary muscle,<xref rid="bib53" ref-type="bibr"> ⁵³ </xref> is a gradual, calcium-related increase in muscle contractility upon exposure to maintained stretch), compared to wild-type controls.<xref rid="bib54" ref-type="bibr"> ⁵⁴ </xref> This suggests that TRPC1 may not be an obligatory and/or exclusive component of the SFR (similar findings were reported for TRPC3 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/7222">http://www.ncbi.nlm.nih.gov/gene/7222</ext-link>)<xref rid="bib55" ref-type="bibr"> ⁵⁵ </xref>). However, as with all knockout experiments, there is always the possibility of compensatory changes in expression of other genes. One way of assessing this would be to use acute knockdown experiments, ideally involving tissue-specific drivers of protein expression. It would also be instructive to explore acute MEF responses that would be expected to precede the SFR in cardiac myocytes or tissue preparations of TRPC1^{&#x02212;&#x000a0;/&#x000a0;&#x02212;&#x000a0;} mice.</p><p> <italic><bold>TRPC6</bold></italic>: Mammalian TRPC6 was initially identified as a mechanosensitive ion channel by Spassova et al.,<xref rid="bib56" ref-type="bibr"> ⁵⁶ </xref> who found that overexpression of TRPC6 in human embryonic kidney cell line 293 (HEK293) cells induced I_SAC,NS. However, a subsequent study by Gottlieb et al.<xref rid="bib50" ref-type="bibr"> ⁵⁰ </xref> found that TRPC6 overexpression in CHO and COS cells had no significant effect. More recently, it has been suggested that TRPC6 is not mechanosensitive, unless co-expressed with the angiotensin II type 1 (AT1) receptor.<xref rid="bib45" ref-type="bibr"> ^45,47 </xref> Data, more directly relevant for cardiac mechanosensitivity, came from Dyachenko et al.,<xref rid="bib58" ref-type="bibr"> ⁵⁸ </xref> who used mouse ventricular myocytes, as opposed to heterologous expression systems. Their whole-cell patch clamp experiments identified a robust I_SAC,NS in response to shear stimuli, which was inhibited by pore-blocking TRPC6 antibodies. TRPC6 knockout blunts the SFR in wild-type murine models, while its genetic down-regulation or pharmacological block returns &#x02018;hyper-responsive&#x02019; murine models of Duchenne muscular dystrophy back to normal SFR levels,<xref rid="bib55" ref-type="bibr"> ⁵⁵ </xref> highlighting the potential clinical relevance of targeted TRPC6 manipulation.</p><p>TRPC6 is among a small number of SAC candidates that is highly expressed in human heart homogenates.<xref rid="bib48" ref-type="bibr"> ⁴⁸ </xref> In murine heart, TRPC6 appears to be localised to T-tubules.<xref rid="bib58" ref-type="bibr"> ⁵⁸ </xref> In agreement with this observation, detubulation inhibits I_SAC,NS in murine cardiomyocytes.<xref rid="bib58" ref-type="bibr"> ⁵⁸ </xref> Interestingly, a recent paper has suggested that the localization of TRPC6 shows marked plasticity in response to sympathetic stimulation via &#x003b1;1A receptors, and that these channels can translocate from T-tubules to the sarcolemma.<xref rid="bib59" ref-type="bibr"> ⁵⁹ </xref> Whether this occurs physiologically is unclear; however, pre-treatment with &#x003b1;1A-agonists might serve as a useful experimental intervention to facilitate single-channel recordings of TRPC6, and potentially other channels localised in T-tubules, in adult ventricular myocytes.</p><p> <italic><bold>Other TRP channels</bold>:</italic> Several other members of the TRP family are mechanosensitive and are expressed in the heart. The TRPC3 protein has been identified in rat ventricular myocytes, also located in T-tubules.<xref rid="bib60" ref-type="bibr"> ⁶⁰ </xref> Mechanical stretch of neonatal cardiomyocytes in a mouse model displays TRPC3 overexpression induced ROS production, similar to TRPC3-activation by 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG is a fairly unspecific activator of TRPC channels, so effects cannot be attributed with confidence to TRPC3.<xref rid="bib60" ref-type="bibr"> ⁶⁰ </xref> The vanilloid TRP channel 2 (TRPV2 (http://www.ncbi.nlm.nih.gov/gene/51393)) has been reported to be mechanosensitive (studied using cell-volume changes and patch pipette suction<xref rid="bib61" ref-type="bibr"> ⁶¹ </xref>) and is expressed in the mouse heart.<xref rid="bib44" ref-type="bibr"> ^44,62 </xref> Using the TRPV2 agonist probenicid in wild-type and TRPV2^{&#x02212;&#x000a0;/&#x000a0;&#x02212;&#x000a0;} constitutive knockout mice, it was shown that this channel appears to contribute to baseline cardiac function, participating in the calcium-handling machinery of heart cells.<xref rid="bib62" ref-type="bibr"> ⁶² </xref> </p><p>TRPV4 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/59341">http://www.ncbi.nlm.nih.gov/gene/59341</ext-link>) mRNA is weakly expressed in cardiac muscle,<xref rid="bib63" ref-type="bibr"> ⁶³ </xref> and TRPV4 protein was located in cultured neonatal rat ventricular myocytes only in the nucleus.<xref rid="bib64" ref-type="bibr"> ⁶⁴ </xref> However, caution is warranted here, regarding mRNA or protein expression in tissue. Firstly, mRNA and protein expression levels do not necessarily correlate<xref rid="bib65" ref-type="bibr"> ⁶⁵ </xref> and secondly, while high expression levels are confirmatory of a significant presence and indicative of functional relevance, low expression in whole tissue homogenates should be interpreted with care. If a protein is present in minority cell populations of the heart (such as Purkinje fibres), it could be of immense functional relevance, even if it was only detected at trace levels. In addition, some SAC, such as TREK, may be expressed at the membrane, but can be strongly inhibited in resting conditions,<xref rid="bib66" ref-type="bibr"> ⁶⁶ </xref> making the assessment of availability of functional channels even more difficult.</p><p>Finally, the melastatin TRP channel 4 (TRPM4) is expressed in cardiomyocytes,<xref rid="bib45" ref-type="bibr"> ⁴⁵ </xref> and has been implicated in stretch-activated responses of vasculature smooth muscle cells.<xref rid="bib67" ref-type="bibr"> ⁶⁷ </xref> Overexpression of TRPM4 may be involved in an inheritable form of progressive familial heart block type I,<xref rid="bib68" ref-type="bibr"> ⁶⁸ </xref> and the identification of a possible stretch-activated component of this disease &#x02013; mediated by TRPM4 &#x02013; would be of pronounced clinical relevance. Thus, in addition to TRPC1 and TRPC6, the ion channels TRPC3, TRPV2, TRPV4 and TRPM4 form translationally-relevant targets for further basic and applied research.</p><sec><title>Piezo1</title><p>The discovery of Piezo1 and Piezo2 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/63895">http://www.ncbi.nlm.nih.gov/gene/63895</ext-link>) by Patapoutian's group in 2010<xref rid="bib46" ref-type="bibr"> ⁴⁶ </xref> represents one of the most important breakthroughs in the field of mechanotransduction in recent years. Piezo1 was initially identified in the neuro-2a neuronal cell line by siRNA knockdown of the expression of membrane proteins with unknown function. Knockdown of the FAM38A (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/415849">http://www.ncbi.nlm.nih.gov/gene/415849</ext-link>) gene inhibited I_SAC,NS and the gene product was named Piezo1. Mechanosensitivity was confirmed by heterologous expression of Piezo1 in HEK cells, which induced a robust I_SAC,NS. Piezo1 is huge, containing approximately 2,500 amino acids, arranged in 24&#x02013;32 transmembrane domains that assemble as a tetramer.<xref rid="bib69" ref-type="bibr"> ⁶⁹ </xref> Intuitively, this massive structure, and the associated large surface area, could be well-adapted for sensing changes in bilayer curvature and/or stretch. In line with this hypothesis, it has been shown that Piezo1 gating is associated with dimensional changes.<xref rid="bib70" ref-type="bibr"> ⁷⁰ </xref> </p><p>Currently, no data has been published directly addressing Piezo1 mechanosensitivity in the heart. However, Piezo1 channel electrophysiological properties are similar to that of endogenous cardiac SAC_NS, including weak voltage dependency, comparable single channel conductance, inactivation, and sensitivity to GsMTx-4.<xref rid="bib71" ref-type="bibr"> ^{71&#x02013;73} </xref> Furthermore, Piezo1 mRNA is expressed in the murine heart<xref rid="bib46" ref-type="bibr"> ^46,74 </xref> albeit at low levels (see comment on whole-tissue expression levels, above). Piezo1 is involved in erythrocyte volume homeostasis. Morpholino-mediated knockdown of Piezo1 results in swelling and lysis of red blood cells and consequent anemia.<xref rid="bib75" ref-type="bibr"> ⁷⁵ </xref> Interestingly, this function is close to that of bacterial mechanosensitive channels of large and small conductance (MscL and MscS).<xref rid="bib76" ref-type="bibr"> ⁷⁶ </xref> </p><p>Undoubtedly this is an exciting and dynamic area of development. Basic science questions concerning structure, protein partners, and regulation of Piezo1 need to be addressed,<xref rid="bib77" ref-type="bibr"> ⁷⁷ </xref> as does the question of whether Piezo1 is present in, and relevant for, the human heart.</p></sec></sec><sec><title>SAC_K </title><p>Whole-cell SAC_K currents (I_SAC,K) were first described by Kim et al.<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> in rat atrial myocytes. In contrast to SAC_NS, SAC_K, are outwardly rectifying, and as such, allow potassium ions to move more easily out of the cell than into it. Compared to SAC_NS, SAC_K tend to have larger single channel conductances. They also inactivate in a time-dependent manner and are generally insensitive to GsMTx-4.<xref rid="bib78" ref-type="bibr"> ⁷⁸ </xref> Being potassium-selective, their reversal potential lies negative to the resting membrane potential of cardiac cells, so activation of SAC_K will generally cause membrane repolarisation or hyperpolarisation.<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> To date, single-channel recordings of I_SAC,K in adult mammalian cardiac myocytes have been obtained from atrial<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> and ventricular myocytes,<xref rid="bib36" ref-type="bibr"> ^36,79,80 </xref> suggesting that their subcellular compartmentalization differs from SAC_NS. Primary molecular candidates for cardiac SAC_K are TREK-1, BK_Ca and K_ATP.</p></sec><sec><title>TREK-1</title><p>TREK-1 is a member of the two-pore domain potassium channel family, which is associated with a &#x02018;leak&#x02019; potassium ion conductance in cardiomyocytes.<xref rid="bib81" ref-type="bibr"> ⁸¹ </xref> TREK-1, however, displays more complex permeation and gating properties than a simple &#x02018;leak&#x02019; channel, and is regulated by a number of factors including pH, temperature, second messenger systems, and membrane deformation/stretch.<xref rid="bib82" ref-type="bibr"> ⁸² </xref> Mechanosensitivity was attributed to TREK-1 by Patel et al.<xref rid="bib39" ref-type="bibr"> ³⁹ </xref> based on single-channel patch clamp recordings from transfected COS cells. Subsequently, Terrenoire et al.<xref rid="bib83" ref-type="bibr"> ⁸³ </xref> demonstrated that I_SAC,K (endogenous to rat atrial myocytes) displays a number of properties that bear striking similarities to recombinant TREK-1 channels. This includes their single-channel conductance, lack of voltage dependency, &#x02018;burst mode&#x02019; activity, and sensitivity to pharmacological interventions, and, in particular, a unique sensitivity to volatile anaesthetics not shared by other cardiac potassium ion channels.<xref rid="bib81" ref-type="bibr"> ⁸¹ </xref> </p><p>A number of studies have identified TREK-1 mRNA expression in rat atria, as well as in left, right, and septal ventricular myocytes.<xref rid="bib83" ref-type="bibr"> ^{83&#x02013;86} </xref> The protein appears to be arranged in longitudinal stripes at the surface of cardiomyocytes: a pattern that might support directional stretch sensing.<xref rid="bib80" ref-type="bibr"> ⁸⁰ </xref> At the tissue level, TREK-1 expression is distinctly heterogeneous, with a gradient of mRNA expression that increases, transmurally, from epicardial to endocardial cells.<xref rid="bib86" ref-type="bibr"> ⁸⁶ </xref> This heterogeneity appears to correlate with transmural changes in MEF sensitivity, where stretch causes the most pronounced action potential shortening in the sub-endocardium.<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> To our knowledge, mRNA analysis thus far has failed to identify TREK-1 expression in the human heart.<xref rid="bib88" ref-type="bibr"> ^88,89 </xref> It has been suggested that the TWIK-related arachidonic acid-activated potassium channel (TRAAK (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/50801">http://www.ncbi.nlm.nih.gov/gene/50801</ext-link>)) or TWIK-related acid-sensitive potassium channel (TASK-1 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/gene/3777">http://www.ncbi.nlm.nih.gov/gene/3777</ext-link>)) of the K2P family, which appear to be expressed in the human heart,<xref rid="bib90" ref-type="bibr"> ⁹⁰ </xref> may act as TREK-1 homologues. Indeed, when TRAAK is expressed in heterologous model systems, it forms a channel with very similar properties to TREK-1.<xref rid="bib91" ref-type="bibr"> ⁹¹ </xref> Characterisation of both the presence and functional relevance of these ion channels in human requires further elucidation.</p><sec><title>BK_Ca </title><p>BK_Ca channels have large conductances, and they respond to voltage changes and alterations in intracellular calcium ion concentration in a manner that allows them to contribute to repolarisation.<xref rid="bib92" ref-type="bibr"> ⁹² </xref> Functionally, BK_Ca channels have been suggested to control heart rate and to offer cardioprotection during ischaemia.<xref rid="bib93" ref-type="bibr"> ⁹³ </xref> Kawakubo et al.<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> identified mechanosensitivity of BK_Ca channels in membrane patches excised from cultured embryonic chick ventricular myocytes. Attempts to measure single-channel activity in post-hatch chick cardiomyocytes have been unsuccessful, although Iribe et al.<xref rid="bib95" ref-type="bibr"> ⁹⁵ </xref> characterised a whole-cell I_SAC,K which was sensitive to iberiotoxin (a BK_Ca inhibitor). Interestingly, this I_SAC,K was also sensitive to extracellular sodium. The authors suggest that BK_Ca activation could occur secondary to mechanical modulation of sodium ion influx (e.g. via SAC_NS), and consequentially shift sodium-calcium exchanger activity towards preservation of intracellular calcium. If this is the case, BK_Ca might not be directly stretch sensitive.</p><p>Whether or not BK_Ca channels are responsible for I_SAC,K in embryonic chick cardiomyocytes, there is little evidence to suggest that BK_Ca channels form cardiac SAC_K in other species. In the human heart, BK_Ca channels are sparsely expressed,<xref rid="bib92" ref-type="bibr"> ⁹² </xref> and they may be confined to cardiac fibroblasts (where BK_Ca was detected using Western blot<xref rid="bib96" ref-type="bibr"> ⁹⁶ </xref>). However, functional fibroblast-myocyte electrical coupling can occur (at least in some areas of the heart<xref rid="bib97" ref-type="bibr"> ^97,98 </xref>) so it is possible that stretch-activation of BK_Ca channels in cardiac fibroblasts could still affect myocyte function via a heterocellular coupling pathway.</p></sec></sec><sec><title>K_ATP </title><p>Although classically considered to be ligand-gated, Van Wagoner et al.<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> obtained single-channel inside-out patch clamp recordings from neonatal rat atrial myocytes which revealed that patch pipette negative pressure increased K_ATP channel ATP-sensitivity.<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> Synergistically, ATP-reduction potentiated stretch sensitivity.<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> Ischaemia, simulated in adult guinea pig ventricular myocytes by application of a metabolic uncoupler, also uncovered K_ATP mechanosensitivity that was absent in control conditions,<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> an observation that was more recently confirmed in rat cardiomyocytes.<xref rid="bib100" ref-type="bibr"> ¹⁰⁰ </xref> The synergism for K_ATP channel activation by metabolic and mechanical stress might explain differences in quantitative aspects of ATP reduction needed to activate K_ATP in isolated cells, compared to the organ level (where K_ATP open at less depleted ATP levels). A reason for this difference could be the fact that isolated cells are not normally subjected to mechanical co-activation, while at the organ level ischaemia is usually associated with decreased shortening, or even stretch, of the tissue affected by reduced availability of ATP.</p><p>In keeping with this notion, &#x02018;stretch-preconditioning&#x02019; has been reported to reduce ischaemia- reperfusion injury, an effect that disappears when K_ATP channels are blocked.<xref rid="bib68" ref-type="bibr"> ⁶⁸ </xref> Moreover, cardiac fibroblasts progressively express functional K_ATP channels in scar and border zone tissue following infarction, suggesting that we must consider the effect of cells other than cardiomyocytes in pre-/post- conditioning of the heart, and the role of SAC in these processes.<xref rid="bib101" ref-type="bibr"> ^{101&#x02013;103} </xref> </p><p>Although there is little evidence to suggest that K_ATP are responsible for mechanosensitivity of the heart in normal beat-by-beat physiology (in healthy cells and tissue, diastolic mechanical stimulation depolarizes cardiomyocytes), the potential role of these ion channels in ischaemic or other disease conditions warrants further research.</p></sec><sec><title>SAC modulators</title><p>Several pharmacological compounds have been identified to modulate SAC activity (<xref ref-type="fig" rid="fig3">Figure 3</xref>),<xref rid="bib104" ref-type="bibr"> ^104,105 </xref> and their potential role as pharmacological tools for heart rhythm management has been previously reviewed by White.<xref rid="bib106" ref-type="bibr"> ¹⁰⁶ </xref> </p><p>Most of the known SAC-modulators are non-specific inhibitors, such as gadolinium ions, amiloride and cationic antibiotics (streptomycin, penicillin, kanamycin). Among the very few specific SAC inhibitors<xref rid="bib107" ref-type="bibr"> ¹⁰⁷ </xref> reported so far is the peptide GsMTx-4. It inhibits TRPC5 when activated by hypo-osmotic and receptor stimulation,<xref rid="bib111" ref-type="bibr"> ^108,109 </xref> as well as TRPC6,<xref rid="bib56" ref-type="bibr"> ⁵⁶ </xref> and Piezo1 channels when applied to the external face of the membrane.<xref rid="bib72" ref-type="bibr"> ^72,73 </xref> GsMTx-4 is active both in its D and L enantiomers, showing the mechanism of action is not stereospecfic or chiral.<xref rid="bib110" ref-type="bibr"> ¹¹⁰ </xref> Instead, the mode of action of GsMTx-4 is thought to involve insertion into the outer membrane leaflet in the proximity of the channel, relieving lipid stress and favouring the closed state of SAC.<xref rid="bib110" ref-type="bibr"> ¹¹⁰ </xref> Counterintuitively, GsMTx-4 sensitizes the bacterial channels, MscS and MscL, to tension,<xref rid="bib111" ref-type="bibr"> ¹¹¹ </xref> while it has no effect on TREK-1 channels,<xref rid="bib72" ref-type="bibr"> ⁷² </xref> so that overall the mode of action of GsMTx-4 still requires further elucidation.</p><p>TREK-1 is poorly responsive to classic potassium channel blockers,<xref rid="bib112" ref-type="bibr"> ¹¹² </xref> but its functions are modified by a variety of pharmacologic agents such as volatile anaesthetics,<xref rid="bib112" ref-type="bibr"> ¹¹² </xref> riluzole,<xref rid="bib113" ref-type="bibr"> ¹¹³ </xref> fluoxetine<xref rid="bib114" ref-type="bibr"> ¹¹⁴ </xref> and spadin.<xref rid="bib115" ref-type="bibr"> ^115,116 </xref> Recently, new modulators of TREK-1 were identified by Bagriantsev et al.<xref rid="bib117" ref-type="bibr"> ¹¹⁷ </xref> They characterized inhibitors and, importantly, activators (which are very rare for SAC channels). Known openers for SAC are amphipathic substances which insert selectively into one membrane leaflet only, locally inducing either concave or convex curvature, which may induce SAC-activating tension.<xref rid="bib118" ref-type="bibr"> ¹¹⁸ </xref> </p><p>Other useful substances include probenicid, which is a TRP agonist that is fairly specific to TRPV2,<xref rid="bib119" ref-type="bibr"> ¹¹⁹ </xref> and 9-phenanthrol, which blocks TRPM4.<xref rid="bib120" ref-type="bibr"> ¹²⁰ </xref> </p><p>It is important to note, though, that not all SAC blockers that work at the level of isolated or cultured cells are equally efficient in native tissue. Streptomycin, for example, may not be able to easily access SAC in whole cardiac tissue,<xref rid="bib121" ref-type="bibr"> ¹²¹ </xref> even though it is an efficient SAC_NS blocker in single cardiomyocytes (an important consideration for cell-culture based work, which often employs media containing streptomycin by default). This will be one of the reasons for which antibiotics, such as streptomycin, can be prescribed to patients without instantaneous side effects on stretch-sensing. Another compound, Gd³⁺, is used clinically in a chelated formulation, which explains the lack of pronounced immediate SAC-effects in radio-contrast studies. As an aside, Gd³⁺ precipitates in physiologically buffered solutions.<xref rid="bib122" ref-type="bibr"> ¹²² </xref> Clearly caution is called for when assessing (potentially false-) negative results on Gd³⁺ effects in physiological solutions, or on streptomycin effects <italic>in vivo</italic>.</p></sec><sec><title>Discussion</title><p>The heart is a superbly mechanosensitive organ. SAC are thought to provide one of the mechanisms underlying cardiac MEF,<xref rid="bib20" ref-type="bibr"> ^20,123,124 </xref> the process by which changes in the mechanical environment of the heart lead to altered cardiac electrical activity. Identification of molecular substrates for cardiac SAC will not only provide novel insight into processes that underlie MEF, but also support the long-term aim of developing SAC-specific drugs for the treatment of mechanically induced cardiac pathologies.<xref rid="bib106" ref-type="bibr"> ¹⁰⁶ </xref> </p><p>In terms of physiological beat-by-beat effects, activation of SAC has been shown to underlie the stretch-induced increase in spontaneous sino-atrial node (SAN) cell pacemaking rate.<xref rid="bib16" ref-type="bibr"> ¹⁶ </xref> Block of SAC using GsMTx-4 terminates this positive chronotropic response in SAN tissue<xref rid="bib121" ref-type="bibr"> ¹²¹ </xref> which, in its guise of a respiratory sinus arrhythmia, persists at whole body level &#x02013; even in heart transplant recipients after additional pharmacological denervation.<xref rid="bib125" ref-type="bibr"> ¹²⁵ </xref> </p><p>At the same time, sustained pressure and/or volume overload favour arrhythmogenesis.<xref rid="bib25" ref-type="bibr"> ^{25,124,126,127} </xref> Application of SAC-blockers such as GsMTx-4 has been shown to reversibly reduce the preload dependent increase in both incidence and duration of burst-pacing induced atrial fibrillation in isolated heart experiments.<xref rid="bib28" ref-type="bibr"> ²⁸ </xref> In patients, it can be difficult to distinguish stretch-induced changes in electrophysiology from other chronically occurring aspects of structural and functional remodelling. However, an impressive illustration of acute effects of ventricular loading has been provided by Waxman et al.,<xref rid="bib128" ref-type="bibr"> ¹²⁸ </xref> who showed that performing the <italic>Valsalva manoeuvre</italic> may terminate ventricular tachycardia by temporary reduction of ventricular filling. The <italic>Valsalva manoeuvre</italic>, an attempt to forcefully exhale against the closed glottis, increases intrathoracic pressure, favouring a net reduction of intravascular volume in the chest (i.e. impeding venous return and favouring arterial drainage to other parts of the body). In this study, the reduction in cardiac dimensions was confirmed radiographically. Cessation of ventricular tachycardia coincided with removal of ventricular strain, while arrhythmia resumption occurred upon refilling after the end of the manoeuvre. Since this type of response can be seen not only in neurologically intact, but also in pharmacologically<xref rid="bib128" ref-type="bibr"> ¹²⁸ </xref> or surgically<xref rid="bib7" ref-type="bibr"> ⁷ </xref> denervated patients (transplant recipients), it is not attributable to a nervous reflex. This highlights how removal of strain may unmask the presence of stretch-induced arrhythmias, even in a chronic setting.</p><p>Various SAC have been implicated in the heart's (patho-)physiological responses to mechanical stimuli, but in the absence of firm identification of molecular substrates for cardiac SAC, successful mechanistic exploration of cardiac mechanosensitivity is a challenging task.</p><p>Conceptually, it is pragmatic to subdivide SAC into two categories, SAC_NS and SAC_K. For both, there are several candidate proteins. SAC_NS were initially thought to be formed by TRP proteins and, most convincingly, TRPC6 antibodies inhibit whole-cell I_SAC,NS in mouse ventricular myocytes.<xref rid="bib58" ref-type="bibr"> ⁵⁸ </xref> However, subsequent heterologous expression studies yielded conflicting results.<xref rid="bib50" ref-type="bibr"> ^50,56 </xref> More recently, attention has turned towards the newly discovered Piezo1 channels.<xref rid="bib46" ref-type="bibr"> ⁴⁶ </xref> Although there is no published data yet on specific electrophysiological effects of Piezo1 in cardiomyocytes, comparative kinetic analysis suggests that these proteins may function as cardiac SAC_NS. In as far as cardiac SAC_K are concerned, recombinant TREK-1 has remarkably similar properties to endogenous SAC_K,<xref rid="bib78" ref-type="bibr"> ⁷⁸ </xref> but the protein has yet to be identified in human heart.<xref rid="bib88" ref-type="bibr"> ⁸⁸ </xref> BK_Ca channels have also received considerable interest, but their stretch-sensitivity, if present,<xref rid="bib95" ref-type="bibr"> ⁹⁵ </xref> may be limited to immature<xref rid="bib129" ref-type="bibr"> ¹²⁹ </xref> and/or non-mammalian cardiomyocytes,<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> or to cardiac connective tissue.<xref rid="bib96" ref-type="bibr"> ⁹⁶ </xref> Finally, K_ATP channels display metabolic and mechanical co-activation,<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> which may help explain some of the differences between experimental and clinical findings on the extent of ATP-reduction needed to activate them. In addition, this insight could shed light on hitherto ill-explored links between ischaemic and mechanical preconditioning.</p><p>As will be apparent from the above, the currently available information on the molecular substrates of cardiac SAC poses more questions than it answers. A number of reasons contribute to this. It is notoriously difficult to control and/or quantify the extent and quality of local mechanical stimuli that an individual ion channel is exposed to.<xref rid="bib130" ref-type="bibr"> ¹³⁰ </xref> Tools to apply strain at whole-cell, tissue, and organ levels exist (including the application of shear stress, axial stretch, or cell volume changes), but there is no commonly implemented &#x02018;gold standard&#x02019; for the stimulation of SAC.<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> Furthermore, these techniques have been used with a wide variety of cellular models from different species and developmental stages, making cross comparison of results challenging. In addition, it is difficult to interrelate macroscopic interventions and observations at cell and tissue levels with molecular substrates: in part because there is no &#x02018;zero-strain&#x02019; reference even in patch clamp studies. Attempts to explore causal links from low-level mechanism to integrated response, and back, include changes in gene expression,<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> pharmacological interventions,<xref rid="bib131" ref-type="bibr"> ¹³¹ </xref> and computational modelling.<xref rid="bib132" ref-type="bibr"> ^{132&#x02013;135} </xref> </p><p>Further challenges arise from the possibility that ventricular SAC may be localised in T-tubules, caveolae, or intercalated discs. This is thought to explain why patch clamping of single SAC is so rare in freshly isolated ventricular cardiomyocytes from adult mammals.<xref rid="bib130" ref-type="bibr"> ¹³⁰ </xref> One possible way around this problem may be to use pre-exposure to &#x003b1;1A receptor stimulation, to aid SAC translocation from T-tubules to the sarcolemma.<xref rid="bib59" ref-type="bibr"> ⁵⁹ </xref> Another would be pre-stretching of the cardiac tissue prior to cell isolation, as this can cause surface membrane incorporation of caveolae.<xref rid="bib43" ref-type="bibr"> ⁴³ </xref> Thirdly, one could isolate the T-tubules using sequential centrifugation of homogenised cardiomyocytes followed by purification of T-tubule membranes by vesicle immuno-isolation and reconstitution into a continuous membrane.<xref rid="bib136" ref-type="bibr"> ¹³⁶ </xref> It might then be possible to directly patch clamp SAC on the isolated T-tubule membrane. Less invasively, scanning ion conductance microscopy, which generates a three-dimensional topographical map of the cell surface prior to patch clamping, has been suggested as a means to directly target the T-tubule ostium where SAC are more likely to be present.<xref rid="bib137" ref-type="bibr"> ¹³⁷ </xref> On the other hand, there is evidence to suggest that SAC may activate indirectly via second messenger signalling cascades.<xref rid="bib138" ref-type="bibr"> ¹³⁸ </xref> Therefore patch clamping single ion channels (where SAC activation probably occurs as a direct result of bilayer deformation<xref rid="bib130" ref-type="bibr"> ¹³⁰ </xref>) may provide only a partial picture of patho-physiological function.</p><p>In addition to SAC in the outer cell membrane, there may be non-sarcolemmal SAC in the sarcoplasmic reticulum<xref rid="bib12" ref-type="bibr"> ¹² </xref> or mitochondria.<xref rid="bib139" ref-type="bibr"> ^139,140 </xref> Cardiac non-myocytes are also mechanosensitive and exhibit electrophysiological properties modulated by mechanical stimuli.<xref rid="bib18" ref-type="bibr"> ^{18,141&#x02013;143} </xref> Channels, such as Nav1.5 and TRPM7, that were initially identified as stretch-modulated in non-cardiac cell types,<xref rid="bib144" ref-type="bibr"> ^144,145 </xref> have now been found in cardiac fibroblasts.<xref rid="bib146" ref-type="bibr"> ^146,147 </xref> </p><p>Finally, there is a growing body of evidence to suggest that many cardiac ion channels, even those that are not classically considered as SAC (e.g. voltage- or ligand-gated channels), are sensitive to mechanical modulation of their gating behaviour.<xref rid="bib148" ref-type="bibr"> ¹⁴⁸ </xref> Future research should therefore focus on characterising the mechanical stimuli experienced by cardiomyocytes <italic>in vivo</italic>, so that they can be more closely replicated <italic>in vitro</italic>. This can be aided greatly by high-resolution imaging of the beating heart,<xref rid="bib149" ref-type="bibr"> ¹⁴⁹ </xref> followed by whole heart histological reconstruction<xref rid="bib151" ref-type="bibr"> ^150,151 </xref> and subsequent computational re-integration of tissue deformation<xref rid="bib152" ref-type="bibr"> ^152,153 </xref> with a granularity that allows identification of local stress-strain dynamics<xref rid="bib154" ref-type="bibr"> ¹⁵⁴ </xref> and prediction of microstructural effects on electrophysiology.<xref rid="bib155" ref-type="bibr"> ¹⁵⁵ </xref> Direct measurement, and validation of modelling predictions, currently suffers from a number of technical limitations, in particular the inability to measure locally acting forces <italic>in situ</italic>. The recent development of F&#x000f6;rster Resonance Energy Transfer (FRET)-based force sensors that can be genetically inserted into intracellular proteins,<xref rid="bib156" ref-type="bibr"> ¹⁵⁶ </xref> may open up a treasure chest of novel insight if they can be applied to heart research. These force sensors are based on the energy transfer between two compatible fluorophores. The efficacy of the energy transfer is inversely proportional to the distance between the donor and the acceptor, multiplied by 10⁶, making the FRET signal very sensitive to small distance changes. Meng and Sachs<xref rid="bib157" ref-type="bibr"> ¹⁵⁷ </xref> have calibrated their probe using DNA to be able to quantify forces from fluorescent signal changes. These sensors constitute a very powerful tool for the assessment of the mechanical state in components of single cells or tissues. Until now little is known about forces within the cell/cytoskeleton, both when cells are at rest, or while mechanically stimulated. In addition, intracellular force reporters would be very useful to improve our understanding of the interplay of SAC with other mechanosensors, like integrins and the cytoskeleton.</p><p>Armed with a more thorough understanding of physiological mechanical stimuli, and novel techniques, we expect to improve our understanding of the molecular substrate of cardiac SAC, and to better predict their pathophysiological roles for the regulation of heart rate and rhythm in the mechanosensitive heart (<xref ref-type="fig" rid="fig4">Figure 4</xref>).</p></sec><sec><title>Conclusion</title><p>The heart is an integrated electro-mechanical system (<xref ref-type="fig" rid="fig1">Figure 1</xref>). SAC (<xref ref-type="fig" rid="fig2">Figure 2</xref>) are key contributors to cardiac mechanosensitivity. SAC_K generally cause re- (or even hyper-) polarisation, while SAC_NS depolarise resting tissue and have differential effects on activated cells (speeding up early, delaying late, repolarisation; <xref ref-type="fig" rid="fig3">Figure 3</xref>). Systemic effects of SAC activation therefore depend on timing (relative to the cardiac cycle), intensity (e.g. relative to excitation threshold), and location (relative to the different components of cardiac tissue; <xref ref-type="fig" rid="fig4">Figure 4</xref>). Thus, diastolic stretch may accelerate heart rate, or even trigger extra beats, depending on whether pacemaker cells or working myocardium drive the response. This is believed to underlie mechanical pacing by pre-cordial percussion, and the occasionally observed termination of re-entrant arrhythmias upon application of pre-cordial thump.<xref rid="bib160" ref-type="bibr"> ^{160&#x02013;162} </xref> Systolic stretch will alter repolarisation dynamics which, in particular in the context of spatially heterogeneous mechanical stimuli and/or regionally varying expression of SAC, may act as an arrhythmia-sustaining mechanism for mechanically-induced rhythm disturbances.</p><p>We are on the brink of obtaining an improved understanding of the molecular substrates of cardiac SAC, and of the way in which macroscopic mechanical interventions translate into stimuli at subcellular levels. This is hoped to lead to improved insight into the physiological relevance of SAC, their involvement in acute and chronic diseases, and to guide the development of novel means to targeting cardiac SAC for therapeutic benefit.</p></sec><sec><title>Funding</title><p>This work has been supported by the UK Biotechnology and Biological Sciences Research Council, the European Research Council, and the Magdi Yacoub Institute at Harefield. RP is holder of an Imperial College Junior Research Fellowship; PK is a Senior Fellow of the British Heart Foundation.</p></sec><sec><title>Competing interests</title><p>None.</p></sec><sec><title>List of Abbreviations</title><p> <list list-type="simple"><list-item><p>BK_Ca: Large-conductance calcium-activated potassium channel</p></list-item><list-item><p>CHO: Chinese hamster ovary cell line</p></list-item><list-item><p>GsMTx-4: Grammostola spatulata Mechano-Toxin 4</p></list-item><list-item><p>HEK: Human embryonic kidney cell line</p></list-item><list-item><p>I_SAC,K: Potassium-selective stretch-activated current</p></list-item><list-item><p>I_SAC,NS: Cation non-selective stretch-activated current</p></list-item><list-item><p>K_ATP: ATP-sensitive potassium channel</p></list-item><list-item><p>MEF: Mechano-electric feedback</p></list-item><list-item><p>MSC: Mechanosensitive ion channels</p></list-item><list-item><p>OAG: 1-oleoyl-2-acetyl-sn-glycerol</p></list-item><list-item><p>SAC: Stretch-activated channels</p></list-item><list-item><p>SAC_K: Potassium-selective SAC</p></list-item><list-item><p>SAC_NS: Cation non-selective SAC</p></list-item><list-item><p>SFR: Slow force response</p></list-item><list-item><p>TASK: TWIK-related acid-sensitive potassium channel</p></list-item><list-item><p>TRAAK: TWIK-related arachidonic acid-activated potassium channel</p></list-item><list-item><p>TREK: TWIK-related potassium channel</p></list-item><list-item><p>TRP: Transient receptor potential</p></list-item><list-item><p>TRPC: Canonical TRP channel</p></list-item><list-item><p>TRPM: Melastatin TRP channel</p></list-item><list-item><p>TRPV: Vanilloid TRP channel</p></list-item><list-item><p>TWIK: Tandem of two-pore K⁺ domains in a weak inwardly rectifying K⁺ channel</p></list-item><list-item><p>VAC: Volume-activated ion channels</p></list-item></list> </p></sec>

Macitentan in pulmonary arterial hypertension: The SERAPHIN trial

<p>Major limitations of pulmonary arterial hypertension (PAH) drug trials include the small number of enrolled patients, short term follow up (12-16 weeks), and lack of morbidity and mortality primary endpoints. The recently published SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary arterial Hypertension to Improve cliNical outcome) trial represents an important landmark in the history of clinical trials in PAH being the largest and longest clinical study conducted thus far in PAH patients with morbidity and mortality events as primary endpoint. SERAPHIN trial investigated whether long-term treatment with the new endothelin receptor antagonist macitentan would reduce the risk of mortality and morbidity in PAH patients.</p>

<contrib contrib-type="author"><name><surname>Said</surname><given-names>Karim</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Background</title><p>Current clinical research in pulmonary arterial hypertension (PAH) focuses on the development of more potent and less toxic drugs that target pathophysiologic pathways known to be important in PAH with special emphasis on endothelin, nitric oxide and prostacyclin pathways.</p><p>Endothelin is one of the most potent vasoconstrictor ever identified with additional proliferative and profibrotic activities. Endothelin exerts its effects by binding to 2 distinct receptor isoforms in the pulmonary vascular smooth muscle cells, endothelin-A and -B receptors.</p><p>Until recently, only two endothelin receptor antagonists (ERAs) have been approved for the treatment of PAH: bosentan (an oral active dual endothelin-A and -B receptor antagonist) and ambrisentan (a selective for the endothelin-A receptor blocker). A third agent, sitaxsentan, was withdrawn from the market in December 2010 after cases of potentially drug-induced fatal hepatotoxicity had been reported</p><p>ERAs are associated with important adverse events including elevation of hepatic transaminases and peripheral edema. Approximately 3% of patients will need to discontinue bosentan due to these adverse effects on hepatic function.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Another limitation of available ERAs is drug-drug interaction. Of interest are the interactions of bosentan with sildenafil, a frequently used combination therapy, where sildenafil plasma levels are reduced by about 50% while bosentan concentrations rise by approximately 50%.<xref rid="bib2" ref-type="bibr"> ^2&#x02013;3 </xref> </p><p>Recently, the US Food and Drug Administration has approved a new ERA macitentan to treat PAH in adults. Support for approval of macitentan comes from the recently published SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary arterial Hypertension to Improve cliNical outcome) trial.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> </p></sec><sec><title>Macitentan</title><p>Macitentan is a dual ERA that was developed by modifying the structure of bosentan to increase efficacy and safety. Macitentan is characterized by slow receptor dissociation kinetics and enhanced tissue penetration.<xref rid="bib5" ref-type="bibr"> ^5,6 </xref> The receptor occupancy half-life of mecitentan is 15-times greater than bosentan<xref rid="bib6" ref-type="bibr"> ⁶ </xref> allowing for a once-a-day dosing regimen, as ambrisentan, whereas bosentan is dosed twice daily. In contrast to other ERAs, macitentan has a low propensity for drug&#x02013;drug interactions.<xref rid="bib7" ref-type="bibr"> ^7&#x02013;8 </xref> </p></sec><sec><title>Seraphin Trial</title><p>The SERAPHIN study is double-blind, randomized, placebo-controlled study that was designed to evaluate the efficacy and safety of long term treatment with macitentan. The study involved 742 patients with PAH in 151 centers in 39 countries all over the world. Patients were randomized 1:1:1 to placebo (<italic>n</italic>&#x000a0;=&#x000a0;250), macitentan 3&#x000a0;mg (<italic>n</italic>&#x000a0;=&#x000a0;250) or macitentan 10&#x000a0;mg (<italic>n</italic>&#x000a0;=&#x000a0;242) once daily. The mean duration of study treatment was: 85.3 weeks, 99.5 weeks, and 103.9 weeks for the placebo, the 3-mg dose, and the 10-mg dose, respectively.</p><p>The study recruited patients with PAH (confirmed by right-heart catheterization) of almost any etiology with WHO functional class II&#x02013;IV. Patients were allowed to receive PAH background therapy throughout the study; hence 64% of all patients were receiving concomitant treatment with oral phosphodiesterase type 5 inhibitors (61.4%) or oral or inhaled prostanoids (5.4%).</p><p>The primary end point was the time from the initiation of treatment to the first occurrence of a composite end point of death, atrial septostomy, lung transplantation, initiation of treatment with IV or SC prostanoids, or worsening of PAH. Worsening of PAH was defined by the occurrence of all three of the following: a decrease in the 6-minute walk distance (6MWD) of at least 15%; worsening of symptoms; and the need for additional treatment for PAH. Secondary efficacy endpoints were: change from baseline to month 6 in 6MWD, change from baseline to month 6 in WHO functional class and time to either death due to PAH or hospitalization due to PAH.</p><p>The results showed that over the study period macitentan 10&#x000a0;mg reduced the risk of primary end point by 45% (<italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.0001) compared with those who received placebo. This corresponds to an absolute risk reduction of 16% and a number-needed-to-treat of 6 patients. For macitentan 3&#x000a0;mg, risk of primary endpoint was reduced by 30% (<italic>p</italic>&#x000a0;=&#x000a0;0.0108) relative to placebo. Risk reduction was driven primarily by reductions in PAH worsening. Worth mentioning, the benefit in the primary end point was the same with PAH-drug-therapy-naive patients as with patients treated with combination therapy.</p><p>Compared to placebo group, the composite risk of PAH-related death or hospitalization was significantly reduced by 34% for the 3&#x000a0;mg macitentan dose and 50% for the 10&#x000a0;mg dose. When death was considered alone, there was a trend toward reduction in the rate of death due to PAH (<italic>p</italic>&#x000a0;=&#x000a0;0.07) with the 10-mg dose of macitentan as compared with placebo.</p><p>Relative to the placebo group, the 6MWD at 6 month had increased by 16.8&#x000a0;m (<italic>p</italic>&#x000a0;=&#x000a0;0.01) in the group that received 3&#x000a0;mg macitentan and by 22&#x000a0;m (<italic>p</italic>&#x000a0;=&#x000a0;0.008) in the group that received 10&#x000a0;mg macitentan. The WHO functional class improved from baseline to month 6 in 13% of the patients in the placebo group, as compared with 20% of those in the group that received 3&#x000a0;mg of macitentan (<italic>p</italic>&#x000a0;=&#x000a0;0.04) and 22% of those in the group that received 10&#x000a0;mg of macitentan (<italic>p</italic>&#x000a0;=&#x000a0;0.006)</p><p>Macitentan was generally well tolerated with similar rates of patients discontinuing treatment due to adverse events across all groups. Rates of elevated hepatic transaminases or peripheral edema were similar across the three study groups. In particular, 4.5% of patients in the placebo group experienced elevations of hepatic transaminases aminotransferases (&#x0003e;3 times the upper limit of normal) compared with 3.6% of patients in the 3&#x000a0;mg macitentan group and 3.4% in the 10&#x000a0;mg macitentan group. Importantly, a hemoglobin level &#x0003c;&#x000a0;8&#x000a0;gm/dl was encountered more frequently among patients receiving 10&#x000a0;mg or 3&#x000a0;mg macitentan (4.3% and 1.7% respectively) compared to placebo group (0.4%).</p></sec><sec><title>What have we learned?</title><p>SERAPHIN trial may represent an important landmark in the history of clinical trials in PAH for several reasons. First, it is the largest randomized, controlled study in PAH patients; second, it is the first randomized PAH trial to include morbidity and mortality events as primary endpoint; third, it is the first randomized PAH study with a predefined long-term treatment follow up (median duration of more than 2 years).</p><p>In pivotal studies of PAH, clinical endpoints had been secondary or exploratory endpoints without adjudication and with very low event rates. The traditional primary endpoint in these studies has been the 6MWD and, accordingly, nearly all available treatments for PAH have been approved based on change in 6MWD. However the prognostic relevance of 6MWD to long-term outcomes is questionable. In a recent meta-analysis of 3,112 patients from 22 clinical trials, changes in 6MWD were not predictable of the favorable effects of pharmacological treatments on clinical events including all-cause death, hospitalization for PAH, transplant, initiation of rescue therapy, and composite outcome.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> In addition, improvement in 6MWD may not be noticed in patients who are already on effective background therapy or in patients with less severe symptomatic disease who have high baseline walk distances but, nevertheless, may have substantial pathology (ceiling effect).<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref> Accordingly, current guidelines suggest that the primary end point in phase 3 trials of new treatments for PAH should be morbidity and mortality.<xref rid="bib11" ref-type="bibr"> ^11,12,13 </xref> In accordance with this, SERAPHIN used a robust definition of morbidity and mortality as a primary end-point to capture clinically relevant events which reflect the true progression of PAH. The success of SERAPHIN study demonstrates that such trials are feasible in the field of PAH.</p><p>One of the important limitations of phase 2 and 3 PAH trials, as is the case with orphan diseases in general, is the small sample size. The large number of patients (<italic>n</italic>&#x000a0;=&#x000a0;742) enrolled in SERAPHIN trial was possible only with the contribution of 151 centers in 39 countries all over the world. This highlights the importance of multicentre international design for future PAH studies.</p><p>Besides recruiting large number of patients, PAH trials should be long enough in duration to enable enough events to occur to allow adequate statistical powering of the study. However, currently available PAH-targeted therapies have been approved for the treatment of PAH on the basis of short-term trials (12 to 16 weeks). Importantly, patients in the SERAPHIN trial were followed with an average duration of 2 years; this is important to assess the effect of therapy on a chronic progressive disease such that of PAH.</p><p>In the SERAPHIN trial, about two thirds of patients were on background therapy (mostly phosphodiesterase type 5 inhibitor). This high rate of combination therapy is important for several reasons:</p><p>(1) With the progressive nature of PAH disease, many patients will need the introduction of additional treatments. Accordingly, permitting combination therapy in the majority of patients in SERAPHIN trial reflects everyday practice in treating real PAH patients and increases the validity of the trial. The use of background therapy in the placebo group eliminates ethical concerns regarding depriving these patients from an effective therapy. With the long term follow up the SERAPHIN trial, it would have been difficult to maintain the randomized patients on a single PAH-targeted therapy because of disease progression. The positive results of the study practically eliminate the concern that the inclusion of patients on a background effective therapy may reduce the ability to demonstrate a statistically significant difference between the placebo and the active treatment groups. Given the low likelihood of drug-drug interaction (specifically with sildenafil and warfarin), macitentan may be the appropriate ERA drug to be used in combination therapy.</p><p>Although there was a trend for a macitentan-related reduction in death, this was not statistically significant. The SERAPHIN study was not powered to detect difference in mortality outcome. In addition, since PAH is a progressive disease and clinical deterioration is likely to precede death, it was unlikely that death was recorded as the first event.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> </p><p>In the SERAPHIN study, &#x0201c;worsening of PAH&#x0201d; was more likely to be the driver of the primary endpoint. However, this endpoint was very precisely defined, and an expert adjudication committee confirmed each event in a blinded fashion, emphasizing the robustness and clinical relevance of this endpoint</p><p>In the SERAPHIN trial, the 6MWD had increased by a mean of 22&#x000a0;m among patients on 10&#x000a0;mg macitentan, relative to placebo. This change in 6MWD parallels those reported in other trials. In a pooled analysis of 10 randomized placebo-controlled trials previously submitted to the FDA, active PAH treatment was associated with associated with change of 6MWT at 12 week of 22.4 m (95% CI: 17.4&#x02013;27.5&#x000a0;m) relative to placebo.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> Nevertheless, the change in 6MWD is less than 41.8 meters, a value that was previously reported to correspond to a statistically significant reduction in clinical events.<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref> This again challenges the use of 6MWD as a surrogate endpoint in PAH trials.</p><p>Macitentan was well tolerated in the SERAPHIN trial and, remarkably, rates of adverse events commonly associated with the ERA drug class (elevated liver aminotransferases and peripheral edema) were similar in the placebo and macitentan groups. Compared with placebo, a higher proportion of macitentan-treated patients had headache and respiratory adverse events, particularly those affecting the upper respiratory tract, mainly nasopharyngitis. These adverse events are known with ERAs and thought to be the results of vasodilatation.</p><p>In terms of liver test abnormalities, macitentan appears to have a better safety profile compared with bosentan and similar to amrisentan. Results of European post-marketing surveillance of bosentan in pulmonary hypertension showed elevated transaminases in 8% of patients with a discontinuation rate of 3% in bosentan-naive patients.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Accordingly, liver function test should be performed monthly in patients receiving bosentan or ambresntan.<xref rid="bib15" ref-type="bibr"> ¹⁵ </xref> It has been reported that bosentan inhibits the bile salt export pump, which may lead to cholestatic liver injury due to the intracellular accumulation of bile salts.<xref rid="bib16" ref-type="bibr"> ¹⁶ </xref> Macitentan has no significant inhibitory effects on hepatic bile salt transport and, therefore, has the potential for a favorable liver safety profile.<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref> </p><p>Reduction in blood hemoglobin &#x0003c;&#x000a0;8&#x000a0;g/dl was observed in 4.3% of patients receiving 10&#x000a0;mg of macitentan compared to only 0.4% of patients in the placebo group. Due to an as yet incompletely identified mechanism, potentially related to vasodilatation and decreased vascular permeability with subsequent fluid shift producing haemodilution, all ERAs are associated with a modest dose-dependent and partially transient reduction in haemoglobin levels. The significance of this hemoglobin reduction noticed with macitentan can only be firmly established postmarketing.</p></sec>

Riociguat: PATENT-1 Study

<p>Stimulators of soluble guanylate cyclase (sCG) are novel pharmacological agents that directly stimulate sGC. Ongoing research on sGC stimulators led to the development of the more potent and more specific sGC stimulator, riociguat.</p><p>Recently, the US Food and Drug Administration has approved riociguat to treat pulmonary arterial hypertension in adults. Support for the approval of riociguat comes from the recently published PATENT-1 (Pulmonary Arterial Hypertension Soluble Guanylate Cyclase&#x02013;Stimulator Trial 1) study.</p>

<contrib contrib-type="author"><name><surname>Said</surname><given-names>Karim</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Background</title><p>Pulmonary arterial hypertension (PAH) is associated with the impairment of the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway, supporting a role for therapeutic interventions which target this pathway.<xref rid="bib1" ref-type="bibr"> ^1&#x02013;2 </xref> </p><p>Until recently, the only practical therapeutic strategy to enhance the NO&#x02013;sGC&#x02013;cGMP pathway was the use of phosphodiesterase-5 (PDE-5) inhibitors, such as sildenafil, tadalafil, and vardena&#x0fb01;l to slow cGMP degradation. The clinical benefits associated with the PDE-5 inhibitor have led to interest in testing whether other agents that modulate NO signaling might be similarly beneficial in PAH. This is important considering the finding that up to 60% of patients with PAH do not respond to therapy with the PDE-5 inhibitor sildenafil, with some indication that pulmonary cGMP production is markedly impaired.<xref rid="bib3" ref-type="bibr"> ^3&#x02013;4 </xref> </p><p>Stimulators of sCG are novel pharmacological agents that directly stimulate sGC, both independently of NO and in synergy with NO. Ongoing research on sGC stimulators led to the development of the more potent and more specific sGC stimulator, riociguat.<xref rid="bib5" ref-type="bibr"> ⁵ </xref> </p><p>Recently, the US Food and Drug Administration has approved riociguat to treat PAH in adults. Support for approval of riociguat comes from the recently published PATENT-1 (Pulmonary Arterial Hypertension Soluble Guanylate Cyclase&#x02013;Stimulator Trial 1) study.<xref rid="bib6" ref-type="bibr"> ⁶ </xref> </p></sec><sec><title>Soluble guanylate cyclase as a therapeutic target in PAH</title><p>sGC is a dimeric, heme-containing, redox-sensitive enzyme that catalyzes the synthesis of the second messenger cGMP, which produces (through a number of downstream mechanisms) numerous biological effects, including vasorelaxation and inhibition of fibrosis, smooth muscle proliferation, apoptosis, leukocyte recruitment, and platelet aggregation.<xref rid="bib5" ref-type="bibr"> ^5&#x02013;8 </xref> </p><p>NO binds to sGC only when the heme group on sGC is in the reduced ferrous state. Notably, binding of NO to the reduced heme group leads to an approximately 200-fold increase in the conversion of GTP to cGMP.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> Alternatively, oxidation of this heme group results in its dissociation from the enzyme and the generation of NO-insensitive sGC.<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref> </p><p>In the presence of an intact heme-moiety, the sGC is a constitutively active enzyme that basally releases cGMP.<xref rid="bib11" ref-type="bibr"> ¹¹ </xref> However in PAH, although the total sGC expression is increased, alteration of the redox state of sGC through oxidative stress may lead to reduced levels of the NO-sensitive form of sGC.<xref rid="bib12" ref-type="bibr"> ¹² </xref> </p><p>sGC agonists are divided in two different categories according to their mechanism of action<xref rid="bib5" ref-type="bibr"> ^5&#x02013;13 </xref>:<list list-type="simple"><list-item><label>(1)</label><p>sGC stimulators sensitize sGC to NO by stabilizing the binding site on sGC. Accordingly, action of sGC stimulators is dependent on the presence of a reduced heme (heme-dependent compounds such as riociguat)</p></list-item><list-item><label>(2)</label><p>sGC activators preferentially and effectively activate sGC when it is in an oxidized (heme-independent compounds such as cinaciguat)</p></list-item></list> </p><p>Riociguat is the first drug approved in the new class of sGC stimulators. Riociguat acts through a dual mechanism: (1) direct stimulation of sGC in a NO independent fashion, and (2) by sensitization of sGC to low endogenous NO levels.<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref> In experimental studies, riociguat stimulated recombinant sGC up to 73-fold, and in the presence of a NO-releasing agent, increased the activity of sGC 112-fold above baseline.<xref rid="bib15" ref-type="bibr"> ¹⁵ </xref> Pre-clinical studies with sGC stimulators have shown vasodilatory, antiproliferative, antifibrotic, and antiinflammatory effects.<xref rid="bib5" ref-type="bibr"> ^5&#x02013;16 </xref> </p></sec><sec><title>Patent-1</title><p>PATENT-1<xref rid="bib6" ref-type="bibr"> ⁶ </xref> is a double-blind, randomized, placebo-controlled trial of 443 patients with PAH at 124 centers in 30 countries. Patients were randomly assigned in 2:4:1 ratio to; placebo, riociguat in individually adjusted doses up to 2.5&#x000a0;mg three times daily (2.5&#x000a0;mg maximum group), or riociguat in individually adjusted doses that were capped at 1.5&#x000a0;mg three times daily (1.5&#x000a0;mg maximum group). The 1.5-mg maximum group was included for exploratory purposes (to provide information about lower riociguat doses), and the data from that group were not included in the efficacy analyses.</p><p>The primary endpoint was change from baseline to the end of week 12 in the 6-minute walk distance (6MWD). Secondary endpoints included pulmonary vascular resistance changes, N-terminal prohormone brain-type natriuretic peptide (NT-proBNP), WHO functional class, time to clinical worsening, Borg scores, EuroQoL 5-dimensional Classification Component scores, and Living with Pulmonary Hypertension scores.</p><p>At week 12, 6MWD had increased from baseline by a mean of 30&#x000a0;m in the 2.5&#x000a0;mg&#x02013;maximum group and had decreased by a mean of 6&#x000a0;m in the placebo group (least-squares mean difference, 36&#x000a0;m; 95% confidence interval: 20 to 52; <italic>P</italic>&#x000a0;&#x0003c;&#x000a0;0.001).</p><p>Significant benefits were seen in the 2.5&#x000a0;mg&#x02013;maximum group, as compared with the placebo group, with respect to a range of secondary end points including pulmonary vascular resistance (<italic>P</italic>&#x000a0;&#x0003c;&#x000a0;0.001), NT-proBNP (<italic>P</italic>&#x000a0;&#x0003c;&#x000a0;0.001), WHO functional class (<italic>p</italic>&#x000a0;=&#x000a0;0.003), time to clinical worsening (<italic>p</italic>&#x000a0;=&#x000a0;0.005), and score on the Borg dyspnea scale (<italic>p</italic>&#x000a0;=&#x000a0;0.002).</p><p>Notably, patients who were receiving endothelin-receptor antagonists or non-intravenous prostanoids were permitted into the study and, accordingly, half of patients were on background therapy for PAH: 44% with endothelin-receptor antagonists and 6% with nonintravenous prostanoids. Pre-specified subgroup analysis showed that riociguat improved the 6MWD in patients who had not received other PAH-targeted therapies and also in those who had been on endothelin-receptor antagonists or prostanoids.</p><p>Concerning the safety profile, riociguat was well tolerated with a discontinuation rate of 3% in the 2.5&#x000a0;mg&#x02013;maximum group versus 7% in the placebo group. Syncope occurred less frequently in the 2.5-mg maximum (1%) compared to placebo (4%). The 2.5&#x000a0;mg maximum group had increased rates of hypotension (10%) and anemia (8%) compared to placebo group (2% for each), though without statistical significance.</p></sec><sec><title>What have we learned?</title><p>Both PDE-5 inhibitors and sGS stimulants target the NO-sGC-cGMP pathway. From a mechanistic point of view, sGC stimulators may have several advantages over PDE-5 inhibitors:<list list-type="simple"><list-item><label>[3]</label><p>The therapeutic action of PDE-5 inhibitors is dependent on baseline NO availability (which is typically reduced in PAH).<xref rid="bib13" ref-type="bibr"> ¹³ </xref> In contrast, owing to its NO-independent mode of action, sGC stimulators are effective even when NO production is markedly reduced.</p></list-item><list-item><label>[4]</label><p>PDE-5 inhibitors acts by prevention of cGMP degradation; accordingly in diseases where cGMP levels are low (as in PAH), the effectiveness of PDE-5 inhibitors is expected to be markedly limited. Furthermore, when PDE-5 is inhibited, the activity of other PDEs may compensate for it.</p></list-item><list-item><label>[5]</label><p>In PAH, sGC is upregulated in small pulmonary arteries<xref rid="bib15" ref-type="bibr"> ¹⁵ </xref> (as a compensatory mechanism) with increased opportunity for enhanced therapeutic actions of sGC stimulants.</p></list-item></list> </p><p>Compared with other PAH-targeted therapies, the position of riociguat within the therapeutic armamentarium in the management of PAH depends on several factors including: efficacy, safety, suitability for use in drug combination, drug-drug interaction, number of daily doses, and cost.<list list-type="simple"><list-item><label>[6]</label><p> <italic><bold>Efficacy:</bold></italic> In the PATENT-1 trial, the overall difference in the 6MWD with riociguat as compared with placebo, was 36&#x000a0;m at 12 weeks. This change in 6MWD is consistent with the increases observed in previous studies (22.4&#x000a0;m; 95% confidence interval: 17.4&#x02013;27.5&#x000a0;m).<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref> In comparison with PDE-5 inhibitors, this change in 6MWD is less than that observed with sildenafil in the SUPER trial,<xref rid="bib18" ref-type="bibr"> ¹⁸ </xref> where the mean placebo-corrected treatment on 6MWD was 45&#x000a0;m, 46&#x000a0;m, and 50&#x000a0;m for patients receiving 20, 40, and 80&#x000a0;mg of sildenafil, respectively. On the other hand, the improvement in 6MWD reported in PATENT-1 is close to that reported with tadalafil in the PHIRST trial, where tadalafil in 40&#x000a0;mg was associated with 33&#x000a0;m increase in 6MWD relative to placebo.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> Improvement in WHO functional class in PATENT-1 is modest where 21% of patients moved to lower class. In SUPER trial, the proportions of patients with an improvement of at least one functional class were 7%, 36%, and 42% for patients receiving 20, 40, and 80&#x000a0;mg of sildenafil, respectively.<xref rid="bib18" ref-type="bibr"> ¹⁸ </xref> In the PHIRST trial, no significant differences in the proportions of patients with and without improvement of WHO functional class were observed with tadalafil compared with placebo.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> </p><p>Importantly, many variables should be considered when comparing changes in 6MWD or WHO functional class among different studies (e.g., population characteristics, baseline 6MWD and WHO functional class, duration of study, proportion of patients on background therapy). For example, in the PHIRST study,<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> about half of patients were receiving bosentan as a background therapy, while in SUPER background therapy was not permitted.<xref rid="bib18" ref-type="bibr"> ¹⁸ </xref> This is important since the use of background effective therapy may reduce the ability to demonstrate a statistically significant difference in 6MWD or WHO functional class between the placebo and the active treatment groups.</p></list-item><list-item><label>[7]</label><p> <italic><bold>Safety:</bold></italic> Riociguat was well tolerated and had a favorable safety pro&#x0fb01;le. Two adverse events appear to be common among patients receiving the highest tolerated dose of riociguat: hypotension (10%) and anemia (8%).<xref rid="bib6" ref-type="bibr"> ⁶ </xref> The risk of hypotension should be minimized by a gradual individual dose titration to the highest tolerated dose (in PATENT-1, riociguat was titrated over 8 weeks), and by contraindicating concomitant use with other drugs affecting the NO-sGC-cGMP pathway (e.g., PDE-5 inhibitors, nitrates). The apparent increased risk of bleeding has been addressed by means of a prominent warning and a description of bleeding events in the adverse reactions section of the Product Monograph.</p></list-item><list-item><label>[8]</label><p> <italic><bold>Drug-drug interaction:</bold></italic> So far, the interaction potential of riociguat with other drugs is virtually unknown. Recent <italic>in vitro</italic> study suggests that riociguat is a P-glycoprotein substrate and might therefore act as a victim drug when co-administered with strong P-glycoprotein inducers or inhibitors.<xref rid="bib20" ref-type="bibr"> ²⁰ </xref> </p></list-item><list-item><label>[9]</label><p> <italic><bold>Use in combination therapy:</bold></italic> There is a growing trend to combine drugs that target multiple pathologic pathways in an attempt to increase efficacy and optimize outcomes in PAH patients. In one retrospective analysis, 56% of patients required additional therapy within 2 years.<xref rid="bib21" ref-type="bibr"> ²¹ </xref> Half of patients in PATENT-1 were on background therapy for PAH with significant improvement in the 6MWD in PAH-drug-therapy-naive patients as well as patients treated with combination therapy. Endothelin receptor antagonists were the most common drug class combined with riociguat. The combination between riociguat and PDE-5 inhibitor is contraindicated. PATENT-plus trial investigated the effects of riociguat on supine systolic blood pressure in patients receiving sildenafil over 12 weeks. In this study, riociguat was associated with a high rate of discontinuation due to hypotension with no evidence that this combination exerts a beneficial effect.<xref rid="bib22" ref-type="bibr"> ²² </xref> </p></list-item></list> </p><p>Preserving right ventricular (RV) function is one of the current targets of PAH therapy.<xref rid="bib23" ref-type="bibr"> ²³ </xref> The significant reduction of NT-proBNP in PAH patients receiving riociguat may denote a favorable effect on RV performance. However, the precise mechanisms underlying this positive effect remain uncertain. Possible mechanisms may include: reduction of RV afterload induced by pulmonary vasodilatation; reversing remodeling of pulmonary vasculature mediated by antiproliferative and antifibrotic effect; or direct effect on the RV. This possibility is supported by the results of experiments in a mouse model of chronic RV pressure overload, in which riociguat treatment reduced the collagen content of the RV and improved the RV ejection fraction.<xref rid="bib16" ref-type="bibr"> ¹⁶ </xref> </p><p>One of the major limitation of PAH trials is the short duration. Accordingly, long-term open-label extension study for patients who completed PATENT-1 was performed (PATENT-2).<xref rid="bib24" ref-type="bibr"> ²⁴ </xref> After 1 year of treatment, 6MWD further improved by 48&#x000a0;m over the original baseline of PATENT-1, WHO functional class also continued to improve and 68% of the overall cohort were in functional class I/II after 1 year of treatment.</p><p>In conclusion, riociguat, the first drug approved in the new class of sGC stimulators, represents an advance within the available therapeutic armamentarium for PAH with an efficacy that is expected to be comparable to PDE-5 inhibitors. Since combination therapy is gaining more ground in the management strategy of patients with PAH, large-scale and long term studies with clinical endpoints should be planned in order to further evaluate the role of the combination between riociguat and other PAH-targeted therapies with special emphasis on endothelin receptor antagonists.</p></sec>

3D-modelling of transient left bronchial obstruction following bidirectional superior cavopulmonary shunt

<p>Extrinsic compression of airways is one the most important causes of respiratory insufficiency in the perioperative period in children with congenital heart disease. This is especially true of pathologies that involve surgery of the aortic arch or conduit replacement of the right ventricular outflow tract. However bronchial obstruction is uncommon in the setting of bidirectional cavopulmonary shunt alone.</p><p>We report the case of an infant with a functionally univentricular heart who had a bidirectional superior cavopulmonary shunt and disconnection of the main pulmonary artery from the ventricular mass with oversewing of pulmonary valve. Post-operatively the patient desaturated due to compression of left main bronchus by the left pulmonary artery anteriorly and the descending aorta posteriorly. This was clearly defined by CT based on 3D-modelling of the airways and great vessels. The child was managed conservatively by ventilator support, selective bronchial suctioning and systemic steroids with a successful outcome.</p>

<contrib contrib-type="author"><name><surname>Lone</surname><given-names>Reyaz A</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib><contrib contrib-type="author"><name><surname>Chapron</surname><given-names>Julien</given-names></name><xref ref-type="aff" rid="aff2"> ² </xref></contrib><contrib contrib-type="author"><name><surname>Faris</surname><given-names>Aslam</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib><contrib contrib-type="author"><name><surname>John</surname><given-names>Jiju</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib><contrib contrib-type="author"><name><surname>Samaan</surname><given-names>Sandra Abou</given-names></name><xref ref-type="aff" rid="aff3"> ³ </xref></contrib><contrib contrib-type="author"><name><surname>Bhat</surname><given-names>Akhlaque Nabi</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>The current approach to the surgical management of patients with univentricular hearts is staged repair, which includes neonatal surgery to establish a source of controlled pulmonary blood flow and eliminate systemic outflow obstruction, followed successively by bidirectional superior cavopulmonary shunt (BSCPS) and a Fontan completion. Respiratory compromise is an important cause of desaturation following a BSCPS and is usually due to consolidation or collapse of the lung parenchyma and/or collections of fluid or air in the pleural space. Respiratory compromise due to bronchial obstruction is uncommon in this setting.</p><p>We present a patient with a functionally univentricular heart who had a normal airway. Following a BSCPS, she developed desaturation with inability to wean from ventilator. Brochoscopy and CT angiography revealed compression of left main bronchus by pulmonary artery anteriorly and descending aorta posteriorly. The site and cause of obstruction was clearly defined by CT-based 3D-modelling of the trachea, bronchi and great vessels. The patient improved with conservative management and was extubated and discharged home without any residual airway obstruction.</p></sec><sec><title>Clinical report</title><p>A full-term baby was diagnosed with double inlet left ventricle (DILV), levo-transposition of great arteries (L-TGA), large unrestrictive ventricular spetal defect (VSD), and an atrial septal defect (ASD). Aorta originated from the non-dominant anterior ventricle and pulmonary artery came from the dominant posterior ventricle. A small patent ductus arteriosus (PDA) was also present. The main pulmonary artery was banded and the PDA ligated in the neonatal period. Follow up echocardiography showed pulmonary artery band gradient of 71mm Hg with no sub-aortic obstruction.</p><p>At 5 months of age a bidirectional superior cavopulmonary shunt was performed. The main pulmonary artery was disconnected from the ventricular mass and the pulmonary valve was oversewn. The child was extubated soon after surgery, but had respiratory distress, requiring reintubation. Auscultation of the chest showed diminished air entry into the left lung, which was attributed the position of the endotracheal tube. Chest x-rays were normal.</p><p>Echocardiography showed unobstructed shunt, unobstructed branch pulmonary arteries, good ventricular function and no atrioventricular valve regurgitation. Echo-imaging injection of agitated saline in the right upper limb vein was not suggestive of pulmonary arteriovenous malformations. Ventilator strategy to maintain relative hypercarbia to improve superior venacaval return did not improve saturations. Inhaled nitric oxide also showed no improvement.</p><p>Cardiac catheterization showed patent BSCPS and branch pulmonary arteries and no decompressing veins. Femoral arterial saturation was 56%, and the left and right pulmonary artery saturations 37% superior venacaval, right and left pulmonary artery pressures were 17&#x000a0;mm Hg. Mean left and right atrial pressures were 4&#x000a0;mm Hg and the left ventricular end diastolic pressure was 5&#x000a0;mm Hg.</p><p>During cardiac catheterization it was observed from chest screening that left lung expansion was poor. The position of the tube was optimized but there was no improvement in the left lung expansion. The endotracheal tube was maneuvered into the left main bronchus and hand ventilation attempted, but it was too difficult to inflate the left lung, and this was clearly observed on screening. This raised a strong possibility of bronchial obstruction. Bronchoscopy was therefore performed which showed extrinsic pulsatile compression of the left main bronchus. CT angiography confirmed impingement of the left main bronchus between pulmonary artery anteriorly and descending aorta posteriorly (<xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><p>The site and cause of obstruction was clearly defined by CT-based 3D-modelling of the airways and great vessels. The patient was managed conservatively with ventilator support, selective bronchial suctioning and mucolytic installation under bronchoscopic guidance and systemic steroid were given for one week, the child was successfully extubated to nasal CPAP and was subsequent discharged home with oxygen saturation in 80s.</p></sec><sec><title>Method for 3D modelling</title><p>CT scans were obtained via a Siemens Sensation 64 with a slice thickness of 1.0&#x000a0;mm and a slice increment of 0.8&#x000a0;mm. DICOM were imported into Mimics (Materialise, Leuven, Belgium) for 3D reconstruction of the blood volumes in the single ventricle, aorta and pulmonary artery. The processed files were exported as STL files into 3-matic (Materialise, Leuven, Belgium) to create the various images of interest.</p></sec><sec><title>Discussion</title><p>Causes of desaturation flowing bidirectional superior cavopulmonary shunt include anastamotic obstruction, presence of decompressing vein from the cavopulmonary circuit to the inferior vena cava territory or to the atrium, high pulmonary vascular resistance, ventricular dysfunction, and, in rare cases, acute pulmonary arteriovenous malformations. Bronchial obstruction has been commonly described following aortic arch repair and conduit reconstruction of the pulmonary outflow tract<xref rid="bib1" ref-type="bibr"> ^1,2 </xref> but it is uncommon following BSCPS. Compression of the left main bronchus between the left pulmonary artery anteriorly and the descending aorta posteriorly has been described in a 3-month-old child following patch augmentation of aortic arch and closure of VSD.<xref rid="bib3" ref-type="bibr"> ³ </xref> They called it the &#x0201c;pincer effect&#x0201d;. In their patient, augmented aortic arch was the main culprit, which was surgically elongated to relieve the obstruction. A 13-year-old patient in their series was the only case of a functionally univentricular heart, who, after an extra cardiac Fontan operation, developed left bronchial compression by pincer effect between the posterior side of the ascending aorta and the anterior side of the descending aorta. This patient needed aortopexy and placement of a stent in the left main bronchus to relieve the obstructions. We believe that in our patient disconnection of the main pulmonary artery from the ventricular mass caused the branch pulmonary arteries to fall back into the posterior mediastinum where the left pulmonary artery caught the left main bronchus between itself and the posteriorly placed descending aorta (<xref ref-type="fig" rid="fig2">Figure 2</xref>). In combination with inflammatory edema that follows surgery, the left bronchus was trapped between two big vessels was obstructed. With time, conservative management, and steroids, the edema subsided and the compression on the bronchus was relieved.</p><p>Bronchoscopy is helpful in visualizing luminal obstruction to the left main bronchus. 3D reconstruction based on medical imaging is an effective method of defining the cause of respiratory obstruction. Computed tomography scanning is particularly useful for demonstration changes in airway caliber, in addition to the location, degree and extent of the airway narrowing.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> CT angiography was successful in confirming the mechanism of airway obstruction and planning therapeutic intervention in 17 patients who developed airway obstruction following operations that involved reconstruction of the aortic arch or the right ventricular outflow tract.<xref rid="bib3" ref-type="bibr"> ³ </xref> </p></sec><sec><title>Conclusion</title><p>Transient left bronchial obstruction following a BSCPS is described as a cause of desaturation. Conservative management was successful, leading to full recovery. The use of 3D modelling described here represents a major refinement for accurately determining the site and cause of the obstruction, and can be repeated using MRI if necessary to determine the response to therapy.</p></sec>

STOP-HF: Expanding the role of HF programs into the community

<p>The St Vincent's Screening TO Prevent Heart Failure (STOP-HF) study is a recently published trial that assessed the use of brain natriuretic peptide (BNP) as a screening tool for HF in an at-risk population in reducing newly-diagnosed heart failure and prevalence of significant left ventricular (LV) systolic and/or diastolic dysfunction. The study provides an excellent model to the global community on how to integrate primary care simple screening with secondary and tertiary level targeted diagnostic and therapeutic system. This integration includes screening of high-risk groups, use of a sensitive screening tool, early diagnostic modalities, early therapeutic interventions, and proper assessment of the hard clinical outcomes. However, more studies are needed across multiple sites around the world with different levels of health care services and variable biomarkers to identify higher-risk groups.</p>

<contrib contrib-type="author"><name><surname>ElMaghawry</surname><given-names>Mohamed</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>ElGuindy</surname><given-names>Ahmed</given-names></name></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Prologue</title><p>The last fifty years have witnessed remarkable improvement in the morbidity and mortality trends of most cardiovascular diseases. However, heart failure (HF) remains a notable exception. HF is a growing global health problem in both industrialized and developing nations. In fact, HF is the second most common cause for hospital admissions; the first cause is normal delivery.<xref rid="bib1" ref-type="bibr"> ¹ </xref> In the United States, the number of people with HF is expected to rise 46 percent from 5 million in 2012 to 8 million in 2030. The rise in patient numbers will double the costs of HF treatment, from $31 billion in 2012, to a staggering $70 billion in 2030.<xref rid="bib2" ref-type="bibr"> ² </xref> These facts have stimulated the search for new effective methods to combat HF. An attractive strategy is to integrate the early detection of high-risk patients at the primary care level with advanced diagnostic and therapeutic strategies at the tertiary care level.</p><p>The St Vincent's Screening TO Prevent Heart Failure (STOP-HF) study is a recently published trial in the Journal of American Medical Association (JAMA) assessing the use of brain natriuretic peptide (BNP) as a screening tool for HF in an at-risk population in reducing newly-diagnosed heart failure and prevalence of significant left ventricular (LV) systolic and/or diastolic dysfunction.<xref rid="bib3" ref-type="bibr"> ³ </xref> </p></sec><sec><title>STOP-HF trial design and results</title><p>In this &#x0201c;first-of-its-type&#x0201d; study, the investigators recruited 1374 participants with various cardiovascular risk factors from a nurse-provided primary care cardiovascular screening program in the catchment area of St Vincent's University Hospital, Dublin, Ireland, between January 2005 and December 2009.Eligibility criteria were age older than 40 years (mean age, 64.8 [SD, 10.2] years) and a history of one or more of the following; hypertension, hypercholesterolemia, obesity, vascular disease, diabetes mellitus, arrhythmias, moderate to severe valvular heart disease. Exclusion criteria included refusal to consent, established evidence of LV systolic dysfunction, history of symptomatic HF, or a diagnosis compromising survival over the study period.</p><p>The participants were randomly assigned to receive the usual primary care (control condition; <italic>n</italic>&#x000a0;=&#x000a0;677) or screening with BNP testing (<italic>n</italic>&#x000a0;=&#x000a0;697) and followed up until December 2011 (mean follow-up, 4.2 [SD, 1.2] years). Intervention-group participants, with BNP levels of 50&#x000a0;pg/mL or higher, underwent echocardiography and collaborative care between their primary care physician and specialist cardiovascular service.</p><p>The primary end point was prevalence of asymptomatic systolic LV dysfunction, with or without newly diagnosed heart failure. Due to the slower than expected recruitment rates, the investigators extended the study period and redefined the primary endpoint to include significant LV diastolic dysfunction as determined by a ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E') greater than 15.It is important to note that this change did not alter the validity of the study design. Secondary end points included emergency hospitalization for arrhythmia, transient ischemic attack, stroke, myocardial infarction, peripheral or pulmonary thrombosis/embolus, or heart failure. The inclusion of asymptomatic LV systolic dysfunction or significant diastolic dysfunction as a component of the primary endpoint reflect the heightened risk status of these abnormalities, specifically to the later development of HF.</p><p>A total of 263 patients (41.6%) in the intervention group had at least 1 BNP reading of 50&#x000a0;pg/mL or higher. Of the risk factors included in the study, this finding was consistent with the increasing age of the population. As expected, the intervention group underwent more cardiovascular investigations and received more renin-angiotensin-aldosterone system&#x02013;based therapy at follow-up. The primary end point of left ventricular dysfunction with or without HF was met in 59 patients (8.7%) in the control group and 37 patients (5.3%) in the intervention group (odds ratio [OR], 0.55; 95% CI, 0.37&#x02013;0.82; <italic>P</italic>&#x000a0;=&#x000a0;0.003). Asymptomatic LV dysfunction was found in 45 (6.6%) of 677 control-group patients and 30 (4.3%) of 697 intervention-group patients (OR, 0.57; 95% CI, 0.37&#x02013;0.88; <italic>P</italic>&#x000a0;=&#x000a0;0.01). HF occurred in 14 (2.1%) of 677 control-group patients and 7 (1.0%) of 697 intervention-group patients (OR, 0.48; 95% CI, 0.20&#x02013;1.20; <italic>P</italic>&#x000a0;=&#x000a0;0.12). The incidence rates of emergency hospitalization for major cardiovascular events were 40.4 per 1000 patient-years in the control group versus 22.3 per 1000 patient-years in the intervention group (incidence rate ratio, 0.60; 95% CI, 0.45&#x02013;0.81; <italic>P</italic>&#x000a0;=&#x000a0;0.002).<xref rid="bib3" ref-type="bibr"> ³ </xref> </p></sec><sec><title>Critique</title><p>STOP-HF is the first prospective, randomized trial to demonstrate reduction in adverse cardiovascular clinical outcomes using BNP guided collaborative care in a broad community cohort. BNP blood level has long been established as an important diagnostic and prognostic tool in the management of HF. BNP is stored in the ventricles, and to a lesser extent in the atria. High ventricular filling pressures stimulate the release of BNP which has a diuretic, natriuretic, and antihypertensive effect by inhibiting the renin-angiotensin-aldosterone system. The recent HF guidelines recommend that BNP screening may have some value in populations with certain risk factors such as previous ischemic heart disease, diabetes, and/or hypertension. What was unique about STOP-HF is that it reached beyond the simple confirmation of BNP as a risk predictor of HF. The investigators' aim was to prevent HF through risk factor modifications using medical, dietary and lifestyle interventions in a high-risk group defined by BNP.</p><p>The STOP-HF study raises some interesting points. First, the study highlights the importance of dedicated HF programs to adequately address the global burden of HF. The reduction in LV dysfunction, HF, and HF hospitalization rates observed in the intervention group must be interpreted in the light of the integrated approach utilized in STOP-HF. This multifaceted approach included many risk factors modifiers such as repeated echocardiography and early use of angiotensin receptor blockers. The HF program implemented in St Vincent's hospital includes specialized clinics with a team of specially trained registered nurses, nurse practitioner, pharmacists, dietician, palliative care specialists and cardiologists. The study results would not be reproducible in other less-than-ideal health care settings.</p><p>Second, STOP-HF draws the attention to the importance of including patients with asymptomatic LV systolic dysfunction and significant LV diastolic dysfunction when assessing the overall burden of HF in a population. These two entities may be overlooked in a non-dedicated primary health care set up.</p><p>Third, more population-based studies are needed to identify the optimum mean to screen for HF. Other than BNP, many novel markers have proven their efficacy in detecting pathological process associated with early HF such as myocardial stretch (ST2 protein),<xref rid="bib4" ref-type="bibr"> ⁴ </xref> myocyte injury (high sensitivity troponin assay),<xref rid="bib5" ref-type="bibr"> ⁵ </xref> and profibrotic process (procollagen type I amino terminal propetide (PINP).<xref rid="bib6" ref-type="bibr"> ⁶ </xref> Future trials should also target approaches such as genomics, epigenomics, metabolomics and transcriptomics for the discovery of novel biomarkers and disease pathway underlying HF in high-risk populations. For example, high mortality rates have been reported in Indian Asians due to coronary artery disease. In the UK, Indian Asians have two-fold higher coronary heart disease mortality compared to Europeans. The prospective Indian Asian cohorts such as the London Life Sciences Population Study (LOLIPOP) incorporate the &#x0201c;omics&#x0201d; approach to provide an excellent opportunity for the identification of new factors underlying coronary artery disease in this high risk population.<xref rid="bib7" ref-type="bibr"> ⁷ </xref> </p><p>Fourth, a limitation to STOP-HF is that it was underpowered for determination of mortality and all-cause hospitalization outcomes. However, longer follow up analysis (10&#x02013;15 years) could possibly demonstrate a significant decrease in the death rates in the intervention group. In addition, there were too few events for further sub-group analysis according to age or risk factors. Future larger studies should focus on recognising those who would benefit the most from this proactive screening strategy.</p><p>Finally, the cost-effectiveness analysis of this strategy has not yet been reported. A critical question that needs to be answered before adopting a similar screening program on a wider scale is how much this strategy cost? Hopefully, with even more specific target population and widely available screening tools, this strategy may further reduce its cost and optimize its efficacy.</p></sec><sec><title>What have we learnt?</title><p>STOP-HF provides an excellent model to the global community on how to integrate primary care simple screening with secondary and tertiary level targeted diagnostic and therapeutic system. This integration includes screening of high-risk groups, use of a sensitive screening tool, early diagnostic modalities, early therapeutic interventions, and proper assessment of the hard clinical outcomes. However, to reach for the Holy Grail of reducing the global HF burden, more studies are needed across multiple sites around the world with different levels of health care services. More specific, higher-risk groups may show more benefit from this approach with a lesser cost to the public health systems.</p></sec>

Is there an increased cardiovascular risk in metabolically healthy obese individuals? Lessons from the HUNT (Nord-Tr&#x000f8;ndelag Health) study

<p>Obesity and metabolic syndrome frequently co-exist and are major health problems worldwide. Prior research has questioned whether obesity without cardiometabolic abnormalities &#x0201c;metabolically healthy obesity&#x0201d; (MHO), has adverse effects on overall cardiovascular disease risk (CVD). The association between MHO and the first development of acute myocardial infarction and heart failure (HF) was evaluated in the second HUNT (Nord-Tr&#x000f8;ndelag Health).</p>

<contrib contrib-type="author"><name><surname>Al Suwaidi</surname><given-names>Jassim</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>Atherosclerosis, obesity, and metabolic syndrome are closely linked and constitute, arguably, the most menacing three conditions to modern society.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Obesity adversely affects almost all of the major cardiovascular risk factors, including blood pressure, dyslipidemia and insulin sensitivity leading to metabolic syndrome and type-2 diabetes mellitus. It is not surprising that obesity increases the risk of almost of all cardiovascular disorders including hypertension, coronary artery disease, heart failure, atrial fibrillation and peripheral arterial disease. However, previous research has questioned whether obesity without cardiometabolic abnormalities &#x0201c;metabolically healthy obesity&#x0201d; (MHO) has adverse effects on overall cardiovascular risk.<xref rid="bib2" ref-type="bibr"> ² </xref> Using data from the second HUNT (Nord-Tr&#x000f8;ndelag health) study M&#x000f8;rkedal B, et al attempts to evaluate this association.<xref rid="bib3" ref-type="bibr"> ³ </xref> </p></sec><sec><title>The study</title><p>Inhabitants 20 years of age and older in Nord-Tr&#x000f8;ndelag County in Norway were invited to participate in the second HUNT from August 1995 to June 1997. Of the 93,898 individuals eligible to participate, 64,726 (69%) accepted the invitation, and attended a clinical examination conducted by trained nurses. Exclusion criteria were; missing information on body-mass index (BMI) or history of acute myocardial infarction (AMI), heart failure (HF) or cerebral stroke at baseline. Thus 61,299 participants (28,255 men and 33,044 women) were included in the main analyses of BMI and metabolic health with risk of AMI and HF. Furthermore, 21,796 of participants had information about their BMI from prior analysis; the tuberculosis screening (conducted between 1966 and 1969) and from HUNT-1 (conducted between 1984 and 1986). Thus, for the latter proportion of participants, BMI measurements' were available approximately 10 and 30 years before baseline for the present study.</p><p>The investigators used a modified definition of metabolic health as described by the International Diabetes Federation. Participants were categorized as metabolically unhealthy if they had elevated waist circumference (&#x0003e;94&#x000a0;cm for men, &#x0003e;80&#x000a0;cm for women) or BMI &#x02265;&#x000a0;30&#x000a0;kg/m2 in addition to 2 or more of the following criteria: elevated nonfasting triglycerides (&#x000a0;&#x02265;&#x000a0;1.7&#x000a0;mmol/l), reduced high-density lipoprotein cholesterol (&#x000a0;&#x0003c;&#x000a0;1.03&#x000a0;mmol/l for men, &#x0003c;&#x000a0;1.29&#x000a0;mmol/l form women), elevated blood pressure (&#x000a0;&#x02265;&#x000a0;130/85&#x000a0;mmHg) or use of blood pressure medication, elevated nonfasting glucose (&#x000a0;&#x02265;&#x000a0;11.1&#x000a0;mmol/l), or diabetes diagnosis. Patients were subdivided into three categories according to their BMI; 25&#x000a0;&#x0003c;&#x000a0;kg/m2 (normal), 25 to 29.9&#x000a0;kg/m2 (overweight) and &#x02265;&#x000a0;30&#x000a0;kg/m2 (obese) and metabolically healthy or unhealthy.</p><p>The investigators also performed sub-analysis using 6 categories of BMI (underweight &#x0003c;&#x000a0;18.5&#x000a0;kg/m2, normal weight 18.3-24.9&#x000a0;kg/m2, overweight 25-29.9&#x000a0;kg/m2, class I obese 30-34.9&#x000a0;kg/m2, class II obese 35-39.9, and class III obese&#x000a0;&#x02265;&#x000a0;40&#x000a0;kg/m2). A separate analysis was also made for the duration of obesity among participants whom previous BMI measurements had been conducted. In this analysis, the participants were divided into 5 categories: long-term normal weight; long-term overweight; long-term obese; recent development of obesity; and variable body mass (any other combination of BMI categories). Analysis of abdominal obesity (waist-hip ratio &#x0003e;0.9 for men and &#x0003e;0.85 for women) instead of BMI and outcome was also made. Endpoints: first AMI or for HF.</p></sec><sec><title>Results</title><p>Among the overall participants, 10,059 (16.4%) were classified as obese and 15,576 (25.4%) were classified as metabolically unhealthy. Among the obese, the proportion of metabolically healthy (MHO) was 26.4%. Obese and metabolically healthy participants were more likely to be women younger, and unmarried compared with obese and metabolically unhealthy participants (MUO).</p><sec><title>Acute myocardial infarction (AMI)</title><p>During a median follow-up of 12.2 years, 2,547 participants had a first AMI. The age- and sex-adjusted HR among obese men and women who were metabolically healthy was 1.0 (95% CI: 0.8-1.2) compared with normal weight and metabolically healthy participants. The corresponding HR for obese and metabolically unhealthy men and women was 1.7 (95%: 1.5-1.9). Furthermore, the risk of AMI was consistently higher among metabolically unhealthy participants across the range of BMI, including the severe obese, compared with metabolically healthy participants. Neither long-term obesity nor recently developed obesity was associated with substantial risk for AMI among metabolically healthy participants.</p></sec><sec><title>Heart failure (HF)</title><p>During a median follow-up of 12.3 years 1,201 participants developed HF. There was a stronger risk of HF associated with long-term obesity, regardless of metabolic status, compared with normal-weight and metabolically healthy participants. There was also a higher risk of HF among metabolically healthy participants who had recently developed obesity. The results of using abdominal waist circumference were similar to those obtained in he primary analyses using BMI.</p></sec></sec><sec><title>Discussion</title><p>The investigators concluded that the metabolic status and not obesity was the main determinant risk of AMI. In contrast, the risk of HF was similarly increased in MHO and MUO participants compared with normal-weight participants with healthy metabolic status, suggesting that metabolic health may not play a central role for these associations. The results of using abdominal waist circumference were similar to those obtained in he primary analyses using BMI for AMI &#x00026; HF. This increased risk of HF has been explained in an accompanying editorial by the fact that increased adiposity increases total blood volume, stroke volume, cardiac output and cardiac work leading to significant abnormalities on both the right and left sides of the heart.<xref rid="bib2" ref-type="bibr"> ² </xref> The complexity of the association between obesity and cardiovascular diseases is further complicated by the current understanding of the various physiologic functions of adiposity. Adipose tissue in addition to its role in thermogenesis and energy storage, it is a complex endocrine organ and is believed to have a role in the evolution of human brain as well as in myocardial regeneration and repair.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> </p><p>The findings of the current study are <italic>not</italic> concordant with a recently published meta-analysis<xref rid="bib5" ref-type="bibr"> ⁵ </xref> as well as a number of recent studies<xref rid="bib6" ref-type="bibr"> ^6,7 </xref> (see <xref ref-type="table" rid="tbl1">Table 1</xref>). A meta-analysis by Kramer et al<xref rid="bib5" ref-type="bibr"> ⁵ </xref>, which included 8 previously published studies, demonstrated a significant association between being overweight and cardiovascular events. The Whitehall II study<xref rid="bib6" ref-type="bibr"> ⁶ </xref>, an ongoing prospective cohort study, included 7122 participants aged 39-63 years who were enrolled between 1991 and 1993 and followed up for 17.4 years. Cardiovascular diseases risk was comparable between metabolically healthy and unhealthy obese participants, although the risk of type-2 diabetes was lower among MHO compared to MUO.</p><p>Another recent study from Korea by Chang and colleagues<xref rid="bib7" ref-type="bibr"> ⁷ </xref>, which involved 14,828 metabolically healthy individuals who took part in a comprehensive regional health-screening program compared coronary calcium scores (CAC) between MHO versus metabolically normal weight participants. Across a series of analyses adjusting for potential confounding variables, the MHO group had a significantly greater prevalence of coronary atherosclerosis compared with the metabolically normal weight group. However following additional adjustment of metabolic risk factors and LDL-C levels, this difference no longer remained significant. The authors concluded obesity even among metabolically healthy individuals is associated with greater prevalence of subclinical CAD. Furthermore, this association appears to be determined by components of metabolic parameters that fell below specific threshold levels. Rush Puri, MD in an accompanying editorial<xref rid="bib8" ref-type="bibr"> ⁸ </xref> suggested that it is probably time to dispel the concept of metabolically healthy obesity. Finally, the interaction between obesity / metabolic syndrome and cardiovascular risk is further complicated by the dietary &#x0201c;habit&#x0201d; in the community, for example in Norway there is higher consumption of fish which may play a protective role when compared to that in the Middle East, where the high consumption of red meat needs to be studied.</p><p>In conclusion, even with these recent studies including that of HUNT-2<xref rid="bib3" ref-type="bibr"> ³ </xref>, the association between metabolically healthy obesity cardiovascular disease risks (specifically coronary artery disease) remains controversial and needs further study.</p></sec><sec><title>What we have learned?</title><p>Obesity and metabolic syndrome are major public health problems. The incidence of obesity-related metabolic disturbances varies widely among obese individuals. Whether MHO is associated with reduced risk of cardiovascular disease is controversial.</p></sec>

Anticoagulation in pulmonary arterial hypertension: Contemporary data from COMPERA registry

<p>The use of anticoagulant therapy in patients with pulmonary arterial hypertension (PAH) has been controversial for decades. Recommendations for anticoagulation in these patients are often derived from small, retrospective, and single centre studies without any placebo-controlled randomized study. Furthermore, uncertainties exist regarding a number of issues such as patient selection, risk stratification for bleeding, the intensity of anticoagulation, appropriateness of anticoagulation in different types of PAH, and the potential use of new oral anticoagulants.</p><p>Recently, the database of the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) has been analyzed to assess the effect of anticoagulation on the long-term outcome of patients with various forms of PAH. This analysis is the largest to date to assess anticoagulant therapy in PAH patients in a prospective design with long observation period. The results of COMPERA lend support to current recommendations for the use of anticoagulant therapy in patients with idiopathic PAH, but not in other forms of PAH. Also, the study confirmed the previously reported concern that anticoagulant therapy may be harmful in patients with scleroderma-associated PAH.</p>

<contrib contrib-type="author"><name><surname>Said</surname><given-names>Karim</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Background</title><p>The exact role of chronic thrombosis in the pulmonary arteries in patients with pulmonary arterial hypertension (PAH) is controversial. One view suggests that thrombosis is an epiphenomenon related to stasis and endothelial dysfunction. Another view holds that chronic organized thrombotic pulmonary vascular lesions are an integral part of pulmonary vascular remodeling leading to progressive luminal narrowing with increased pulmonary vascular resistance and progression of PAH.<xref rid="bib1" ref-type="bibr"> ^1&#x02013;2 </xref> </p><p>Irrespective of whether thrombosis is a cause or consequence of PAH, anticoagulants have been used for decades in PAH patients. The main rationales for the use of anticoagulant therapy in PAH are:<list list-type="simple"><list-item><label>(1)</label><p>Pathological evidences that thrombi are a common finding in idiopathic PAH patients. In two retrospective studies evaluating histopathology in idiopathic PAH patients (formerly called primary pulmonary hypertension), the prevalence rates for chronic organized pulmonary vascular thromboses were 56% and 57%.<xref rid="bib3" ref-type="bibr"> ^3&#x02013;4 </xref> </p></list-item><list-item><label>(2)</label><p>Evidence that PAH is associated with prothrombotic abnormalities, causing <italic>in situ</italic> thrombosis. These abnormalities include all components of coagulation cascade: coagulation factors, platelet function, and fibrinolytic system (for a review, see <xref rid="bib2" ref-type="bibr"> ² </xref>).</p></list-item><list-item><label>(3)</label><p>Evidence from observational studies that showed better outcomes in idiopathic PAH patients receiving anticoagulant therapy. In a systematic review of seven observational studies, survival benefit was demonstrated in five studies, while two did not support these findings.<xref rid="bib5" ref-type="bibr"> ⁵ </xref> </p></list-item></list> </p><p>However, the use of anticoagulants in patients with PAH has been a controversial subject for decades for several reasons. First, there has been no placebo-controlled randomized trial that has assessed the effectiveness of anticoagulant therapy in patients with PAH. Available data were derived from small, retrospective, and single centre studies. Second, available literature is restricted to idiopathic PAH with almost no published evidence for other types of PAH. Accordingly, the generalizability of survival benefit reported in idiopathic PAH patients to other types of PAH (eg, scleroderma associated PAH) remains controversial. Third, there is lack of data on the added benefit of anticoagulant therapy in patients receiving modern PAH-target therapy. Fourth, little data exist regarding the risk stratification of bleeding in PAH patient receiving anticoagulant therapy.</p><p>Currently, the European Society of Cardiology and the European Respiratory Society recommend that anticoagulant treatment should be considered in patients with idiopathic PAH, heritable PAH, and PAH due to use of anorexigens (Class IIa), with a lower level of recommendation in patients with associated PAH (Class IIb).<xref rid="bib6" ref-type="bibr"> ⁶ </xref> The American College of Chest Physicians clinical guidelines support the use of anticoagulation with a grade &#x0201c;B&#x0201d; recommendation (a moderate recommendation) based on fair level of evidence in idiopathic PAH patients, and weak recommendation based on expert opinion only for other PAH types.<xref rid="bib7" ref-type="bibr"> ⁷ </xref> </p></sec><sec><title>Anticoagulation In Pah: Data From Compera Registry</title><p>The database of the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA),<xref rid="bib8" ref-type="bibr"> ⁸ </xref> was recently analyzed to assess the effect of anticoagulation on the long-term survival in patients with various forms of PAH. COMPERA is an ongoing prospective European pulmonary hypertension registry that began in 2007 with the contribution of 41 pulmonary hypertension centers from 7 European countries.</p><p>The study analyzed the data of 1283 patients with newly diagnosed PAH based on right heart catheterization. Patients who received anticoagulation at any time during the registry were grouped into the anticoagulation group (<italic>n</italic>&#x000a0;=&#x000a0;738; 58%), whereas patients who never received anticoagulation were grouped into the no anticoagulation group (<italic>n</italic>&#x000a0;=&#x000a0;545; 42%). According to type of PAH, anticoagulation was used in 66% of 800 patients with idiopathic PAH, and in 43% of 483 patients with other forms of PAH. Vitamin K antagonists were used in 93% of patients followed by heparins (6%) and new oral anticoagulants (1%).</p><p>In idiopathic PAH patients, during the 3-year follow-up period, the mortality rate in anticoagulation group was 14.2%, versus 21% in the no anticoagulation group (survival advantage, <italic>p</italic>&#x000a0;=&#x000a0;0.006). This survival benefit occurred despite the fact that patients in the anticoagulation group had worse baseline hemodynamics. In a matched-pair analysis (based on sex, age, functional class, and pulmonary vascular resistance) the survival difference between both study groups remained statistically significant (<italic>p</italic>&#x000a0;=&#x000a0;0.017). Furthermore, multivariable regression analysis confirmed the beneficial effect of anticoagulation on survival of idiopathic PAH patients (hazard ratio, 0.79; 95% confidence interval, 0.66&#x02013;0.94)</p><p>In patients with other forms of PAH, during the 3-year follow-up period, mortality rate in anticoagulation group was 21.9% versus 15% in the no anticoagulation group without statistically significant survival difference (<italic>p</italic>&#x000a0;=&#x000a0;0.156).</p><p>Among the 208 patients with scleroderma-spectrum of disease associated with PAH, 26.9% of patients in the coagulation group died, compared to 17.3% in the no anticoagulation group without statistically significant survival difference (<italic>p</italic>&#x000a0;=&#x000a0;0.28). However, the use of anticoagulants in these patients was associated with a non-significant trend toward a worse survival in the single predictor analysis (HR, 1.82; 95% CI, 0.94 to 3.54; <italic>P</italic>&#x000a0;=&#x000a0;0.08)</p><p>As regards bleeding risk, the COMPERA database was not designed to systematically capture all bleeding events. Available data denote that, among the 219 deaths, bleeding was attributed as a cause of death in 4 patients (2%). In addition, there were 3 nonfatal but serious bleeding events resulting in hospital admission. Of note, among these 7 bleeding events, 6 occurred in the anticoagulation group.</p></sec><sec><title>What have we learned?</title><p>Data of the COPMERA registry lend support to current recommendations for the use of anticoagulant therapy in patients with idiopathic PAH, but not in other forms of PAH. Also, the data substantiated the previously reported concern that anticoagulant therapy may be harmful in patients with scleroderma-associated PAH.</p><p>The importance of the COMPERA lies in: (1) being the largest study so far assessing the effects of anticoagulation therapy in patients with PAH; (2) the prospective design; (3) the 3-year observation period; (4) the low number of patients lost to follow-up (&#x000a0;&#x0003c;&#x000a0;3%); and (4) the use of modern PAH-targeted therapy including combination therapy in 45% of all patients, reflecting the current real-world practice.</p><p>Results of the COMPERA registry open the gate for several unanswered questions related to criteria that should be used to select patients for anticoagulant therapy; risk stratification for bleeding; the optimum target international normalized ratio (INR); the potential role of new oral anticoagulants; and the need for further randomized controlled trials.</p><sec><title>Patient selection</title><p>The decision of anticoagulant therapy in a patient with PAH should consider the balance between the risk of PAH-related mortality versus the risk of bleeding related to anticoagulant therapy in this particular patient.</p><sec><title>Risk of PAH-related mortality</title><p>Mortality risk in PAH patients can be assessed by focusing on parameters with established prognostic importance. These prognostic factors can be categorized into clinical risk factors (WHO function class, 6 minutes walk test, clinical evidences of right ventricular failure, and rapid progression of symptoms); echocardiographic risk factors (right ventricular dysfunction, pericardial effusion); hemodynamic parameters (elevated right atrial pressure, low cardiac output index); or laboratory risk factors (elevated NT-pro BNP).<xref rid="bib6" ref-type="bibr"> ^6,9 </xref> In this regard, the population of COMPERA are considered to be at intermediate risk for worse outcome (WHO function class III in 75% of patients, mean 6 minutes walk test of 294&#x000a0;m; mean right atrial pressure of 8.8&#x000a0;mmHg and mean cardiac output index of 2.2 L/min/m²).</p><p>Another point to be considered is how early anticoagulant therapy should be initiated in PAH patients. Introduction of anticoagulant therapy at an early stage of the disease may carry the possible advantage of slowing the progression of luminal narrowing in PAH. However, this strategy may be associated with increased life-time exposure to anticoagulant therapy with increased bleeding risk. Alternatively, the use of anticoagulant therapy in patients in an advanced stage of the disease is expected to offer more protection, since these patients have low cardiopulmonary reserve that cannot withstand further arterial obstruction. Nevertheless, these patients may be also at increased bleeding risk related to hepatic and gastrointestinal congestion.</p></sec><sec><title>Risk of bleeding</title><p>Bleeding in PAH patients is important for two reasons: it occurs in relatively higher rates compared with other diseases; and it may be associated with serious sequelae. In a retrospective single centre study, major bleeding ranged from 2.4 per 100 patient-years for chronic thromboembolic pulmonary hypertension and 5.4 per 100 patient-years for idiopathic PAH, to 19 per 100 patient-years for PAH associated with connective tissue disease.<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref> These rates are considered high compared to the reported rates of major bleeding in patients with atrial fibrillation receiving oral anticoagulants (2.0 per 100 patient-years).<xref rid="bib11" ref-type="bibr"> ^{11&#x02013;12} </xref> The occurrence of an otherwise mild bleeding can be a catastrophic event in PAH patients. These patients are volume sensitive and acute blood loss may induce a fatal vicious circle of cardiopulmonary decompensation that leads to irreversible cardiogenic shock. Chronic blood loss will impair cardiopulmonary reserve and in severe anemia both tissue hypoxia and lactic acidosis contribute to increase pulmonary artery pressure.</p><p>A number of factors should be considered to assess risk of bleeding in these patients.<list list-type="simple"><list-item><label>[4]</label><p>Type of PAH: bleeding risk is increased in 3 groups of patients with PAH: (a) patients with connective tissue diseases, specially patients with scleroderma in whom the risk of gastrointestinal bleeding is increased due to the presence of luminal telangiectasia<xref rid="bib13" ref-type="bibr"> ¹³ </xref>; (b) patients with PAH related to congenital heart disease and (c) patients with portopulmonary hypertension with increased risk for gastrointestinal bleeding owing to the presence of varices and abnormal coagulation profile.</p></list-item><list-item><label>[5]</label><p>Comorbidities including chronic kidney disease, chronic liver disease, unexplained anemia, and peptic ulcer.</p></list-item><list-item><label>[6]</label><p>Drug-drug interaction: caution should also be exercised with the concomitant use of PAH-target therapies and warfarin. Bosentan partially induces the cytochrome P450 system, thereby increasing warfarin metabolism and the required dose. The platelet-inhibiting effect of prostacyclin analogues and sildenafil is widely acknowledged, yet its clinical relevance is still unclear, with respect to concomitant use of warfarin.</p></list-item><list-item><label>[7]</label><p>Age: elderly patients are at increased bleeding risk while on anticoagulant. At the same time increased age is associated with increased mortality risk in PAH patients. In the COMPERA,<xref rid="bib8" ref-type="bibr"> ⁸ </xref> age was an independent predictor of mortality among patients with idiopathic PAH (HR: 1.35; 95% CI: 1.14 to 1.61). Similarly, in the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL), male patients &#x0003e;60 year was an independent predictor of increased mortality (HR, 2.2; 95% CI, 1.6 to 3.0).<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref> Data on the risk-benefit ratio of anticoagulant therapy in pediatric PAH population is lacking.</p></list-item></list>Unfortunately, the COMPERA database was not designed to systematically capture all bleeding events. All the study could state was that bleeding complications were responsible for &#x0223c;2% of the deaths in all cohorts, and that serious bleedings occurred predominantly in the anticoagulation group. No data were available regarding less severe bleeding or the development of iron deficiency anemia. Risk factors for increased bleeding were not systematically assessed in COMPERA; the presence of these risk factors might have affected the decision to use (or not to use) anticoagulants, as well as survival.</p></sec></sec><sec><title>Target INR</title><p>Generally, the target INR in PAH patients varies, from 1.5&#x02013;2.5 in most centers of North America, to 2.0&#x02013;3.0 in European centers.<xref rid="bib6" ref-type="bibr"> ⁶ </xref> Unfortunately, data regarding INR in the COPMERA study were deficient; it was mentioned that the INR was 2&#x02013;3 in all but one center and that about 58% of patients in the anticoagulation group had received anticoagulants for the entire observation period. Furthermore, COMPERA did not provide data regarding the frequency and duration of INR values inside and outside the target range, or reasons for anticoagulant discontinuation.</p></sec><sec><title>New oral anticoagulants</title><p>In COMPERA, 6% of patients in the anticoagulant group were receiving new oral anticoagulants. In atrial fibrillation and venous thromboembolism studies, new oral anticoagulants were, on the whole, non-inferior for efficacy and, to different degrees, superior for some bleeding endpoints compared with vitamin K antagonist. However, the use of new oral anticoagulants in PAH patients cannot be recommended because of the lack of evidence on efficacy and safety in addition to the difficulty to reverse the anticoagulant effect in emergency situations and the potential vulnerability to drug-drug interactions with PAH-targeted therapies.</p></sec><sec><title>Future research</title><p>Given the high mortality in idiopathic PAH and the 20% relative risk reduction in mortality among idiopathic PAH patients in the COMPERA, it would be possibly difficult &#x02013; from ethical point of view &#x02013; to have placebo-controlled randomized trial in the future to further assess the efficacy and safety of anticoagulants in idiopathic PAH. Rather, new randomized controlled trials designed for the evaluation of newer PAH drugs should stratify patients according to anticoagulant use. More studies are needed to answer previous questions related to patients' selection and risk stratification, target INR, and role of new oral anticoagulants.</p></sec></sec><sec sec-type="conclusions"><title>Conclusion</title><p>Contemporary data from the COMPERA registry support the use of anticoagulant therapy in patients with idiopathic PAH, but not in other types of PAH. Importantly, the data substantiated the previously reported concern that anticoagulant therapy may be harmful in patients with scleroderma-associated PAH. Further research into the role of anticoagulation in PAH is needed to establish best practice recommendations.</p></sec>

RAAFT-2: Radiofrequency ablation vs antiarrhythmic drugs as first-line treatment of paroxysmal atrial fibrillation

<p>Radiofrequency ablation (RFA) for the treatment of paroxysmal Atrial Fibrillation (pAF) has a class 1 indication in patients who have not tolerated or responded to antiarrhythmic medications. Antiarrhythmic medications (AAM) are, however, limited not only by modest efficacy, but also by significant side effects. Discontinuation rates for AAM range from 11-40% in trials. The RAAFT-2 trial evaluates the use of RFA as a first line treatment for pAF compared to optimal pharmacological management (1).</p>

<contrib contrib-type="author"><name><surname>Kaba</surname><given-names>Riyaz A</given-names></name><xref ref-type="aff" rid="aff1 aff2 aff3 aff4"> ^1,2,3,4 </xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>Cannie</surname><given-names>Douglas</given-names></name><xref ref-type="aff" rid="aff2"> ² </xref></contrib><contrib contrib-type="author"><name><surname>Ahmed</surname><given-names>Omar</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>RAAFT-2 trial</title><p>The RAAFT-2 trial was a multicentre randomised clinical trial that was sponsored and co-ordinated by the Population Health Research Institute at McMaster University and an unrestricted research grant from Biosense Webster. It randomised 127 drug- &#x00026; ablation-naive patients aged 18&#x02013;75 with pAF to either first line catheter ablation (<italic>n</italic>&#x000a0;=&#x000a0;66), or medical therapy (<italic>n</italic>&#x000a0;=&#x000a0;61). Subjects were randomised in a 1:1 ratio to either treatment if they were symptomatic with recurrent pAF, and had &#x02264;&#x000a0;4 episodes within the previous 6 months, one of which had to be documented by surface electrocardiography (ECG). All patients had normal systolic function and no history of heart failure or hypertension.</p><p>At baseline there were two significant differences between the study group characteristics; previous electrical cardioversion (33.3% RFA group vs 52.5% AAM group, <italic>p</italic>&#x000a0;=&#x000a0;0.03) and use of oral anticoagulation (53% RFA group vs 31.1% AAM, <italic>p</italic>&#x000a0;=&#x000a0;0.01).</p><p>After randomisation patients entered a 90-day blanking period during which medications were titrated or ablation was performed. After this period, primary outcome events were recorded. Patients were followed up at 1, 3, 6, 12 and 24 months. The study also utilised transtelephonic monitoring (TTM) to assess the cardiac rhythm of patients biweekly and whenever subjects experienced symptoms of possible AF.</p><p>RFA involved circumferential isolation of the pulmonary veins with confirmation of entrance block. Additional lesions were left to investigator discretion. AAM's and cardioversions were allowed during the 90-day blanking period only.</p><p>Patients randomised to the AAM group had their medications selected according to investigator discretion, with doses being based on guidelines.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Patients in this group were able to undergo RFA after the 90-day treatment period if AAM had failed. This was demonstrated by either drug discontinuation due to intolerance, adverse events or inefficacy (recurrence of pAF or atrial tachyarrhythmia lasting &#x0003e;30 seconds).</p><p>The primary efficacy outcome was time to first recurrence of symptomatic or asymptomatic atrial arrhythmia lasting more than 30 seconds, as documented by ECG or TTM. Secondary outcomes included first documented recurrence of AF-related atrial arrhythmia, repeated episodes of AF-related atrial arrhythmia, and quality of life at the 1-year follow-up. The study was powered to test the superiority of RFA over AAD using cox regression analysis, stratified by clinical site.</p><p>The Primary Safety Outcome was defined as the comparison of the proportion of patients with an occurrence of a cluster of serious complications in the RFA or AAM arms.</p></sec><sec sec-type="results"><title>Results</title><p>Primary outcome analysis demonstrated significantly lower recurrence of atrial tachyarrhythmia in the ablation group (54.5%) than in the medically treated group (72.1%) in the 2 years of follow-up (HR, 0.56; 95% CI 0.35-0.90, <italic>P</italic>&#x000a0;=&#x000a0;0.02). Recurrence of symptomatic atrial tachyarrhythmia was also lower in the ablation group (40.9% vs. 57.4%; HR 0.52 95% CI 0.30&#x02013;0.89 <italic>P</italic>&#x000a0;=&#x000a0;0.02).</p><p>Quality of life, as assessed by the EQ5D score, was significantly improved at 12 months in the RFA group (<italic>P</italic>&#x000a0;=&#x000a0;0.03) but not in the AAM group (<italic>P</italic>&#x000a0;=&#x000a0;0.22), although there was no statistically significant difference between the groups at 12 months (<italic>P</italic>&#x000a0;=&#x000a0;0.25). There were no deaths or strokes in either group.</p><p>In the AAM group, flecainide was prescribed to 69% of patients at a mean dose of 175.8&#x000a0;mg/d and and 25% received propafenone at a mean dose of 487.7&#x000a0;mg/d. More than one type of drug was received by 16.4% of patients during the 90-day blanking period. Fifty-nine per cent of the AAM group had to discontinue at least one AAM, and 47.5% of patients underwent RFA during the 2-year follow up period.</p><p>In the ablation group, complete pulmonary vein isolation (PVI; defined as entrance block) was achieved in 87% of the cases. In addition to PVI, sets of ablation in other regions of the left atrium were performed in at least 21.3%. During the 2-year follow up period, 13.6% required an additional ablation and 9.09% received AAM therapy. Adverse events occurred in 9% of those in the RFA group; 6% experiencing pericardial effusion with tamponade.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>The results in the RAAFT-2 trial add to an increasing body of evidence showing potential benefits of ablation therapy as a primary treatment for paroxysmal atrial fibrillation in certain patients.<xref rid="bib2" ref-type="bibr"> ^2,3 </xref> </p><p>The study demonstrated a significantly decreased rate of recurrent atrial tachyarrhythmias in patients treated with radiofrequency ablation. Freedom from symptomatic AF was also lower in the RFA arm. However, the complication rate was unexpectedly high in the RFA group, given that the operators in the trial were highly skilled and the patient population was relatively healthy. Furthermore, although all patients were reported to have pAF, a large proportion (more than 21%) of patients underwent sets of ablation beyond pulmonary vein isolation; such ablation-sets are likely to have played a role, at least in part, in development of recurrent atrial tachyarrhythmias, and thereby potentially diluted the results of outcomes following ablation therapy.</p><p>The study's strengths include the frequent assessments by TTM and the multi-institutional, international patient cohort. Limitations include the small sample size and its bias towards young, healthy patients. The baseline characteristics of the study groups were not identical; there was a statistically significantly increased rate of electrical cardioversions in the AAM group.</p><p>When removing TTM, the significance of ablation over AAM disappeared, highlighting the importance of frequent ECG monitoring. The authors highlighted the difficulty in estimating the burden of AF, even with TTM. Studies such as RAAFT-2 remain limited without the use of implantable cardiac monitors to identify the incidence of asymptomatic AF more accurately.<xref rid="bib3" ref-type="bibr"> ³ </xref>.</p><p>In conclusion, according to this study, RFA appears to be modestly superior to AAM, reducing recurrence of symptomatic and asymptomatic atrial tachyarrhythmia in patients with pAF; ablation therapy does however carry risks and patients require careful counselling before embarking on ablation as first-line therapy for pAF.</p></sec>

STREAM and FAST-MI &#x02013; Pharmacoinvasive therapy: A continued role for fibrinolysis in the primary PCI era

<p>Data from the Strategic Reperfusion Early After Myocardial Infarction (STREAM) trial⁶ and 5-year results from the French Registry of Acute ST-Elevation and Non-ST-Elevation Myocardial Infarction (FAST-MI)⁷ are evaluated for further evidence on the effectiveness and safety of a pharmacoinvasive approach for patients presenting with acute ST-segment elevation myocardial infarction (STEMI).</p>

<contrib contrib-type="author"><name><surname>ElGuindy</surname><given-names>Ahmed M.</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>Primary percutaneous coronary intervention (PPCI) is currently the preferred reperfusion therapy for patients presenting with acute ST-segment elevation myocardial infarction (STEMI) when it can be performed by an experienced team in a timely fashion.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Current practice guidelines also recommend the transfer of patients presenting to non-PCI capable hospitals to hospitals offering PPCI services if the first medical contact (FMC)-to-device time is kept to less than 120 minutes. When this is not feasible, as is the case in many areas around the world, a pharmacoinvasive strategy consisting of early fibrinolysis followed by transfer to a PCI-capable hospital for either immediate (rescue) PCI for patients with failed thrombolysis, or for non-urgent coronary angiography to determine the need for additional revascularization within 3&#x02013;24 is a reasonable alternative.<xref rid="bib2" ref-type="bibr"> ^2,3 </xref> This approach should not to be confused with <italic>facilitated PCI</italic> where thrombolysis (full- or half-dose) is followed by immediate pre-planned PCI to mitigate the delay associated with PCI. The latter strategy, while being intuitively appealing, is not recommended owing to increased risk of adverse events including death, intracranial hemorrhage, and paradoxically, ischemic events (likely due to fibrinolysis-induced platelet activation).<xref rid="bib4" ref-type="bibr"> ^4,5 </xref> </p><p>Data from the Strategic Reperfusion Early After Myocardial Infarction (STREAM) trial<xref rid="bib6" ref-type="bibr"> ⁶ </xref> and 5-year results from the French Registry of Acute ST-Elevation and Non-ST-Elevation Myocardial Infarction (FAST-MI)<xref rid="bib7" ref-type="bibr"> ⁷ </xref> provide further evidence on the effectiveness and safety of a pharmacoinvasive approach.</p></sec><sec><title>Stream Trial</title><p>This open-label, multicenter, prospective, randomized trial was designed to test whether fibronlytic therapy &#x02013; administered before arrival to hospital, or early after admission &#x02013; coupled with early coronary angiography provides outcomes similar to PPCI in patients presenting with acute STEMI. Patients were eligible for enrollment if they presented within 3 hours from onset of symptoms, had evidence of acute STEMI on their initial electrocardiogram (ECG), and were unable to undergo primary PCI within one hour after the first medical contact (FMC). Over a period of 4 years, 1915 patients were enrolled from 99 sites in 15 countries. 1892 ultimately underwent randomization (81% in the ambulance setting) to either receiving tenecteplase along with antiplatelet and anticoagulant therapy, followed by coronary angiography within 6&#x02013;24 hours (pharmacoinvasive group) or to primary PCI (PPCI group). According to the investigator's judgment, urgent coronary angiography (and PCI when appropriate) in the pharmacoinvasive group was allowed at any stage in the presence of hemodynamic or electrical instability, worsening ischemia or sustained/progressive ST-segment elevation. The primary end-point was a composite of death from any cause, shock, congestive heart failure or reinfarction at 30 days. Safety end-points included ischemic stroke, intracranial and non-intracranial hemorrhage bleeding. Upon the advice of the data and safety monitoring board, the trial protocol was amended after 21% of the study population had been enrolled: the dose of tenecteplase was reduced by 50% in patients 75 years of age or older because of an excess rate of intracranial hemorrhage observed in that group.</p><p>At 30 days, the primary end-point occurred in 116 patients (12.4%) in the pharmacoinvasive group and 135 patients (14.3%) in the PPCI group (relative risk in the pharmacoinvasive group, 0.86; 95% CI, 0.68&#x02013;1.09; <italic>p</italic>&#x000a0;=&#x000a0;0.21). The incidence of the primary end-point in the prespecified subgroups (grouped by age, gender, weight, systolic blood pressure, infarct location, Killip class, TIMI risk score, and diabetes) was generally similar to the overall results. 36% of patients in the pharmacoinvasive group required &#x0201c;rescue&#x0201d; PCI. Significantly more open vessels were found during coronary angiography (before PCI) in the pharmacoinvasive group compared to the PPCI group (TIMI flow grade 0 in 16% vs. 59.3% respectively, <italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.001). Overall, 80% of patients in the pharmacoinvasive group and 90% in the PPCI group eventually underwent PCI (<italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.001). However, significantly more patients in the pharmacoinvasive group underwent coronary artery bypass surgery (4.7% vs. 2.1 %, <italic>p</italic>&#x000a0;=&#x000a0;0.002).<xref rid="bib6" ref-type="bibr"> ⁶ </xref> </p><p>An important evaluation of the rates of aborted myocardial infarction (prespecified secondary end-point) was recently published in a separate communication.<xref rid="bib8" ref-type="bibr"> ⁸ </xref> Aborted myocardial infarction was defined as ST-elevation resolution &#x02265;&#x000a0;50% (90 minutes post-initiation of tenecteplase in the pharmacoinvasive group or 30 minutes post-PCI in the PPCI group) with minimal biomarker rise; defined as CK-MB &#x02264;&#x000a0;2 times the upper limit of normal (ULN) or cardiac troponin T/I &#x02264;&#x000a0;5 times the ULN. Amongst the patients who fulfilled these criteria, those who developed new pathological Q-waves on their discharge ECGs were excluded. Overall, 99 patients (11.1%) in the pharmacoinvasive group had aborted MI compared to 59 patients (6.9%) in the PPCI group (<italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.01), a finding most likely driven by the significantly shorter time delay from onset of symptoms to start of reperfusion therapy in the formed group (100 minutes vs. 178 minutes for the pharmacoinvasive and PPCI groups respectively, <italic>p</italic>&#x000a0;&#x0003c;&#x000a0;0.001). The difference in aborted MI rates observed between both groups did not however have a significant interaction with the primary composite end-point.</p></sec><sec><title>Fast-mi Registry</title><p>The FAST-MI Registry was designed to evaluate the &#x0201c;real world&#x0201d; management of patients with acute MI, and to assess their in-hospital, medium- and long-term outcomes. Patients were recruited consecutively at the end of 2005 from 223 centers across France over a period of one month. Physicians participating in the study cared for their patients according to their usual practice, completely independent from the study.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> The investigators recently reported the 5-year survival rates of patients with STEMI who sought medical attention within 12 hours from the onset of symptoms.<xref rid="bib7" ref-type="bibr"> ⁷ </xref> Of the 1492 patients whose data was available, 447 (30%) received fibrinolysis (two thirds of whom had pre-hospital fibrinolysis), 583 (39%) were referred for PPCI, and 462 (31%) received no reperfusion therapy. Patients who did not receive reperfusion therapy were older, more likely to have history of cardiovascular disease and other comorbidities, as well as an overall higher risk profile. On the other hand, patients treated with fibrinolysis and those referred for PPCI had mostly similar risk profiles, including Global Registry of Acute Coronary Events (GRACE) score, but one important difference was significantly shorter time delays before seeking medical attention in the fibrinolysis group. The latter group of patients also received clopidogrel, low molecular weight heparin, or glycoprotein IIb/IIIa inhibitors less frequently than the group referred for PPCI. Among patients treated with fibrinolysis, 96% underwent subsequent coronary angiography (38% within 3 hours of fibrinolysis, 23% between 3 and 24 hours, and 39% beyond 24 hours), with most of them (84%) undergoing PCI. 32% of patients in the fibrinolysis group required urgent referral for &#x0201c;rescue&#x0201d; PCI.</p><p>Survival at 5 years was 88% in patients receiving fibrinolysis and 84% for those undergoing PPCI (HR, 0.73; CI, 0.50&#x02013;1.06; <italic>p</italic>&#x000a0;=&#x000a0;0.1). When the timing of administration of fibrinolysis was considered, prehospital fibrinolysis was associated with lower 5-year mortality (HR, 0.57; CI, 0.36&#x02013;0.88), while in-hospital fibrinolysis was associated with a trend toward increased 5-year mortality (HR, 1.19; CI, 0.72&#x02013;1.96) compared to PPCI. The investigators also studied the subgroup of patients who sought medical attention within 180 minutes from the onset of symptoms (STREAM-like population). 5-year survival in this population was 88% and 81% in the fibrinolysis and PPCI groups respectively (HR, 0.63; CI, 0.41&#x02013;0.98; <italic>p</italic>&#x000a0;=&#x000a0;0.039). However, in a propensity score-adjusted matched analysis, the benefit seen with prehospital fibrinolysis and with fibrinolysis (pre- or in-hospital) in the STREAM-like population did not remain statistically significant.</p></sec><sec><title>Discussion</title><p>In agreement with several recent studies<xref rid="bib10" ref-type="bibr"> ^{10&#x02013;13} </xref> as well as the current American and European practice guidelines,<xref rid="bib2" ref-type="bibr"> ^2,3 </xref> both STREAM and FAST-MI support the current recommendation of performing a coronary angiogram within 3&#x02013;24 hours after successful fibrinolysis when timely PPCI is unavailable. However, extrapolating these findings to other healthcare systems around the world should be done with caution for the following reasons:<list list-type="simple"><list-item><label>&#x02022;</label><p>STREAM randomized a very specific group of STEMI patients, namely those with a symptom onset-to-FMC of less than 3 hours. It is well recognized that the fibrinolytic agents are more effective early in the course of STEMI because of the absence of fibrin cross-linking in the fresh thrombus, and this effect progressively declines after the first 3 hours.<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref> Similarly, two-thirds of the FAST-MI patients receiving fibrinolysis did so prior to hospital admission. It remains unclear whether these finding are also applicable to late presenters.</p></list-item><list-item><label>&#x02022;</label><p>The fibrinolytic agent used in STREAM and in the majority of FAST-MI patients was tenecteplase (TNK) which has an extended half-life allowing for a single bolus administration. TNK is more fibrin-specific, is associated with less intracranial hemorrhage, and higher rates of infarct artery patency compared to streprokinase &#x02013; which remains the most frequently administered fibrinolytic agent worldwide.<xref rid="bib15" ref-type="bibr"> ¹⁵ </xref> It is unknown whether a pharmacoinvasive approach utilizing streptokinase as the fibrinolytic agent would yield similar results.</p></list-item><list-item><label>&#x02022;</label><p>The emergency medical system is France is very well established and often includes physicians. This has undoubtedly contributed not only to the high prehospital fibrinolysis rate (66% of patients), but also to the early initiation of treatment. As a result, PCI-related delay (defined as FMC-to-fibrinolysis time subtracted from FMC-to-PPCI time) was considerable (105 minutes compared to 78 minutes in STREAM) and might have contributed to the favorable outcomes observed in the fibrinolysis group. This setup and high rate of prehospital fibrinolysis is clearly difficult to reproduce in many countries/regions.</p></list-item></list> </p></sec><sec><title>What have we learned?</title><p>Timely PPCI remains the reperfusion strategy of choice in patients with acute STEMI. Findings from STREAM and FAST-MI lend further support to the adoption of a pharmacoinvasive strategy in areas where this cannot be achieved. In this setting, concerted efforts to improve emergency medical services is essential. Prehospital fibrinolysis should probably be considered in remote areas where transport time to a hospital is unacceptably long. Besides proper training of EMS personnel, this can be facilitated by wireless transmission of 12-lead ECGs to an offsite cardiologist, a practice which is currently adopted in many areas around the world.<xref rid="bib16" ref-type="bibr"> ¹⁶ </xref> Standardized inter-hospital transfer protocols should be established to allow for routine post-fibrinolysis coronary angiography (and PCI when appropriate) within the recommended time frame, as well as urgent rescue PCI for patients with failed thrombolysis. It is still unclear whether late presenters (&#x0003e;3 hours) and elderly patients derive a similar benefit from such approach.</p><p>Finally, while system-related delays have been the focus of numerous studies and scrutiny, which have resulted in remarkable improvements in emergency medical services response, transfer times, door-to-needle and/or door-to-device times;<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref> one should not forget that the ultimate objective in patients with acute STEMI is reducing the <italic>total ischemic time</italic> which also includes the time delay to FMC. The latter has received significantly less attention, which in part is related to difficulties in accurate measurement, given its susceptibility to recall bias and the fact that symptoms may be vague or intermittent in a considerable number of STEMI patients. It is worth noting that this patient-related delay &#x02013; on average &#x02013; constituted approximately 60% and 30% of the total ischemic time in STREAM's pharmacoinvasive and PPCI populations respectively, while one third of FAST-MI's population had a time-to-FMC of more than 120 minutes (which on its own exceeds the maximum allowed system-related delay). This delay is almost certainly longer in less developed regions/countries where emergency services and public awareness/education programs are not well-established. Further research and efforts aiming at effective reduction of patient-related delays (in addition to system-related delays) are urgently needed, and carry the potential of driving significant improvements in the short- and long-term outcomes of patients with acute STEMI.</p></sec>

Editors' page

Could not extract abstract

<contrib contrib-type="author"><collab>The Editors</collab><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<p>This issue of the Journal witnesses the introduction of a new feature, relating to in-depth analysis of mechanisms of disease. This section will include a series of commissioned articles by experts in the field. Each series will deal with different molecular mechanisms and clinical implications of specific signaling pathways contributing to an important disease process. The inaugural series deals with pulmonary arterial hypertension, starting with the Role of Endothelin Receptors, by Adrian Chester and colleagues, published in this issue, to be followed by the role of equally important pathways contributing to this important disease to be published in future issues. It is hoped that these series will contribute to our aim of presenting the latest knowledge regarding science and practice which will be of interest to a wide spectrum of our readership.</p>

The role of endothelin-1 in pulmonary arterial hypertension

<p>Pulmonary arterial hypertension (PAH) is a rare but debilitating disease, which if left untreated rapidly progresses to right ventricular failure and eventually death. In the quest to understand the pathogenesis of this disease differences in the profile, expression and action of vasoactive substances released by the endothelium have been identified in patients with PAH. Of these, endothelin-1 (ET-1) is of particular interest since it is known to be an extremely powerful vasoconstrictor and also involved in vascular remodelling. Identification of ET-1 as a target for pharmacological intervention has lead to the discovery of a number of compounds that can block the receptors via which ET-1 mediates its effects. This review sets out the evidence in support of a role for ET-1 in the onset and progression of the disease and reviews the data from the various clinical trials of ET-1 receptor antagonists for the treatment of PAH.</p>

<contrib contrib-type="author"><name><surname>Chester</surname><given-names>Adrian H.</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>Yacoub</surname><given-names>Magdi H.</given-names></name><xref ref-type="aff" rid="aff2"> ² </xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>The regulation of vascular tone in the pulmonary circulation is a complex and multifactorial process that involves the dispensability of the pulmonary vasculature, the function of the heart, concentration of oxygen in the blood and the capacity of the endothelium to release vasoactive substances. All these mechanisms combine to determine pulmonary vascular resistance and to ensure that the pulmonary circulation is maintained as a low pressure, high blood flow circuit. This prevents the passage of fluid into the interstitial space and allows the right ventricle to operate under optimal conditions. Changes in the pulmonary vascular resistance, which is defined as difference between mean pulmonary artery pressure and left atrial pressure, divided by the cardiac output, can lead to changes in the function of the lungs and eventually the right ventricle. Pulmonary arterial hypertension (PAH) is defined as a pulmonary artery pressure greater or equal to 25&#x000a0;mmHg at rest.<xref rid="bib1" ref-type="bibr"> ¹ </xref> The increased pressure in the lung has a knock-on effect on the right ventricle, leading to right ventricular hypertrophy and eventually right heart failure.</p><p>Symptoms of the condition include shortness of breath, fatigue, a non-productive cough, angina pectoris, syncope and peripheral oedema. While this is a rare condition affecting 15-50 people per million of the population, its incidence is associated with other morbidities such as HIV (0.5% of patients), systemic sclerosis (7&#x02013;12% of patients), sickle cell anaemia (2&#x02013;3.75% of patients) mixed connective tissue disease (10&#x02013;45% of patients) and systemic lupus erythematosus (1&#x02013;14% of patients).<xref rid="bib2" ref-type="bibr"> ^2&#x02013;9 </xref> Despite the apparent rareness of the condition, PAH has been classified by the World Health Organistaion (WHO) into 5 distinct categories based on the current understanding of the disease (<xref ref-type="table" rid="tbl1">Table 1</xref>).<xref rid="bib1" ref-type="bibr"> ¹ </xref> </p><p>Each of these different categories of PAH have a number of common pathological changes in the pulmonary circulation, which include vasoconstriction of the pulmonary vessels, remodelling of the vessel wall, plexiform lesions characterised by intimal and medial thickening by smooth muscle cells and endothelial cell proliferation, fibrotic changes in the vessel wall, thrombus formation and regions of neovascularisation (<xref ref-type="fig" rid="fig1">Figure 1</xref>).<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref> </p><p>In the absence of targeted therapies the prognosis of these patients is extremely poor. However with the development of therapies targeted on the pulmonary vasculature the survival of these patients has improved. However this benefit is not seen across all the patient groups, with those who suffer with connective tissue disease or scleroderma fairing much worse than those with an idiopathic cause.<xref rid="bib9" ref-type="bibr"> ⁹ </xref> </p><p>PAH is multifactorial disease and a number of different mechanisms have been proposed to contribute to its onset and progression. There are a number of risk factors associated with the disease which relate to the use of drugs such as aminorex, fenfluramine, dexfenfluramine, cocaine, phenylpropanolamine, St. John's Wort, chemotherapeutic agents, serotonin re-uptake inhibitors amphetamines, methamphetamines and L-tryptophan or exposure to chemicals such as toxic rapeseed oil.<xref rid="bib11" ref-type="bibr"> ¹¹ </xref> In addition, mutations in bonemorphogenic protein receptor 2, systemic sclerosis, HIV infection, portal hypertension, congenital heart disease with left-to-right shunts, recent acute pulmonary embolism and sickle cell disease are all conditions for which PAH is a risk factor.<xref rid="bib12" ref-type="bibr"> ¹² </xref> Although potential causative agents and other diseases associated with PAH represent an apparently diverse range of clinical conditions, there is general agreement that at the cellular level the disease is mediated by dysfunction of the endothelial cells that line the pulmonary vasculature.</p></sec><sec><title>Endothelial regulation of the pulmonary circulation</title><p>In common with all other surfaces in the body over which the blood flows, a continuous layer of endothelial cells covers the pulmonary circulation. While all these cells share the same phenotypic markers (expression of CD31 and/or von Willebrand factor) they cannot be considered as a homogeneous population of cells. Evidence exists that blood vessels of differing size and anatomical locations respond in specific ways, often determined by their different physiological roles.<xref rid="bib13" ref-type="bibr"> ^13,14 </xref> A common feature between endothelial cells is however, the ability to release a range of vasoactive molecules that interact with blood elements and the underlying vascular smooth muscle. These mediators include nitric oxide (NO), prostacyclin and endothelin-1 (ET-1).</p><p>These three mediators act to regulate the diameter of the pulmonary vessel by inducing either vasodilatation (NO and prostacyclin) or vasoconstriction (ET-1). In reality a balancing act exists whereby all three mediators may be present to maintain pulmonary vascular tone at an optimal level. Increases or decreases in the amounts of any one agent produced or changes to the receptors/signaling pathway they stimulate may therefore alter the balance towards vasodilation or vasoconstriction. The Hagen-Poiseuille law states that the resistance to flow in a tube is equal to the product of the length of the tube, the viscosity of the fluid, divided by &#x003c0; and the fourth power of the internal radius of the tube. Thus it can be seen that a small change in the radius of the vessel wall will have a significant change to the resistance to flow.<xref rid="bib15" ref-type="bibr"> ¹⁵ </xref> Under physiological conditions, such as exercise, this allows for changes in pulmonary vascular resistance due to dilation of pulmonary vessels as well as the recruitment of previously closed capillaries.</p><p>In PAH, the profile of endothelium-derived vasoactive factors is changed, with reduced production of vasodilator agents NO and prostacyclin.<xref rid="bib16" ref-type="bibr"> ^{16&#x02013;18} </xref> In addition to their action on vessel diameter, these agents also have an inhibitory effect on the regulation of smooth muscle cell proliferation and platelet activation.<xref rid="bib19" ref-type="bibr"> ¹⁹ </xref> Both prostacyclin and NO systems have therefore been the target of potential pharmacological interventions for the treatment of the disease. NO-releasing agents were shown to not be long-acting enough and also had the potential to stimulate a reflex tachycardia due to any effect on peripheral vessels. Agents that target the phosphodiesterase-5 enzyme, the predominant isoform of the phosphodiesterase enzymes that are responsible for the breakdown of cyclic guanosine monophosphate (cGMP), the second messenger for NO, have shown some encouraging long-term benefits.<xref rid="bib20" ref-type="bibr"> ^{20&#x02013;22} </xref> Agents that can directly activate cGMP have also been a focus of attention and are the subject of clinical trials that are currently in progress.<xref rid="bib23" ref-type="bibr"> ^23,24 </xref> Similarly, there are pharmacological agents that stimulate the IP receptors and mimic the effects of prostacyclin that have been used for the treatment of the disease. However, difficulties in the administration of these drugs (they need to be given by inhalation, subcutaneous injection or continuous intravenous infusion), their short half-life and their relative non-specific action at other receptors have limited their use and effectiveness in the treatment of PAH patients.</p><p>In contrast to trying to stimulate vasodilation in order to resolve PAH, targeting the vasoconstrictor peptide ET-1 is an alternative or additional strategy that has also been explored. This review will focus on the role of ET-1 in the lung, its biosynthesis, pharmacology, and the evidence for its participation in the pathogenesis of the disease. It will then look at the clinical evidence for efficacy of compounds that block the effects of ET-1 and are currently being used, or are in development, for the treatment of patients with PAH.</p></sec><sec><title>Endothelin-1</title><p>The endothelins are a family of 3 isopeptides that share a similarity in structure to the sarafotoxins, which are found in the venom of Israeli Mole Viper (Atractaspis engaddensis). Termed ET-1, ET-2 and ET-3 they are all 21 amino acid peptides with a high level of homology and similar structure<xref rid="bib25" ref-type="bibr"> ²⁵ </xref> (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The genes for ET-1, ET2, and ET-3 are all located on different chromosomes, with the gene for ET-1 being located on chromosome 6p. While principally found in endothelial cells, a range of other cells types have also been shown to express endothelins including cardiac myocytes, lung epithelium, glomerular kidney cells, mesangial cell, leukocytes and macrophages.<xref rid="bib26" ref-type="bibr"> ²⁶ </xref> ET-1 is the predominant endothelin isoform that is expressed in the cardiovascular system.<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> </p><sec><title>Biosynthesis</title><p>ET-1 is not stored in endothelial cells. Its release is dependent upon transcription of the gene, with the rate of transcription being responsive to stimulants and inhibitors to allow rapid changes in the amounts released. Transcription of the ET-1 gene is regulated by a number of factors including c-fos, c-jun, acute phase reactant regulatory elements and nuclear factor-1, AP-1 and GATA-2.<xref rid="bib28" ref-type="bibr"> ^{28&#x02013;30} </xref> The gene encodes for a larger 203 amino acid precursor peptide called preproendothelin. Preproendothelin is cleaved to a smaller 38 amino acid peptide, big-ET-1 by the enzyme furin convertase.<xref rid="bib31" ref-type="bibr"> ³¹ </xref> Mature ET-1 is then produced by the action of a further enzyme, endothelin-converting enzyme (ECE) to produce the active 21 amino acid peptide (<xref ref-type="fig" rid="fig3">Figure 3</xref>). ECE exists in 3 isoforms, with ECE-1 and 2 being responsible for the formation of ET-1. ECE-1 itself exists as four additional isoforms termed a, b, c and d.<xref rid="bib32" ref-type="bibr"> ³² </xref> </p><p>There are multiple factors that can affect the synthesis of ET-1 which include mechanical force (shear stress or pulsatile stretch), hypoxia, oxidised LDL cholesterol, low levels of estrogens, glucose, thrombin, other vasoconstrictors, growth factors, cytokines and adhesion molecules.<xref rid="bib33" ref-type="bibr"> ³³ </xref> In contrast, NO, prostacyclin atrial natriuretic peptides and estrogen can all reduce the amounts of ET-1 released. The release of ET-1 from endothelial cells appears to occur preferentially towards the underlying vascular smooth muscle, possibly due to stoichiometric binding of ET-1 to its receptors.<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> This may explain why only low levels of the peptide can be detected in the circulation, which can act as a guide to the amounts being released in certain conditions, but is not indicative to the concentrations present at the receptors in the vessel wall.</p></sec><sec><title>Endothelin receptors</title><p>The range of effects mediated by ET-1 is achieved via the activation of specific G-protein coupled cell surface receptors. There are two subtypes of ET-1 receptors that have been characterised (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Termed ET_A- and ET_B-receptors, these binding sites consist of single sub-units with a molecular mass in the region of 45-70&#x000a0;kDa and are recognised by ET-1 and when activated transduce the signal to intra-cellular signalling pathways that mediate the response of the cell.<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> However, recent evidence and proposed models of receptor signalling have suggested that the ET-receptor might exist as a heterodimer.<xref rid="bib36" ref-type="bibr"> ³⁶ </xref> Emerging concepts such as receptor cooperation and heterodimerisation are currently being investigated to explain how the dual effects of ET-1 are mediated by its receptors.<xref rid="bib37" ref-type="bibr"> ³⁷ </xref> </p><p>ET-receptors are found on vascular smooth muscle cells and myocytes, while ETB-receptors are also located on vascular smooth muscle cells and endothelial cells.<xref rid="bib33" ref-type="bibr"> ³³ </xref> The receptors on the vascular smooth muscle cells both mediate vasoconstrictor responses via the activation of phospholipase C, an increase in inostitol triphosphate and diacylglycerol and a subsequent increase in intra-cellular calcium, leading to contraction of the cell. In contrast, the mitogenic effects of the peptide are mediated by the stimulation of protein kinase C by diacylglycerol and calcium.<xref rid="bib38" ref-type="bibr"> ^38,39 </xref> Those ETB-receptors that are located on endothelial cells stimulate the release of nitric oxide and prostacyclin. This effect has a small influence on inducing relaxation of the vessel wall (<xref ref-type="fig" rid="fig5">Figure 5</xref>). Additional effects of ETB-receptors are linked to a reduction in ECE expression and inhibition of apoptosis.<xref rid="bib40" ref-type="bibr"> ^40,41 </xref> Endothelial ETB-receptors are also believed to be involved with the clearance of ET-1 from the circulation by internalising the receptor complex once ET-1 has bound. Due to the high surface area of the pulmonary vasculature the lung therefore acts to clear ET-1 from the circulation, with an estimated removal of 50% of the circulating ET-1 as the blood passes across the lung.<xref rid="bib42" ref-type="bibr"> ^42,43 </xref> This may explain why circulating levels of ET-1 are kept at very low levels (in the picomolar range) and why most of the ET-1 released by the endothelium is directed towards to the underlying smooth muscle cells. In addition to contraction of the vessel wall stimulation of ETA and ETB-receptors may also lead to activation of signalling pathways that mediate cell migration, proliferation, apoptosis or cell survival (<xref ref-type="fig" rid="fig6">Figure 6</xref>).</p></sec></sec><sec><title>Pulmonary effects of endothelin-1</title><p>ET-1 is able to affect numerous tissues and organs throughout the body. ET-1 is highly expressed in the lung, with levels of ET-1 mRNA being at least 5 times greater than in any other organ.<xref rid="bib44" ref-type="bibr"> ⁴⁴ </xref> In a similar manner to its actions in other vascular beds, ET-1 in the pulmonary circulation is able to produce an intense and protracted vasoconstriction of the pulmonary arteries and veins at very low concentrations, with its efficacy and potency being greater than 5-hydroxytryptamine, noradrenaline and the thromboxane A2 mimetic, U46619.<xref rid="bib45" ref-type="bibr"> ^45,46 </xref> In addition to its effects on pulmonary vascular tone, ET-1 also has a weak mitogenic effect on pulmonary vascular smooth muscle cells and to stimulate matrix production by the vessel wall. These effects are enhanced by the presence of other growth factors such as TGF-b1 and platelet-derived growth factor.<xref rid="bib26" ref-type="bibr"> ^26,47 </xref> ET-1 has also been shown to be able to stimulate the proliferation of pulmonary fibroblasts. In addition to these effects in the lung, ET-1 has been shown to be able to have a positive inotropic and chronotropic effect in the myocardium and to stimulate the production of cytokines, growth factors and matrix proteins in a variety of other tissues.<xref rid="bib26" ref-type="bibr"> ^26,33,48-52 </xref> </p></sec><sec><title>Role of endothelin-1 in pulmonary arterial hypertension</title><p>The abundance of ET-1 in the lung makes dysregulation of the ET system a prime candidate for involvement in the onset and progression of increased pulmonary vascular resistance (PVR) and pulmonary vascular remodelling. The muscular arteries seen in PAH and vascular endothelial cells have been shown to express greater levels of ET-1 and preproendothelin-1 compared to normal lungs.<xref rid="bib53" ref-type="bibr"> ⁵³ </xref> Expression of ET-1 is also evident in the plexiform lesions that are characteristic of the disease. The levels of expression of ET-1 correlated with the increased levels of PVR, as did the severity of the structural abnormalities found in distal pulmonary arteries (measured by intravascular ultrasound).<xref rid="bib53" ref-type="bibr"> ^53,54 </xref> In support of this apparent increased ability of the lung to release ET-1 is the observation that PAH patients have increased circulating levels of ET-1 and that there are increased levels of ET-1 exiting the lung compared to the levels that enter the lung. This effect is most likely due to a combination of increased production and reduced clearance.<xref rid="bib55" ref-type="bibr"> ⁵⁵ </xref> </p><p>Those patients who have conditions associated with PAH, such as connective tissue disease, congenital heart defects, pulmonary fibrosis (without connective tissue disease) with left-to-right shunts have elevated levels of plasma ET-1.<xref rid="bib56" ref-type="bibr"> ^{56&#x02013;59} </xref> However, some of these patient groups elevated levels of ET-1 occurred in the absence of PAH or did not correlate with haemodynamic changes.<xref rid="bib56" ref-type="bibr"> ^56,60 </xref> </p><p>ET-1 also interacts with ligands at the bone morphogenetic protein receptor-2 (BMPR2). Mutations in BMPR2 have been linked to the familial form of the disease, with inactivating heterozygous mutations, including frameshifts, nonsense and missense mutations, and deletions that could truncate the protein or alter conserved regions, interfering with ligand binding to the BMPR2 or kinase activity.<xref rid="bib61" ref-type="bibr"> ^{61&#x02013;64} </xref> The BMPR2 ligand, BMP7, and in part BMP4, were shown to regulate the balance between vasoconstrictor and vasodilator mechanisms via their ability to suppress ET-1 release from smooth muscle cells and inhibit the contractile response of the vascular wall to the peptide. Over-expression of BMPR2 in rats has been shown to protect against the development of PAH in response to hypoxia. Changes in the function of BMPR2 could either directly or indirectly influence the response of different BMPs and thereby the release of and response to ET-1.</p><p>This body of evidence identifies the ET-1 system as a possible pharmacological target for the management of patients with PAH. At the forefront of this effort is the quest to identify ET-1 receptor antagonists that have the required potency and efficacy to be effective in patients with PAH.</p></sec><sec><title>Endothelin receptor antagonists</title><p>The profile of ET receptors in the pulmonary vasculature presents a dilemma for devising the best strategy for pharmacological modulation of the effects of ET-1. The effects mediated by ETB-receptors on the endothelium and smooth muscle cells have opposing actions. Those of the smooth muscle, along with the ETA-receptors, contribute to the contractile and remodeling effects of the peptide, which would be advantageous to block in patients with PAH. However, the ETB-receptors on the endothelial cells mediate potentially beneficial effects, namely the release of nitric oxide and prostacyclin and possible removal of endothelin from the circulation.<xref rid="bib40" ref-type="bibr"> ^40,42 </xref> The efficacy of compounds designed non-selectively to block all ET-receptors would therefore be limited by the fact they would block endothelial ETB-receptors. Conversely, a selective ETA-receptor antagonist would leave the ETB-receptors on the smooth muscle cells functional and therefore not block all the contractile/remodelling effects of ET-1 on the pulmonary vessel wall. In practice, the success of drug discovery programmes is governed by the ability to identify compounds with selectivity for either of the two receptors. There are currently two ET-receptor antagonists that are in clinical use, Bosentan and Imbrisentan, while drugs like Sitaxsentan, which initially showed favourable results have now been withdrawn due to issues relating to hepatic toxicity. Macitentan is currently in phase III clinical trials (<xref ref-type="fig" rid="fig7">Figure 7</xref>).</p><sec><title>Bosentan</title><p>Bosentan (Tracleer&#x000ae;) is a mixed ETA/ETB- receptor antagonist and was the first ET-receptor antagonist to be used clinically. It has a higher affinity for ETA-receptors compared to that for ETB-receptors. Bosentan has a half-life of approximately 7 hours and a 50% bioavailability.<xref rid="bib65" ref-type="bibr"> ⁶⁵ </xref> Therapy is accompanied with routine liver function tests. It is metabolised by the CYP2C9 and 3A4 isoenzymes of cytochromeP450 and may therefore interact with drugs such as warfarin and digoxin, although their use together in not contraindicated, closer monitoring is recommended.<xref rid="bib4" ref-type="bibr"> ^{4,66&#x02013;68} </xref> </p><p>The first clinical trial with bosentan contained 32 patients treated for 12 weeks, showed in patients with idiopathic PAH or scleroderma-associated PAH to improve performance in the 6-minute walk test by 70&#x000a0;m, improve the cardiac index and reduce the PVR after 8 weeks of treatment.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> Just under half the patients (49%) improved their NYHA function from class III to class II, while the remaining 51% stayed at class III. This was then followed by the BREATHE-1 (Bosentan Randomised trail of Endothelin Antagonist Therapy) study, which studied effects for 16 weeks in 213 patients (69 in the placebo group and 144 in the bosentan group) with idiopathic PAH or connective tissue-associated PAH and was able to demonstrate a 44 minute improvement in the six-minute walking distance, the Borg dyspnea index and WHO functional class. Patients also saw an increase in the time to clinical worsening.<xref rid="bib66" ref-type="bibr"> ⁶⁶ </xref> The BREATH-1 study also saw 9% of the patients exhibit liver toxicity, which was associated with the higher dose of the drug (250&#x000a0;mgs compared to 125&#x000a0;mgs). The BREATHE-2 trial studied the effects of bosentan (62.5&#x000a0;mgs b.i.d for 4 weeks followed by 125&#x000a0;mgs b.i.d for the next 12 weeks) in combination with intravenous therapy with epoprostenol (2&#x000a0;ng/kg/min starting dose, titrated up to a maximum dose of 12 to 16&#x000a0;ng/kg/min for up to 16 weeks) in 33 patients (11 in the placebo groups and 22 in the treatment group) with either idiopathic PAH or connective tissue-associated PAH. While improvements were seen in haemodynamics, exercise capacity and functional class in both groups at week 16, the combination of treatment with the two drugs showed no additional significant effect.<xref rid="bib68" ref-type="bibr"> ⁶⁸ </xref> The BREATHE-3 study provided safety and efficacy data for bosentan in children with PAH treated with or without concomitant prostanoid therapy.<xref rid="bib69" ref-type="bibr"> ⁶⁹ </xref> Bosentan at a target dose of between 31.25&#x02013;125&#x000a0;mg twice daily was well tolerated and gave a reduction in mean pulmonary artery pressure of 8.0&#x000a0;mm Hg and a reduction in PVR of 300&#x000a0;dyne.s.m2/cm5. The study concluded that bosentan had a similar pharmacokinetic profile in paediatric patients with PAH as it did in adults with the disease. The BREATHE-4 and BREATHE-5 trials went on to examine the effect of bosantan in patients whose PAH is related to their infection with the human immunodeficiency virus or patients who had Eisenmenger's syndrome (PAH associated with a congenital heart defect).<xref rid="bib70" ref-type="bibr"> ^70,71 </xref> The BREATHE-4 trial showed an improvement in exercise capacity, WHO functional class, quality of life and cardiopulmonary haemodynamics, while in the BREATH-5 trial, which contained 54 patients (17 in the placebo group and 37 in the bosentan group), bosentan decreased pulmonary vascular resistance and improved exercise capacity.</p><p>Trials in addition to the BREATHE series of studies have also been carried out with bosentan. These have compared the effects of bosentan with the phosphodiesterase-5 inhibitor sildenafil (SERAPH trial, which included idiopathic PAH and connective tissue disease- associated PAH patients) and the selective ETA-receptor antagonist sitaxentan (STRIDE-2 trial, which included idiopathic PAH, connective tissue disease- associated PAH patients and congenital heart disease-associated PAH patients).<xref rid="bib72" ref-type="bibr"> ^72,73 </xref> These trials show that while sildenafil and sitaxsentan both show improvements with a range of clinical parameters, there was no significant difference between their effects and those of bosentan.</p><p>The major limitation of the use of bosentan is the incidence of hepatatic toxicity. In the BREATHE-1 trial there was a 14% incidence in the elevation of alanine aminotransferase and aspartate aminotransferase with the higher (250&#x000a0;mg) dose used.<xref rid="bib66" ref-type="bibr"> ⁶⁶ </xref> It is now recommended that with clinical use of the drug, liver enzymes should be monitored on a monthly basis. Indeed, there has been a reported case of a patient developing cirrhosis of the liver after taking bosentan.<xref rid="bib74" ref-type="bibr"> ⁷⁴ </xref> Other side effects also include a reduction in haemoglobin levels immediately after commencement of therapy, a drop in blood pressure with the intravenous preparation (but not the oral therapy) and peripheral oedema.<xref rid="bib4" ref-type="bibr"> ^{4,61,66,68,71,75&#x02013;77} </xref> </p><p>While the experience of using bosentan is greater than any other ET-receptor antagonist, the profile of adverse side effects is greater than that with other therapies. Thus, as experience grows we will be in a better position to determine which patients groups derived the maximum benefit from the drug and to what extent the side effects of bosentan limit the clinical benefit that can be derived from the drug.<xref rid="bib78" ref-type="bibr"> ⁷⁸ </xref> </p></sec><sec><title>Imbrisentan</title><p>Imbrisentan (Letairis&#x000ae;, Volibris&#x000ae;) is an ET-receptor antagonist that preferentially blocks the ETA-receptor. It has &#x0003e;4000 times greater affinity at the ETA-receptor compared to that at the ETB-receptor.<xref rid="bib79" ref-type="bibr"> ⁷⁹ </xref> Imbrisentan has a half life in the region of 15 hours, allowing daily dosing to be used.<xref rid="bib80" ref-type="bibr"> ⁸⁰ </xref> Unlike bosentan, it is tolerated by the liver, being metabolised via glucuronidation and it has no interaction with warfarin.</p><p>The clinical trials with imbrisentan have shown it to be effective in the treatment of patients with PAH.<xref rid="bib81" ref-type="bibr"> ^81,82 </xref> The first trial to demonstrate the effect of imbrisentan was conducted on patients with idiopathic PAH or PAH associated with collagen vascular disease, anorexigen use or human immunodeficiency virus infection (HIV). The study was able to show improvements in the 6-minute walk, Borg dyspnea index and WHO functional class test for a concentration range of 1&#x02013;10&#x000a0;mg for 12 weeks. These clinical benefits were associated with a reduction in mean pulmonary artery pressure of 5&#x000a0;mmHg and increase in cardiac index of 0.33/min/m2.<xref rid="bib82" ref-type="bibr"> ⁸² </xref> This study was followed by the ARIES series trials. The ARIES-1 study, which contained 201 patients (67 in the placebo group and 67 who received either 5&#x000a0;mg or 10&#x000a0;mg of the drug for 12 weeks) studies 2 doses of imbrisentan (5&#x000a0;mg and 10&#x000a0;mg) in patients with idiopathic PAH, connective tissue disease-associated PAH and a small number of patients with HIV and anorexigenic drug use-associated PAH. The ARIES-2 study was performed over the same time period as the ARIES-1 study and contained 192 patients (65 in the placebo group, 64 who received 2.5&#x000a0;mg and 63 who received 5&#x000a0;mg of the drug). The causes of the PAH were similar to those in the ARIES-1 study.</p><p>All treatment groups in the ARIES studies improved their 6-minute walk test by 31&#x000a0;m and 51&#x000a0;m for 5&#x000a0;mg and 10&#x000a0;mg respectively in the ARIES-1 study and 32&#x000a0;m and 59&#x000a0;m for the 2.5&#x000a0;mg and 5&#x000a0;mg respectively in the ARIES-2 study. Improvements in Borg dyspnoea score and BNP levels were seen in both trials, while NYHA functional class improved in ARIES-1, quality of life improvement and a delay in clinical worsening were seen in ARIES-2. As with other ET-receptor antagonists, peripheral oedema was also seen with imbrisentan, but to a greater degree than with bosentan.<xref rid="bib83" ref-type="bibr"> ⁸³ </xref> </p></sec><sec><title>Sitaxsentan</title><p>Sitaxsentan (Thelin) is a highly selective ETA-receptor antagonist with up to 6500 times greater affinity for the ETA-receptor compared to that for ETB-receptors. Like imbrisentan, it has a long half-life (between 5-7 hours). However its interaction with Cytochrome P450, it inhibits CYP2C9, lead to an interaction with drugs such as warfarin. This has been shown to lead up to a 80% reduction in the dose of warfarin needed to maintain the desired INR.</p><p>The STRIDE studies have investigated the efficacy of sitaxsentan in the treatment of PAH. The STRIDE-1 included 178 patients and the study involved giving patients with idiopathic PAH and PAH associated with connective tissue disease or congenital heart disease 100&#x000a0;mg or 300&#x000a0;mg daily for 12 weeks. Both doses of sitaxsentan improved the 6-minute walk distance, improved the NYHA functional class, cardiac index and PVR at each dose used. However there was not significant change in the peak VO₂ of the patients.<xref rid="bib73" ref-type="bibr"> ^73,84 </xref> The STRIDE-2 trial went on to compare 50&#x000a0;mg and 100&#x000a0;mg of sitaxsentan to placebo and patients receiving bosentan over an 18-week period. 247 patients with a similar profile of causes of PAH as studied in the STRIDE-1 trial. As before, sitaxsentan improved the 6-minute walk distance to a degree comparable with the bosentan group. However, there was not sufficient power in the study to make a direct comparison between the two drugs. Sitaxsentan also elevated hepatic transaminase (levels to over 3 times the normal range) in 3-5% of the patients. There was a similar increase in 6% of the placebo group and 11% of patients who were receiving bosentan. This was in contrast to the STRIDE-1 study where 3% and 10% of patients increased their transaminase levels after taking 100&#x000a0;mg or 300&#x000a0;mg respectively. The study concluded that 100&#x000a0;mg daily was the optimal dose of sitaxsentan for the treatment of PAH.</p><p>In an extension to the STRIDE-2 trial, the STRIDE-2X trial followed the patients for 52 weeks.<xref rid="bib85" ref-type="bibr"> ⁸⁵ </xref> Patients receiving sitaxsentan at 100&#x000a0;mg had 96% overall survival and a 34% risk for a clinical worsening and a 15% risk of discontinuation due to adverse events after a year of treatment. Bosentan in comparison had an 88% overall survival, a 40% risk of a clinical worsening and 30% risk of discontinuation due to adverse events. The risk of elevated aminotransferase and/or alanine aminotransferase was 6% of the sitaxsentan group and 14% for the bosentan group. It has been suggested that based on the two STRIDE-2 trials the use of a highly selective ETA-receptor antagonist such as sitaxsentan is advantageous compared to a non-selective ETA/B receptor antagonist such as bosentan.<xref rid="bib86" ref-type="bibr"> ⁸⁶ </xref> </p><p>There have been a number of other STRIDE trials. The STRIDE-6 trial compared the effectiveness of sitaxsentan in a group of patients who had to discontinue treatment with bosentan due to a lack of efficacy or skin or liver problems.<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> The 6-minute walk test improved by 15% in 5 out of 15 patients taking 100&#x000a0;mg of sitaxsentan. This was a small study involving only 48 patients and lacked a placebo group. Other STRIDE trials, or long-term extension to trials include the STRIDE-4, STRIDE-1X, STRIDE-3, STRU and STRIDE-X trials.<xref rid="bib88" ref-type="bibr"> ⁸⁸ </xref> </p><p>Despite approval for marketing sitaxsentan in Europe, Canada and Australia in 2006, the FDA refused approval due to concerns about the efficacy of the drug in the STRIDE-2 trial. The STRIDE-5 trial was planned to address these issues, as were a series of trials in the USA, designed to compare the efficacy and safety of sitaxsentan with sildenafil (Clinicaltrials.gov identifiers: NCT00795639, NCT00796666 and NCT00796510). However, in 2010 the manufacturer, Pfizer, due to 2 cases of idiosyncratic, fatal hepatic failure associated with sitaxsentan use, terminated these trials.<xref rid="bib89" ref-type="bibr"> ⁸⁹ </xref> As a consequence the drug was voluntarily withdrawn from worldwide use on the 10th December 2010.</p></sec><sec><title>Macitentan</title><p>Macitentan (Opsumit) is a mixed ETA/ETB receptor antagonist that was developed by modifying the structure of bosentan to make sulphonamide derivatives.<xref rid="bib90" ref-type="bibr"> ⁹⁰ </xref> The tert-butyl benzene sulfonamide part of the initial compound was then replaced by a series of simple alkyl sulfamide moieties (<xref ref-type="fig" rid="fig8">Figure 8</xref>). This yielded a series of compounds with differing efficacy at the ETA and ETB receptor. One of these compounds, macitentan, emerged has having improved tissue penetration and high affinity for both receptors.<xref rid="bib91" ref-type="bibr"> ⁹¹ </xref> Doses required to achieve the similar effect of bosentan were an order of magnitude lower for macitentan. The efficacy of the drug is contributed to by the formation of its metabolite ACT-132577, formed by oxidative depropylation, which also has antagonistic activity at ETA and ETB receptors.<xref rid="bib91" ref-type="bibr"> ⁹¹ </xref> Macitentan is slowly absorbed and has a half-life of between 6&#x02013;8.5 and 14&#x02013;18.5 hours depending on dose. In contrast, ACT-132577 has a half-life of approximately 48 hours, indicating that daily dosing is applicable. There was no effect of macitentan on bile salts indicating no detrimental effect on hepatic function.<xref rid="bib92" ref-type="bibr"> ^92,93 </xref> </p><p>The clinical benefit of macitentan was recently demonstrated in the SERAPHIN trial that involved 742 patients. The placebo group of 250 patients were compared to 250 patients who received 3&#x000a0;mg daily and 242 patients received 10&#x000a0;mg daily of the drug. Patients with either idiopathic or heritable PAH or PAH associated with connective-tissue disease, congenital left-to-right shunts, HIV infection, or drug/toxin use/exposure participated in the trial. Macitentan significantly improved the morbidity and mortality of patients with PAH irrespective of whether they had previously received treatment for the disease or not. Improvements in the 6-minute walk test, WHO functional class and reductions of PVR were seen for both concentrations of macitentan. A number of patients withdrew from the trial due to adverse effects that included worsening of PAH, upper respiratory tract infection, peripheral oedema and right ventricular failure. Compared to patients in the placebo group, higher percentages of patients in the two macitentan groups had nasopharyngitis, headache and anaemia. There was no significant incidence in elevations of liver enzymes in any of the three groups. The SERAPHIN trial is discussed in greater detail by Karim Said elsewhere in this issue.</p><p>Macitentan is now approved for use in the treatment of PAH in the USA and Canada and it has received a positive opinion from regulatory authorities in Europe.</p></sec></sec><sec><title>Combination therapy with ET-receptor antagonists</title><p>In addition to ET-1 receptor blockade, there are a number of other established and new therapies used for the treatment of PAH. These include prostacyclin analogues (epoprostenol, treprostinil, iloprost, beraprost), phosphodiesterase-5 (PDE-5) inhibitors (sildenafil, tadalafil, vardenafil) and more recently activators of cGMP (Riociguat).<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> </p><p>There have been several small studies that have specifically examined the benefits of combinations of some of these agents. These studies have shown that combining bosentan with sildenafil is safe and effective in patients with PAH and that the beneficial effects of sildenafil are maintained despite the reduced bioavailability of the PDE-5 inhibitor caused by bosentan.<xref rid="bib95" ref-type="bibr"> ^95,96 </xref> Combining bosentan with prostacyclin analogues was also shown to be safe and effective, with additional improvements seen with bosentan when added to poprostenol or treprostinil therapy.<xref rid="bib97" ref-type="bibr"> ^97,98 </xref> One case report showed recovery over a 6-month period of a woman suffering from progressive right heart failure and severe PAH after treatment was commenced with a combination of bosentan, tadalafil, and beraprost.<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> However, more recently the FREEDOM-C trial, which contained 350 patients studied over a 16-week period, compared the addition of oral treprostinil to patients that were stable on background ET-receptor antagonist and/or a PDE-5 inhibitor. Addition of treprostinil therapy gave no additional benefit in the 6-minute walk test or Borg dyspnea score.<xref rid="bib100" ref-type="bibr"> ¹⁰⁰ </xref> The FREEDOM-C study used the same profile of combination therapy as reported for the FREEDOM-C trial, but used a differing dosing regime for treprostinil since it had been shown that the original dosing regime for triprostinil to be sub-optimal.<xref rid="bib101" ref-type="bibr"> ¹⁰¹ </xref> While there was no change in the profile of adverse events, the combination therapy failed to yield any further additional benefits.<xref rid="bib100" ref-type="bibr"> ¹⁰⁰ </xref> It was suggested that this may have been due to the relatively short duration of the study (16 weeks) and that longer-term studies are warranted.</p></sec><sec><title>Future directions</title><p>The data from the clinical trials with ET-receptor antagonists and clinical practice has shown that blocking the effects of ET-1 are beneficial in the treatment of patients with PAH. While the effects of highly selective ETA-receptor antagonists, such as sitaxsentan, are limited by hepatic toxicity, there appears to be no obvious advantage in selectively blocking one receptor subtype compared to the non-selective actions of drugs like bosentan and macitentan. The structure/activity relationship studies made during the development of macitentan represent the most effective way forward in the development of additional compounds with high affinity at ET receptors and potentially and less deleterious side effects.<xref rid="bib90" ref-type="bibr"> ⁹⁰ </xref> </p><p>In identifying the ET system as a therapeutic target in PAH, attempts would be made to assess the efficacy of other targets of pharmacological intervention. In a similar manner to the way angiotensin receptor blockers and angiotensin converting enzyme inhibitors are used to modulate the actions of angiotensin II, inhibitors of ECE represent potential pharmacological tools to limit the effects of ET-1. Compounds such as SLV-306 (daglutril), which inhibit ECE and neutral endopeptidase, has been shown to reduce circulating ET-1 levels in healthy volunteers, reduce systolic blood pressure and reduce pulmonary and right atrial pressures in patients with congestive heart failure.<xref rid="bib102" ref-type="bibr"> ^102,103 </xref> However this latter study failed to show a true dose-response relationship for the drug. As newer ECE inhibitors are developed, time will tell if this represents a viable pharmacological strategy for the treatment of PAH that will rival existing drugs.</p></sec>

MicroRNAs in heart failure: Small molecules with major impact

<p>MicroRNAs (miRNAs) have emerged as potent modulators of mammalian gene expression, thereby broadening the spectrum of molecular mechanisms orchestrating human physiological and pathological cellular functions. Growing evidence suggests that these small non-coding RNA molecules are pivotal regulators of cardiovascular development and disease. Importantly, multiple miRNAs have been specifically implicated in the onset and progression of heart failure, thus providing a new platform for battling this multi-faceted disease. This review introduces the basic concepts of miRNA biology, describes representative examples of miRNAs associated with multiple aspects of HF pathogenesis, and explores the prognostic, diagnostic and therapeutic potential of miRNAs in the cardiology clinic.</p>

<contrib contrib-type="author"><name><surname>Kalozoumi</surname><given-names>Georgia</given-names></name><xref ref-type="aff" rid="aff1"> ¹ </xref></contrib><contrib contrib-type="author"><name><surname>Yacoub</surname><given-names>Magdi</given-names></name><xref ref-type="aff" rid="aff2 aff3"> ^2,3 </xref></contrib><contrib contrib-type="author"><name><surname>Sanoudou</surname><given-names>Despina</given-names></name><xref ref-type="aff" rid="aff1 aff4"> ^1,4,*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec sec-type="intro"><title>Introduction</title><p>Heart failure (HF) is a complex clinical syndrome that may arise as a consequence of a spectrum of structural and functional cardiac abnormalities, as well as rhythm and conduction disorders.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Symptomatic HF has an incidence of 10/1000 for individuals aged over 65, whilst at the age of 40 the life time risk of developing HF is approximately and 1 in 5.<xref rid="bib2" ref-type="bibr"> ² </xref> HF can be both disabling and fatal. Although survival after diagnosis has improved over time, studies show that roughly 50% of diagnosed HF patients will die within 5 years.<xref rid="bib3" ref-type="bibr"> ^3&#x02013;5 </xref> Systolic dysfunction is considered the most common underlying pathology of HF, and it may occur as a result of ischemic cardiac disease (coronary artery disease, CAD, myocardial infarction, MI) and/or hypertension, idiopathic dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and valve lesions.<xref rid="bib1" ref-type="bibr"> ¹ </xref> Extensive molecular studies have revealed multiple genetic and epigenetic factors implicated in the aforementioned primary pathologies.<xref rid="bib6" ref-type="bibr"> ^6&#x02013;10 </xref> MiRNAs are now emerging as important regulators of gene expression that act to modulate numerous biological properties, under normal and pathological conditions. As such, their role in the cardiovascular system and HF in specific is being thoroughly investigated, with direct implications in our understanding of the cardiac molecular physiology/pathophysiology and future molecular therapeutic targeting of HF.</p></sec><sec><title>miRNAs: Important post-transcriptional regulators of gene expression</title><p>MiRNAs are a group of small (18-25 nucleotide-long), non-coding (i.e. not translated to proteins) RNA molecules that have the ability to bind mature mRNA molecules and affect their translation, thus serving as important post-transcriptional modulators of gene expression. MiRNAs are produced through an elaborate molecular mechanism. Initially, the corresponding DNA region (intergenic, intronic or polycistronic) is transcribed to produce hairpin-shaped primary transcripts called pri-miRNAs.<xref rid="bib11" ref-type="bibr"> ^11,12 </xref> Pri-miRNAs are appropriately processed by the microprocessor complex (Dorsha nuclease and Pasha protein) inside the nucleus, to generate 70 nucleotide-long miRNAs called pre-miRNAs.<xref rid="bib12" ref-type="bibr"> ^12,13 </xref> Pre-miRNAs are in turn transported to the cytoplasm by exportin 5, where they are cleaved by the Dicer protein to form mature double-stranded miRNA molecules.<xref rid="bib14" ref-type="bibr"> ^14,15 </xref> These double-stranded molecules are then cut into two single stranded miRNAs, and one of them is selected by the argonaute protein to serve as the &#x0201c;active&#x0201d; one. The chosen single stranded miRNA is then embodied in an active RNA-induced silencing complex (RISC), containing Dicer and many associated proteins, which is also known as a microRNA ribonucleoprotein complex (miRNP). The remaining single stranded miRNA is decomposed (<xref ref-type="fig" rid="fig1">Figure 1</xref>).<xref rid="bib16" ref-type="bibr"> ^{16&#x02013;19} </xref> </p><p>Each of the miRNP complexes targets specific (one or more) mRNAs, dictated by their 3'-UTR (mRNA untranslated region) base-pair complementarity. Once an miRNA binds an mRNA molecule, it leads to suppression of its translation to protein via two distinct routes, depending on the extent of the miRNA-mRNA complementarity.<xref rid="bib20" ref-type="bibr"> ^20,21 </xref> In the case of perfect or near-perfect base-pairing the target mRNA is destroyed, whereas imperfect binding is more likely to result in reduced synthesis of the corresponding protein, with minimum effect on the mRNA levels.<xref rid="bib20" ref-type="bibr"> ^{20&#x02013;22} </xref> Importantly, a single miRNA may regulate the expression of hundreds of genes, and an mRNA may be targeted by multiple miRNAs.<xref rid="bib23" ref-type="bibr"> ^23,24 </xref> Independently of the mechanism and the extent of mRNA degradation and/or translation repression, the overall outcome is post-transcriptional gene silencing (PTGS).</p><p>The scientific evidence available to date suggest that the human genome encodes over a thousand human miRNAs, targeting over 60% of the mammalian genes and more than one third of human protein-coding genes.<xref ref-type="fn" rid="FN0001"> ¹ </xref>,<xref ref-type="fn" rid="FN0002"> ² </xref> ^, <xref rid="bib23" ref-type="bibr"> ^23,25,26 </xref> Thus, it comes as no surprise that miRNAs emerge as regulators of numerous physiological functions and have been also implicated in a broad spectrum of human disorders.</p><p>The key biological functions affected by miRNAs include cell growth, apoptosis, cell- and tissue- specific differentiation and development,<xref rid="bib27" ref-type="bibr"> ²⁷ </xref> whilst dysregulation in miRNA synthesis and function underlies pathological conditions that affect the majority of human tissues.<xref ref-type="fn" rid="FN0003"> ³ </xref> In cardiology, the latest advances in miRNA research techniques have allowed the high-throughput, genome-wide screening of miRNA expression as well as the prediction of new miRNA-mRNA interactions, thus unveiling the multidimensional role of miRNAs in cardiac development, function and disease (reviewed in <xref rid="bib28" ref-type="bibr"> ^{28&#x02013;33,185} </xref>).</p><p>Herein, the latest advances in heart failure (HF) miRNA research are reviewed, starting with the role of miRNAs in normal cardiac development, in HF pathogenesis, and proceeding with their emerging value in early and improved diagnosis and prognosis, as well as the development of new therapeutic approaches.</p></sec><sec><title>miRNAs play a central role in cardiac development</title><sec><title>Discoveries from animal models</title><sec><title>Aberrations in miRNA biosynthesis lead to abnormal cardiovascular development and embryonic death</title><p>Several lines of evidence have indicated an essential role of miRNAs in heart development. Firstly, a series of studies investigating the impact of mutations in the miRNA processing enzyme Dicer have shown that Dicer activity is required for normal cardiovascular development of the embryo. In particular, loss of Dicer in mice resulted in embryonic lethality at embryonic day 7.5,<xref rid="bib34" ref-type="bibr"> ³⁴ </xref> whilst in zebrafish embryos developmental arrest occurred at day 10.<xref rid="bib35" ref-type="bibr"> ³⁵ </xref> In mice, deletion of the first two exons and hypomorphic expression of Dicer have been related to impaired angiogenesis,<xref rid="bib36" ref-type="bibr"> ^36,37 </xref> and neural crest cell-specific deletion of Dicer led to a spectrum of cardiovascular abnormalities resembling congenital heart syndromes (i.e. Type B Interrupted Aortic Arch, IAA-B, Double Outlet Right Ventricle, DORV, Ventricular Septal Defect, VSD).<xref rid="bib38" ref-type="bibr"> ³⁸ </xref> Zebrafish embryos devoid of Dicer function presented with a tubular heart and pericardial edema, lacking the formation of the two chambers, characteristic of the wild-type heart.<xref rid="bib39" ref-type="bibr"> ³⁹ </xref> Moreover, another group reported excessive endocardial cushion formation (impaired heart septation) in mutant Dicer zebrafish embryos, amongst developmental defects in other tissues.<xref rid="bib40" ref-type="bibr"> ⁴⁰ </xref> </p><p>The role of mature miRNAs in the developing heart was further elucidated through cardiac-specific deletion of Dicer in mice. In specific, conditional ablation of Dicer after the initial commitment of cardiac progenitors (from embryonic day 8.5), during heart patterning and differentiation, led to heart failure and embryonic lethality (embryonic day 12.5).<xref rid="bib41" ref-type="bibr"> ⁴¹ </xref> The observed developmental defects included DORV with a concurrent ventricular septal defect, implying an essential role for Dicer in proper chamber septation and cardiac outflow tract alignment. A critical role for Dicer has also been proposed in murine epicardial cell development, and their consequent differentiation into coronary smooth muscle cells. Specifically, when Dicer was deleted from the epicardium of normal mice, neonates presented with severe cardiac defects including impaired coronary vessel development, and experienced early death.<xref rid="bib42" ref-type="bibr"> ⁴² </xref> </p><p>The role of Dicer has also been investigated during the course of postnatal heart development. In specific, conditional Dicer loss in the postnatal myocardium of 3-week-old mice led to premature death within 1 week, with the main histopathological findings including mild ventricular remodeling and dramatic atrial enlargement.<xref rid="bib43" ref-type="bibr"> ⁴³ </xref> The observed cardiac hypertrophy was accompanied by the reactivation of the fetal cardiac gene program. The targeted deletion of Dicer in adult mouse myocardium has also uncovered a critical role for miRNAs in maintaining adult splicing programs, via modulating the expression of alternative splicing regulators.<xref rid="bib44" ref-type="bibr"> ⁴⁴ </xref> The combined findings from these studies on Dicer deletion suggest that mature miRNAs are essential for the successful transition from the prenatal cardiac gene program to the adult expression profile.</p></sec><sec><title>Multiple miRNAs implicated in different aspects of cardiac development</title><p>To date, a wide range of miRNAs has been specifically implicated in different aspects of cardiovascular development. For example, miR-1, -133a, -133b, comprise a subset of skeletal- and cardiac-muscle specific miRNAs that are induced during and regulate muscle differentiation (reviewed in <xref rid="bib28" ref-type="bibr"> ^28,45,47 </xref>). MiR-1 and miR-133 are two highly conserved miRNAs derived from a common precursor transcript, that exhibit cardiac- and skeletal- muscle specific expression during development and adult life.<xref rid="bib46" ref-type="bibr"> ^{46&#x02013;47} </xref> According to studies, miR-1 (miR-1-1, mir-1-2) targets, amongst others,<xref rid="bib46" ref-type="bibr"> ^46,54 </xref> the transcription factor (TF) Hand2, a promoter of ventricular cardiomyocyte expansion, whose levels are critical for normal cardiomyocyte morphogenesis and development.<xref rid="bib46" ref-type="bibr"> ^{46,48&#x02013;52} </xref> Studies utilizing knockout mice of mir-1-2 have reported dysregulation of cardiac conduction, cell cycle and defective heart development in these animals, a subset of which suffered from early lethality,<xref rid="bib53" ref-type="bibr"> ^53,54 </xref> thereby proposing a distinct role of miR-1-1 and mir-1-2 in cardiac development. MiR133a is also critical for cardiac development. Interestingly, miR-133a-1 and miR-133a-2 present with at least partly overlapping roles, since the deletion of either one at a time results in phenotypically normal the mice. However, the double-mutant miR-133a mouse embryos and neonates present with ventricular-septal defects often leading to early lethality, whilst the surviving animals are prone to dilated cardiomyopathy and heart failure. MiR-133a gene targets include Cyclin D2 and Serum Response Factor, the upregulation of which possibly underlies the dysregulation of cell cycle control and the aberrant activation of the smooth muscle gene program, as observed in miR-133a-1/ miR-133a-2 double mutant mice.<xref rid="bib55" ref-type="bibr"> ⁵⁵ </xref> Cyclin D2 is also targeted by miR-29a, and this process has been shown to suppress cardiomyocyte proliferation during postnatal development in rats.<xref rid="bib56" ref-type="bibr"> ⁵⁶ </xref> A recent global microRNA profiling study reported another miRNA, namely miR-27b, displaying a greatly elevated myocardial expression during heart development in mice. Interestingly, the TF Mef2c, which is involved in cardiac morphogenesis, was shown to be a target of miR-27b.<xref rid="bib57" ref-type="bibr"> ⁵⁷ </xref> </p><p>A series of studies in zebrafish has also provided valuable data for miRNAs implicated in heart development. For example, miR-23 has been shown to inhibit Hyaluronan synthase 2 (Has2) expression and extracellular hyaluronic acid production.<xref rid="bib40" ref-type="bibr"> ⁴⁰ </xref> Has2 is an extracellular remodeling enzyme which is required for endocardial cushion and valve formation, and when inhibited by miR-23 the number of endocardial cells that differentiate into endocardial cushion cells during development in zebrafish embryos was restricted.<xref rid="bib40" ref-type="bibr"> ⁴⁰ </xref> Endocardial cushions develop on the atrio-ventricular canal and play a role in proper heart septation during development. MiR-21 appears to control valve formation upon its flow-dependent expression induction in the developing zebrafish heart. Notably, miR-21 exerts its actions by regulating the expression of the same target genes as mouse/human miR-21, namely Sprouty, Pdcd4, and Ptenb.<xref rid="bib58" ref-type="bibr"> ⁵⁸ </xref> MiR-138, which was specifically expressed in the developing ventricular chamber, was shown to be required for establishment of chamber-specific gene expression patterns. MiR-138 acts by targeting multiple members of the retinoic acid signaling pathway, to prevent ventricular expansion of gene expression normally restricted to the atrio-ventricular valve region.<xref rid="bib59" ref-type="bibr"> ⁵⁹ </xref> Last, but not least, a recent study reported a putative mutual cross-regulation mechanism between the TF Tbx5 and miR-218-1, and demonstrated its implication in heart development in zebrafish.<xref rid="bib60" ref-type="bibr"> ⁶⁰ </xref> Of note, Tbx5 gene expression levels have an overt effect on heart development, and their dysregulation has been related with the establishment of congenital heart defects. Similarly, the Tbx5 downstream targets miR-218-1 and its host gene Slit2 are known to be involved in heart development. Specifically, miR-218-1 was shown to suppress the expression of Robo receptors (Robo1,2), which interact with Slit family ligands to facilitate cell guidance during development. Evidently, the miR-218-1 and Slit/Robo form a regulatory loop required for heart tube formation in zebrafish.<xref rid="bib61" ref-type="bibr"> ⁶¹ </xref> The exact role of miR-218-1 in Tbx-5 regulation, though, is still being explored.<xref rid="bib60" ref-type="bibr"> ⁶⁰ </xref> </p><p>Additional information on cardiac development-related miRNAs has emerged from studies in the Mexican axolotl (salamander). Interestingly, a group investigated the role of a human fetal heart microRNA which is thought to be related to the human miR-499 family, and was therefore named miR-499c, in mutant axolotl hearts in organ culture. Accordingly, the axolotl hearts with abnormal development (without tropomyosin expression, sporadically beating etc) were incubated with the miR-499c, which was able to induce expression of cardiac markers (tropomyosin, troponin, &#x003b1;-syntrophin) in these hearts.<xref rid="bib62" ref-type="bibr"> ⁶² </xref> Evidently, miR-499c treatment promoted the formation of cardiac myofibrils in mutant axolotl hearts, thus showing the potential to restore normal embryonic heart development in this species.<xref rid="bib62" ref-type="bibr"> ⁶² </xref> As presented in the following section, miR-499 possibly plays a key role during human cardiomyocyte (CMC) differentiation, and hence the role of the new miR-499c in cardiac development requires further investigation.</p></sec></sec><sec><title>miRNA expression in embryonic stem cell-derived cardiomyocytes</title><p>However informative studies in animal models may be, they still have to be validated in humans. To this end, human embryonic stem cell-derived cardiomyocytes (hESC-derived CMCs) are now providing valuable new insights. The first miRNA profiling study of hESC-derived CMCs led to the identification of 711 unique miRNAs. Amongst them are miR-1 and miR-133 which have been previously related to heart development, miR-208 which has been shown to be implicated in the reactivation of the cardiac fetal gene program by regulating myosin heavy chain gene (&#x003b1;-, &#x003b2;-MHC) expression,<xref rid="bib63" ref-type="bibr"> ⁶³ </xref> as well as the novel miR-499. MiR-499 was found to share several predicted gene targets with miR-208, while its overexpression in hESCs led to elevated protein levels of the cardiac TF MEF2C,<xref rid="bib64" ref-type="bibr"> ⁶⁴ </xref> which is required for cardiac contractile gene activation and for the structural development of the heart.<xref rid="bib65" ref-type="bibr"> ⁶⁵ </xref> Moreover, miR-1 overexpression in hESCs triggered upregulation of the TF GATA4,<xref rid="bib64" ref-type="bibr"> ⁶⁴ </xref> which is essential during early heart development.<xref rid="bib66" ref-type="bibr"> ⁶⁶ </xref> Accordingly, both miRNAs promoted cardiac specification of the hESCs. A consecutive study explored the distinct roles of miR-1 and -499 in the differentiation of hESCs to CMCs, and reported that miR-499 promotes ventricular specification of hESCs, whereas miR-1 facilitates electrophysiological maturation.<xref rid="bib67" ref-type="bibr"> ⁶⁷ </xref> </p></sec></sec><sec><title>miRNAs in HF pathogenesis</title><p>In addition to cardiac physiology, miRNAs are increasingly associated with pathological cardiac phenotypes. In the setting of HF, despite the multitude of molecular factors already implicated, miRNAs are emerging as novel contributors to both the preceding pathologies and to HF itself.</p><sec><title>miRNA signatures of human failing hearts</title><p>To date, miRNA profiling studies conducted in the human failing heart have identified significant miRNA alterations implicated in both pathogenesis and/or progression.</p><p>Numerous miRNome studies have been conducted using microarrays, amongst other methodologies. For example, Ikeda et al measured the expression of 428 miRNAs in the failing left ventricles of patients with ICM, DCM and aortic stenosis (AS), and detected 87 miRNAs, of which 43 were differentially expressed in at least one diagnostic group.<xref rid="bib69" ref-type="bibr"> ⁶⁹ </xref> The pro-hypertrophic miR-214<xref rid="bib70" ref-type="bibr"> ⁷⁰ </xref> appeared upregulated across all disease groups (2- to 2.8-fold), whereas the anti-hypertrophic miR-1<xref rid="bib71" ref-type="bibr"> ^{71&#x02013;76} </xref> was downregulated in DCM and AS. The miR-19 family was the most downregulated (miR-19a and -19b 2&#x02013;2.7 fold in DCM, AS), possibly contributing to the regulation of ECM protein levels in the heart, as supported by recent studies.<xref rid="bib77" ref-type="bibr"> ⁷⁷ </xref> Another microarray study investigated the miRNA expression pattern of the end-stage failing myocardium, by measuring 467 miRNAs.<xref rid="bib78" ref-type="bibr"> ⁷⁸ </xref> Twenty-eight miRNAs were significantly upregulated and eight of these (miR-21, -23a, -24, -26b, -27, -125, -195, -199a-3p) emerged as directly associated with HF pathophysiology.<xref rid="bib78" ref-type="bibr"> ⁷⁸ </xref> In a similar fashion, Sucharov et al assessed 470 miRNAs in idiopathic DCM and ICM hearts<xref rid="bib68" ref-type="bibr"> ⁶⁸ </xref> and found that, amongst other miRs, miR-100 was upregulated and miR-133(-a, b) was downregulated in HF. Further experiments demonstrated that miR-100 over-expression is implicated in the &#x003b2;-adrenergic receptor-mediated repression of &#x0201c;adult&#x0201d; cardiac genes (i.e.&#x003b1;-MHC, SERCA2a), whilst miR-133b overexpression acts to prevent alterations in gene expression that are due to &#x003b2;-adrenergic receptor stimulation.<xref rid="bib68" ref-type="bibr"> ⁶⁸ </xref> These findings imply that the HF-related shift in the expression of these two miRNAs may serve to reactivate the &#x0201c;fetal gene expression program&#x0201d; (see also<xref rid="bib79" ref-type="bibr"> ⁷⁹ </xref>). Overall, although all studies agree that significant miRNA expression changes occur in HF, the fine details thereof remain unclear and, in some cases, even contradictory. These discrepancies may reflect the existence of distinct miRNA signatures in the failing hearts of different etiologies, or to different stages of disease progression.</p><p>More recently, next generation sequencing has also been used for the analysis of human failing left ventricles of HCM or DCM etiology, and demonstrated significant changes in more than 250 of the 800 known human miRNAs,<xref rid="bib33" ref-type="bibr"> ³³ </xref> with approximately twice as many annotated miRNAs expressed in HF than unaffected cardiac tissue. Amongst the ten most abundant miRNAs in the HF samples that have been previously described in CVD studies, four have been shown to promote (miR-23a) or inhibit cardiac hypertrophy (miR-1<xref rid="bib71" ref-type="bibr"> ^{71&#x02013;76} </xref>), or negatively regulate fibrosis (miR-24,<xref rid="bib82" ref-type="bibr"> ⁸² </xref> -133a<xref rid="bib83" ref-type="bibr"> ⁸³ </xref>). Importantly, amongst the top ten overexpressed miRNAs that have not been described in previous profiling studies in HF (miR-23b,-30d, -125a, -143, -145,-193, -197, -342, -365, -455), miR-145 emerges as an important new player in cardiovascular disease, and in left ventricle pathological remodeling, in specific.<xref rid="bib33" ref-type="bibr"> ³³ </xref> </p><p>With regards to the precise miRNA mechanisms impaired in HF, Thum et al demonstrated that 87% of the over-expressed miRNAs and 84% of the under-expressed miRNAs were similar to the miRNA expression profiles of fetal cardiac tissue (e.g. miR-21, -29, -30, -129, -212), suggesting the activation of the &#x0201c;fetal gene expression program&#x0201d;.<xref rid="bib79" ref-type="bibr"> ⁷⁹ </xref> The reactivation of the &#x0201c;fetal gene expression program&#x0201d; is a hallmark of the hypertrophic and failing myocardium, often accompanying pathological left ventricular remodeling. In order to prove this concept, Thum et al showed that simultaneous re-expression of three of the miRNAs overexpressed in both HF and fetal tissue (miR-21, -129, -212) resulted in activation of fetal gene program and HF-related changes, like hypertrophy, in neonatal and adult CMCs. In specific, the miRNA-regulated fetal genes included ANP, BNP, &#x003b2;-MHC, &#x003b1;-skeletal actin and MEF2a, amongst others.<xref rid="bib79" ref-type="bibr"> ⁷⁹ </xref> This study shed light to significant aspects of the reactivation of the cardiac fetal gene program during HF, and revealed possible molecular players of left ventricular pathological remodeling.</p><p>MiR-21, miR-29 and miR-30 are some of the miRNAs whose HF expression parallels this of fetal hearts, and have been studied extensively in the context of HF. miR-21 appears upregulated in cardiac fibroblasts of DCM-related HF, likely following activation of the STAT3 and NfkB transcription regulators.<xref rid="bib84" ref-type="bibr"> ^{84&#x02013;85} </xref> This is consistent with the emerging topic of miRNA participation in a feedback loop with TFs that regulate their transcription.<xref rid="bib86" ref-type="bibr"> ⁸⁶ </xref> MiR-29 targets multiple ECM proteins (collagens, fibrillins etc) and miR-30 targets the pro-profibrotic factor CTGF, with the former being downregulated in the proximal region to myocardial infarcts,<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> and the latter down-regulated during pathological left ventricular hypertrophy caused by AS.<xref rid="bib88" ref-type="bibr"> ⁸⁸ </xref> The differential regulation of those three miRNAs during HF possibly facilitates the extensive ECM remodeling observed in the myocardium (see also <xref rid="bib75" ref-type="bibr"> ^75,87,89 </xref>).</p><p>Other studies have pinpointed miRNAs related to HF-associated pathologies, such as hypertrophy, HCM, DCM and ICM. In specific, studies in left ventricular tissue acquired from HCM patients revealed increased expression of miR-221, which was also upregulated in the hypertrophic (2 weeks) and failing hearts (9 weeks) of TAC mice. Further studies in rat CMCs demonstrated that forced expression of miR-221 by miRNA mimics is capable of inducing hypertrophy and re-expression of fetal genes <italic>in vitro</italic>, whilst knockdown of endogenous miR-221 abolished these effects. Moreover, <italic>in silico</italic> target prediction and experimental assays indicated that miR-221 possibly acts via targeting the suppressor of cardiac hypertrophy p27.<xref rid="bib90" ref-type="bibr"> ⁹⁰ </xref> MiR-499 upregulation in human hypertrophied and failing hearts was associated with decreased expression of an array of predicted targets. Interestingly, studies in mice showed that miR-499 suffices for the induction of HF and acceleration of the pathological remodeling, upon pressure overload. AKT and MAPKs were amongst the miR-499 numerous targets, while miR-499- induced cardiomyopathy was associated with changes in protein phosphorylation (e.g. HSP90, PP1&#x003b1;), thus revealing a spectrum of putative mechanisms via which miR-499 may contribute to cardiac pathophysiology.<xref rid="bib13" ref-type="bibr"> ¹³ </xref> Of particular interest is also the upregulation of miR-24 in cardiac tissue of ICM and DCM-related HF, which seemingly accounts for the under-expression of junctophilin 2 (JP2). JP2 is a structural protein that anchors the sarcoplasmic reticulum (SR) to the transverse tubules (TT) of the plasma membrane, which are the major sites of the excitation&#x02013;contraction coupling. Importantly, transmission electron microscopic imaging revealed a significant reduction in SR-TT junctions in the ICM and DCM specimens, indicating that miR-24 and JP2 dysregulation may ultimately lead to defective excitation-contraction coupling, a characteristic of failing CMCs.<xref rid="bib91" ref-type="bibr"> ⁹¹ </xref> Examples of miRNAs associated with age-related HF include the downregulated miR-18a, -19a and -19b leading to upregulation of the ECM proteins CTGF and TSP1, possibly in the context of ECM remodeling during HF pathogenesis.<xref rid="bib77" ref-type="bibr"> ⁷⁷ </xref> </p></sec><sec><title>miRNAs signatures in animal models of HF</title><sec><title>miRNAs signatures during the development of cardiac pathologies preceding HF: A close up in hypertrophy</title><p>Besides investigations in human HF, a series of animal model studies, predominantly involving transverse aortic constriction (TAC), have provided valuable insights into the miRNA expression alterations contributing to pathogenesis of hypertrophy and HF. For example, Sayed et al measured the expression of 334 miRNAs at days 1, 7 and 14 post TAC operation in mice and observed significant changed expression in 26, 22 and 51 miRNAs, respectively.<xref rid="bib74" ref-type="bibr"> ⁷⁴ </xref> Importantly, miR-1 was downregulated prior to hypertrophy development (1d) and persisted until later stages of hypertrophy (14d), and specifically up to 1 week before the presentations of HF in the TAC model. Moreover, five of the miRNAs that were upregulated during hypertrophy development (7d) (miR-199a, -199a*, -199b, -21, -214) and persisted until day 14 were the ones that exhibited the greatest change (&#x0003e;2 fold).<xref rid="bib74" ref-type="bibr"> ⁷⁴ </xref> These findings indicate a distinct stage-specific role of miR-1 and the latter five miRNAs in the development of hypertrophy in the TAC mouse model.</p><p>Similar miRNA expression changes were observed in another study, utilizing both the TAC mouse model and mice with cardiac-specific expression of activated calcineurin (CnA) (aimed at inducing pathological cardiac remodeling and hypertrophic growth). Accordingly, 42 miRNAs were differentially expressed in TAC hearts and 47 in CnA, with the two groups sharing 21 upregulated and 7 dowregulated miRNAs. Importantly, six of these miRNAs (miR-23, -24, -125, -195, -199a, -214) were consistent with findings in idiopathic end-stage human failing heart tissue, suggesting the conservation of pathogenic processes across species and highlighting their importance in HF.<xref rid="bib70" ref-type="bibr"> ⁷⁰ </xref> </p><p>The comparative study of a preload versus afterload cardiac hypertrophy mouse model, revealed that miRNA expression changes several days post TAC or shunt, suggesting that these mechanisms are involved in the later stages of remodeling post cardiac overload. The hypertrophy related miRNA- 133, -30 and -208, were regulated only in the afterload model, consistently with the direct role of miR-208 in &#x000df;-MHC upregulation.<xref rid="bib73" ref-type="bibr"> ^73,74,92 </xref> The preload hypertrophy model presented with changes in miR-140, -320 and -455. MiR-320 has been associated with apoptosis, while both miR-320 and miR-140 are upregulated in human HF.<xref rid="bib79" ref-type="bibr"> ⁷⁹ </xref> </p><p>Studies conducted in the left ventricles of a rat model of hypertrophy induced by banding of the ascending aorta, detected four upregulated miRNAs (miR-23a, -27b, -125b and -195), 14 days post operation, when the hypertrophy was already established (left ventricle weight/ body weight ratio increased by 65%).<xref rid="bib93" ref-type="bibr"> ⁹³ </xref> Importantly, miR-23a,-27b and -195 are known to favor CMC hypertrophic growth (see section 3.c.i). The observed changes in the expression of miRNAs in this rat model of hypertrophy are in line with previous studies in mice and human tissue, thus strengthening the notion of intra-species conservation of HF-related miRNA mechanisms.</p></sec><sec><title>miRNA signatures change during disease progression to HF</title><p>The expression of miRNA is a highly dynamic process, with different molecules and combinations thereof being implicated in the different stages of conditions leading to HF.</p><p>The most representative example of miRNA expression pattern shift during HF development is that of miR-1 and miR-133. Both miRNAs are downregulated during the development of hypertrophy, but overexpressed in chronic HF.<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> In specific, miR-1 and miR-133a have been found to be downregulated in mouse and rat models of hypertrophy, but upregulated in canine hearts isolated from animals with chronic HF.<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> Moreover, in the chronic HF animals, miR-1 and miR-133 were shown to be implicated in the development of arrhythmogenesis,<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> a characteristic observed in approximately 50% of congestive HF cases.<xref rid="bib95" ref-type="bibr"> ^95,96 </xref> These findings indicate that miR-1 and miR-133 serve distinct stage-specific roles during the course of HF. Their precise mode of action is discussed in subsequent sections.</p><p>The time course of HCM-HF progression has also been explored in the DBL transgenic mouse model of HCM, which bears mutations in troponin I and myosin heavy chain genes (TnI-203/MHC-403) and presents with severe HCM, HF, and premature death.<xref rid="bib75" ref-type="bibr"> ^75,97 </xref> Measurements in 335 miRNAs showed downregulation of miR-1 and miR-133 in a pre-disease state, and this change preceded upregulation of target genes causal of cardiac hypertrophy and ECM remodeling, thus implying a role in early disease development, consistently with other studies.<xref rid="bib71" ref-type="bibr"> ^{71&#x02013;76} </xref> In end-stage HCM the miRNA signature comprised of 16 miRNAs and corresponded to those of cardiac stress and hypertrophy, including downregulation of miR-1, -133, -30 and -150, and overexpression of miR-21, -199 and -214. This group also engaged microarrays to detect differentially expressed mRNAs in end-stage HCM, and bioinformatical analysis to predict mRNA-miRNA interactions amongst the significantly changed transcripts and miRNAs. As a result, some of the altered miRNAs (miR-1, -21, -30, -31, -133, -150, -222, -486) were further associated with hypertrophy, CMC proliferation, cardiac electrophysiology, calcium signaling, fibrosis, and the TGF-&#x003b2; pathway, based on their predicted interaction with the dysregulated transcripts and the Gene Ontology annotations of the latter.<xref rid="bib75" ref-type="bibr"> ⁷⁵ </xref> These findings suggest that miRNAs play a critical role in the cardiac pathophysiology of the DBL mouse model during end-stage HCM.</p><p>In search of the distinct miRNAs implicated in different stages of hypertrophy-induced HF, miRNA expression alterations have also been investigated during the transition from right ventricular hypertrophy (RVH) to HF in mice that underwent pulmonary artery constriction (PAC).<xref rid="bib100" ref-type="bibr"> ¹⁰⁰ </xref> In addition to left ventricular pathological remodeling, which accompanies the majority of failing hearts, RVH may also lead to failure, predominantly in cases with congenital right-sided cardiac defects. Reddy et al used microarrays to measure the expression of 567 miRNAs in the right ventricle of mice at 2, 4, 10 days post-PAC or sham operation, time points which correspond to early compensated hypertrophy, early decompensated hypertrophy and overt HF, respectively. Although no significant changes were detected at 2 days, at 4 and 10 days, 32 and 49 miRNAs, respectively, were deregulated. In addition, following global mRNA expression profiling of the right ventricles at 10 days, this group associated the deregulated miRNAs with their respective altered mRNA targets. Importantly, during decompensated RVH they reported alterations in miRNA expression that can enhance CMC hypertrophic growth (miR-199a-3p, let-7c), abnormal vascular tone (miR-143/145 cluster), resistance to apoptosis (miR-181a, let 7) and increase collagen synthesis (miR-30). At the HF phase, they reported changes that coincided with reactivation of the fetal gene program in HF (miR-208a, -208b), enhanced apoptosis (miR-34b,-34c, miR-144/451 cluster) and inhibition of endothelial cell proliferation and migration (miR-379, -503). Hypertrophy and HF shared 21 miRNA alterations, with some of them associated with CMC survival and adaptation to stress (miR-21, -210, -214, -199a), apoptosis (-34a), upregulation of collagens (miR-26b, -133, -149) and fibrosis (miRs-21, -29c, -150, -499). These findings further support the notion that miRNA expression is a dynamic process during HF development.</p><p>The study by Reddy et al also pointed out the differences between RVH/HF in the PAC model and LVH/HF in the TAC mouse model. Specifically, they compared the miRNA profile of RVH/HF with publically available microarray data for miRNA expression in TAC mice, and found four miRNAs (-34a, -28, -148a, -93) that were upregulated in RVH/HF but downregulated in LVH/HF. Their predicted mRNA targets are known to enhance apoptosis, modulate energy availability and impair calcium handling. The responses of RV and LV to stress differ, and specifically RV is more susceptible to HF when subjected to afterload.<xref rid="bib101" ref-type="bibr"> ^101,102 </xref> The observed alterations may increase the susceptibility of RV to HF under these circumstances. Thus, these differentially regulated miRNAs may be contributing to the differences between the RV and LV response to pressure overload stress.<xref rid="bib100" ref-type="bibr"> ¹⁰⁰ </xref> </p></sec></sec><sec><title>Characterization of the role of specific miRNAs in HF and associated pathologies in an experimental setting</title><p>The miRNA profiling studies in humans and in animal models of HF brought to light several miRNAs with altered expression and putative roles in HF development, many of which were subjected to further investigation. The studies presented below utilized animal model hearts and cell culture (CMCs, CFs) aiming to prove direct relations between miRNAs and HF or HF-associated pathologies.</p><sec><title>Can miRNAs control cardiac hypertrophy?</title><p>Aiming to demonstrate a direct and sufficient role of selected miRNAs in the induction of cardiac hypertrophy, four teams specifically overexpressed putative pro-hypertrophic miRNAs <italic>in vitro</italic> and <italic>in vivo</italic>. Van Rooij et al overexpressed a selected group of miRs (previously found upregulated in mice undergone TAC, in mice with cardiac overexpression of activated calcineurin, and in idiopathic end-stage human failing heart tissue) in primary rat CMCs. These five microRNAs (miR-23a, -23b, -24, -195, and -214) proved to be individually capable of inducing hypertrophic growth <italic>in vitro.</italic> <xref rid="bib70" ref-type="bibr"> ⁷⁰ </xref> Three of those miRNAs, (mir-24, -195, and -214) were then overexpressed individually in the heart of wild type (WT) mice. The miR-195 transgenic animals developed pathological cardiac hypertrophy and HF, thus revealing a direct and sufficient role of miR-195 in the development of HF in mice. Mir-24 transgenic mice died at the embryonic stage, whilst overexpression of mir-214 had no phenotypic effect. Similarly, Ucar et al investigated the role of miR-212 and -132, also upregulated during hypertrophy in the heart of TAC mice.<xref rid="bib103" ref-type="bibr"> ¹⁰³ </xref> Transgenic mice with cardiac specific overexpression of miR-212 and -132 presented with hypertrophic hearts, exhibited signs of severe HF and experienced premature death. <italic>In vitro</italic> experiments from the same group showed that miR-212 and -132 target the anti-hypertrophic TF Foxo3a, whilst overexpression of miR-212 and -132 results in hyperactivation of pro-hypertrophic calcineurin/NFAT signaling.<xref rid="bib103" ref-type="bibr"> ¹⁰³ </xref> MiR-23a levels have also been found elevated in rodent models of hypertrophy, and specifically upon isoproterenol-induced cardiac hypertrophy in mice,<xref rid="bib80" ref-type="bibr"> ⁸⁰ </xref> as well as pressure overload-induced hypertrophy in both mice and rats.<xref rid="bib93" ref-type="bibr"> ⁹³ </xref> In order to elucidate its role, <italic>Wang et al</italic> produced transgenic mice with cardiac overexpression of miR-23a, which presented with exaggerated hypertrophy upon phenylephrine (PE) treatment or pressure overload induced by TAC.<xref rid="bib81" ref-type="bibr"> ⁸¹ </xref> This study also reported that endogenous miR-23a was upregulated by hypertrophic stimuli (PE, endothelin, ET) in cultured CMCs, thereby indicating that miR-23a participates in the transduction of the hypertrophic signal. Moreover, they identified the anti-hypertrophic Foxo3a as a target of miR-23a, and showed that miR-23a and Foxo3a bi-transgenic mice present with an attenuated hypertrophic response, in comparison to the miR-23a transgenic mice alone.<xref rid="bib81" ref-type="bibr"> ⁸¹ </xref> Interestingly, <italic>in vitro</italic> studies by other teams revealed additional molecular players in the putative miR-23a pro-hypertrophic machinery. Specifically, experiments in CMCs showed that the nuclear factor of activated T cells 3 (NFATc3) induces miR-23a, which in turn targets the negative regulator of hypertrophy muscle ring finger1 (MuRF1) thereby triggering cardiac hypertrophy (<xref ref-type="fig" rid="fig2">Figure 2</xref>).<xref rid="bib80" ref-type="bibr"> ^80,104 </xref> </p><p>Mir-27b has been seen upregulated in cardiac hypertrophy, and specifically in the cardiac-specific Smad4 knockout mouse model.<xref rid="bib105" ref-type="bibr"> ¹⁰⁵ </xref> Importantly, cardiac-specific overexpression of miR-27b in transgenic mice was sufficient to induce hypertrophy and heart dysfunction,<xref rid="bib105" ref-type="bibr"> ¹⁰⁵ </xref> thereby implying a direct association. Moreover, <italic>in vitro</italic> studies showed that mir-27b targets the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and is inhibited by TGF-beta1, findings that were also validated in the TAC mouse model of HF.<xref rid="bib105" ref-type="bibr"> ¹⁰⁵ </xref> Overall, miR-195, -212, -132, -27b emerge as potent inducers of cardiac hypertrophy, while miR-23a appears to serve as a contributive factor to the establishment of this pathology.</p><p>In addition to upregulated pro-hypertrophic miRNAs, disruption of anti-hypertrophic miRNAs expression has also been reported in the hypertrophied and failing myocardium. A representative example is miR-1, which was downregulated in a series of studies in rodent models of hypertrophy, HCM and HF (TAC, AKT overexpression, MHC&#x003b1;-CN mice, cardiac specific Dicer deletion, and DBL transgenic mice). Ikeda et al demonstrated that the size of miR-1 deficient neonatal rat CMCs was significantly increased at baseline and after treatment with pro-hypetrophic stimulus (ET), indicating that miR-1 downregulation promotes hypertrophic growth. According to further studies in CMCs, miR-1 inhibits cell growth-related targets (RasGAP, Cdk9, fibronectin, Rheb), reduces protein synthesis and cell size, and its downregulation promotes hypertrophy.<xref rid="bib74" ref-type="bibr"> ⁷⁴ </xref> In addition, <italic>in vitr</italic>o experiments in a series of studies revealed multiple putative mechanisms of action for mir-1-mediated hypertrophy suppression,<xref rid="bib76" ref-type="bibr"> ^{76,71&#x02013;75} </xref> including targeting of Igf-1 and Igf1-r,<xref rid="bib71" ref-type="bibr"> ⁷¹ </xref> calmodulin, Mef2a and Gata4.<xref rid="bib72" ref-type="bibr"> ⁷² </xref> These data indicate that miR-1 targets key regulators of hypertrophic growth, and may thus act as a central suppressor of hypertrophy via a range of downstream effectors in the failing myocardium. Similarly, the newly described miR-378 has been shown to be down-regulated during hypertrophic growth and HF. Studies in rat CMCs have shown that deficiency of this miRNA is sufficient to induce fetal gene expression, thereby suggesting an anti-hypertrophic role in HF. MiR-378 seemingly acts by negatively regulating the MAPKs pathway. In specific, multiple components of this pathway have been identified as miR-378 targets (Mapk1, Igfr1, Grb2, Ksr1) by Ganesan et al.<xref rid="bib108" ref-type="bibr"> ¹⁰⁸ </xref> In addition, recent experiments in rat CMCs showed that miR-378 directly targets Grb2 and blocks Ras activation, resulting in negative regulation of fetal gene expression and cardiac hypertrophy.<xref rid="bib106" ref-type="bibr"> ^106,107 </xref> MiR-9 is also downregulated following hypertrophic treatments, and confers anti-hypertrophic effects in the murine heart. Wang et al utilized the isoproterenol and aldosterone-induced mouse models of hypertrophy to demonstrate that NFATc3 can promote hypertrophy via induction of myocardin expression, while miR-9 targets and suppresses myocardin.<xref rid="bib109" ref-type="bibr"> ¹⁰⁹ </xref> Whether miR-9 is also underexpressed in human HF and may thus provide a target towards pathological hypertrophy HF inhibition, is yet to be determined.</p></sec><sec><title>miRNAs impact on ECM remodeling and fibrosis</title><p>Besides the establishment of hypertrophy and/or dilatation, the failing myocardium is often accompanied by structural remodeling. However, the remodeling processes that take place during HF development are not restricted to the contractile cell component of the heart (CMCs). The non-myocyte cells of the healthy heart account for more than 60% of the cardiac cells, include cardiac fibroblasts (CFs) and endothelial cells (ECs), and are actively involved in the remodeling process.<xref rid="bib110" ref-type="bibr"> ^110,111 </xref> Fibroblasts, which are responsible for the synthesis of ECM components, account for approximately 90% of the non-myocyte cell mass.<xref rid="bib110" ref-type="bibr"> ^110,111 </xref> In the stressed myocardium, fibroblasts differentiate into active myofibroblasts upon a wide range of stimuli (e.g. TGF-&#x003b2;).<xref rid="bib89" ref-type="bibr"> ^89,110 </xref> These activated cells can regulate the secretion of ECM components and ECM degrading-enzymes (matrix metalloproteinases, MMPs) and tend to proliferate and migrate, acting to remodel the cardiac interstitium.<xref rid="bib89" ref-type="bibr"> ⁸⁹ </xref> This process may result in cardiac fibrosis, a hallmark of pathological hypertrophy and HF, which presents with aberrant proliferation of CFs and excessive deposition of ECM proteins in the interstitium and perivascular regions of the myocardium, ultimately impairing cardiac function.<xref rid="bib89" ref-type="bibr"> ⁸⁹ </xref> </p><p>Several lines of evidence indicate that dysregulation of miRNAs during HF occurs in CFs, besides CMCs, thereby contributing to the development of cardiac fibrosis. In particular, the increased miR-21 expression observed in human HF,<xref rid="bib70" ref-type="bibr"> ⁷⁰ </xref> has been attributed mainly to fibroblasts using the TAC mouse model of HF.<xref rid="bib84" ref-type="bibr"> ⁸⁴ </xref> Specifically, miR-21 is selectively upregulated in the fibroblasts of the failing heart and has been shown to target Spry1, a negative regulator of ERK-MAPK pathway, which functions to enhance growth factor secretion and fibroblast survival, thus promoting interstitial fibrosis.<xref rid="bib84" ref-type="bibr"> ⁸⁴ </xref> MiR-21 was also found upregulated in CFs of the infarct zone after ischemia-reperfusion in mice, where it was shown to induce MMP2 (an ECM degrading enzyme) via direct targeting of PTEN, but its role in fibrosis was not further investigated in this model.<xref rid="bib112" ref-type="bibr"> ¹¹² </xref> A more recent study by Liang et al revealed additional evidence supporting a role for mir-21 in fibrosis: miR-21 was upregulated in the border zone of murine hearts after MI, whereas the negative regulator of TGF&#x003b2;, TGF&#x003b2;RIII, was underexpressed. Further experiments in CFs showed that mir-21 overexpression can enhance collagen production, in part through TGF&#x003b2;RIII suppression, and conversely TGF&#x003b2;RIII overexpression can inhibit mir-21 and reduce collagen production in CFs.<xref rid="bib113" ref-type="bibr"> ¹¹³ </xref> Taken together, these studies imply that mir-21 upregulation under pathologic conditions in the myocardium may impair cardiac function by contributing to cardiac fibrosis.</p><p>The miR-29 family has also been found deregulated in the failing heart and associated with the pathological mediator of fibrosis TGF&#x003b2;. The members of the miR-29 family (miR-29a, b, c) are mainly expressed in the CFs of the murine heart and have been found downregulated in response to a variety of remodeling-inducing stresses (TAC, chronic calcineurin signaling, MI). <italic>In vitro</italic> experiments in cultured CFs showed that this reduction in miR-29 levels may be triggered upon TGF&#x003b2; stimulus.<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> Moreover, the miR-29 family was shown to directly target multiple genes encoding ECM components (collagens, elastin, fibrillins), whilst <italic>in vivo</italic> inhibition of miR-29 in healthy murine hearts and <italic>in vitro</italic> in CFs led to increased collagen expression, thus indicating a role of miR-29 in ECM remodeling.<xref rid="bib87" ref-type="bibr"> ⁸⁷ </xref> </p><p>MiR-24 was found to be correlated with ECM remodeling and TGF&#x003b2;, in a mouse model of MI. In particular, miR-24 was reported downregulated in the infarct zone after MI, and miR-24 treatment led to fibrosis attenuation and improved cardiac function. <italic>In vitro</italic> experiments conducted in CFs showed that miR-24 upregulation could specifically decrease the differentiation and migration of CFs, and reduce fibrosis.<xref rid="bib82" ref-type="bibr"> ⁸² </xref> The same team also demonstrated that miR-24 may act via suppressing its target furin, which is essential for TGF-&#x003b2; secretion, whose secretion is reduced upon miR-24 overexpression in CFs.<xref rid="bib82" ref-type="bibr"> ⁸² </xref> In conclusion, miR-24 downregulation in response to MI possibly serves to promote cardiac fibrosis after MI, which has been identified as a contributive factor to the development of HF.</p><p>MiR-133a is observed deregulated in HF and may have a role in ECM remodeling during HF. In specific, miR-133a and miR-30 has been found downregulated in the homozygous Ren2 rat model of hypertension-induced HF, and in rats having undergone TAC. The downregulation of these miRNAs in pathological LVH paralleled the increased expression of the profibrotic protein CTGF.<xref rid="bib88" ref-type="bibr"> ⁸⁸ </xref> <italic>In vitro</italic> experiments in CMCs and CFs showed that both miRNAs target CTGF, the expression of which was associated with increased collagen synthesis.<xref rid="bib88" ref-type="bibr"> ⁸⁸ </xref> Moreover, a recent study in the DBL transgenic mouse model of HCM (described earlier in this review) reported the downregulation of mir-1 and -133 before ECM remodeling and mir-1,-133 and -30 in end-stage HCM, overall suggesting a distinct role for these miRNAs in pathological ECM remodeling throughout the course of LVH development in HF.<xref rid="bib75" ref-type="bibr"> ⁷⁵ </xref> </p><p>In addition to the pool of residing interstitial CFs, recent studies suggest that epicardial mesothelial cells (EMCs) lining the heart and microvascular endothelial cells may also contribute to the injury-induced fibrotic process in the myocardium. In adults, EMCs can undergo epithelial-to-mesenchymal transition (EMT) due to reactivation of the developmental program or during cardiac injury (e.g. MI).<xref rid="bib114" ref-type="bibr"> ^{114&#x02013;118} </xref> Several research groups have provided <italic>in vitro</italic> and <italic>in vivo</italic> ^115-117 evidence that EMT of EMCs occurring in the injured adult myocardium can give rise to fibroblast-like cells, which contribute to the default repair-driven fibrotic response. Interestingly, <italic>Bronnum</italic> and partners showed in 2013 that miRNAs are capable of regulating fibrogenic EMT of the EMCs in the adult heart.<xref rid="bib119" ref-type="bibr"> ^119,120 </xref> In specific, they found that pro-fibrotic TGF-beta treatment promoted EMT progression in EMC cultures, resulting in expression changes of numerous miRs, and especially miR-21. Evidently, the ectopic expression of miR-21 promoted the fibroblast&#x02013;like phenotype resulting from fibrogenic EMT, whereas a miR-21 antagonist abolished this effect.<xref rid="bib119" ref-type="bibr"> ¹¹⁹ </xref> Moreover, this study determined that several miR-21 mRNA targets were differentially expressed during fibrogenic EMT of EMCs, such as PDCD4 and SPRY1, of which miR-21-dependent suppression contributed to the development of the fibroblast-like phenotype of EMCs.<xref rid="bib119" ref-type="bibr"> ¹¹⁹ </xref> Another study of the same group utilized TGF beta-induced EMT in rat-derived adult EMC cultures to investigate the role of Islet-1 (Isl1), a known marker of progenitor cells such as EMCs. They reported that Isl1 promoted the mesenchymal phenotype in untreated EMCs, whilst during TGF beta-induced EMT Isl1 was underexpressed, exerting a negative effect on EMT progression. The observed underexpression of Isl1 was in part attributable to miR-31, which was shown to act as a negative modulator of cardiac fibrogenic EMT, primarily via targeting Isl1.<xref rid="bib120" ref-type="bibr"> ¹²⁰ </xref> Overall, these studies shed light to molecular mechanisms implicated in the contribution of EMCs to cardiac fibrosis, whilst suggesting a regulatory role for miR-21 and miR-31 in the fibrogenic EMT of EMCs.</p><p>According to recent studies, endothelial cells can also provide fibroblast-like cells through endothelial-to&#x02013;mesenchymal transition (EndMT), but the presence of cells of this origin in the adult myocardium occurs only under pathological conditions and is associated with fibrosis.<xref rid="bib121" ref-type="bibr"> ¹²¹ </xref> <italic>Zeisberg</italic> and partners suggested that endothelial cells may undergo (EndMT) and generate CFs, and they showed that EndMT contributes to cardiac fibrosis progression in mouse models of pressure overload and chronic allograft rejection.<xref rid="bib122" ref-type="bibr"> ¹²² </xref> More recently, Ghosh et al reported differential expression of several miRs during cardiac EndMT.<xref rid="bib123" ref-type="bibr"> ¹²³ </xref> Specifically, they treated cultured mouse cardiac endothelial cells (MCECs) with TGFbeta2 to trigger EndMT, and performed microRNA microarrays to measure total microRNA expression in fibroblast-like cells vs MCECs. They reported significant expression changes in a range of miRs in fibroblast-like cells, and amongst them there were many previously associated with CVD (&#x000a0;&#x02191;&#x000a0;miR-125b, -21, -30b,-195, Let-7c, -7g; &#x02193;&#x000a0;miR-122a, -127, -196, -375). The expression of miR-125b was further validated by qPCR, whilst the protein levels of its target p53 were found downregulated in the EndMT-derived fibroblast-like cells. Interestingly, p53 is known to antagonize TGFbeta-induced profibrotic responses,<xref rid="bib124" ref-type="bibr"> ¹²⁴ </xref> therefore miR-125b overexpression may lead to profibrotic signaling upregulation via suppressing its target p53 in these fibroblast-like cells. In conclusion, EndMT-derived fibroblast-like cells emerge as a novel cardiac fibrosis mediators, whilst their disease-specific existence in the adult myocardium may facilitate the development of miRNA based tools to target fibrosis.</p></sec><sec><title>miRNAs impact on calcium cycling</title><p>The dysregulation of miR-1 and -133a appears to serve multiple and distinct roles during HF development and progression. In contrast to their downregulation in certain rodent models of HF (see sections 3.c.i, ii), miR-1 and miR-133a were found to be upregulated in canine hearts isolated from animals with chronic HF accompanied by increased left ventricular dimensions and impaired contractility of the left ventricle.<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> These miRNAs were shown to target the mRNA of the PP2A catalytic subunit of RyR2, which led to increased RyR2 phosphorylation and abnormal spontaneous sarcoplasmic reticulum Ca²⁺ release, thus contributing to arrythmogenesis.<xref rid="bib94" ref-type="bibr"> ⁹⁴ </xref> These <italic>in vivo</italic> findings confirm previous studies in rat CMCs, where miR-1 over-expression was shown to decrease the protein phosphatase PP2A regulatory subunit B56alpha, which in turn resulted in increased phosphorylation of the L-type and RyR2 calcium channels, and ultimately enhanced cardiac excitation-contraction.<xref rid="bib125" ref-type="bibr"> ¹²⁵ </xref> These observations point to miR-1 and miR-133a as regulators of CMC contractility via modulation of calcium signaling, suggesting their implication in arrhythmia manifestation during HF. The role of miR-1 in calcium signaling has been further investigated in additional rodent models of HF. Studies in the cardiomyocyte-specific SRF knock-out mouse model of HF revealed that sodium-calcium exchanger 1 (NCX1) and AnxA5 mRNAs are targets of miR-1.<xref rid="bib126" ref-type="bibr"> ¹²⁶ </xref> This is consistent with previous studies in chronic post-myocardial infarction rat model of HF, where miR-1 expression was restored by SERCA2a gene therapy (AAV9.SERCA2a) in the failing heart and led to normalization of NCX1 expression.<xref rid="bib127" ref-type="bibr"> ¹²⁷ </xref> Of note, miR-1 expression restoration also resulted in improved cardiac function in this model.<xref rid="bib127" ref-type="bibr"> ¹²⁷ </xref> Moreover, studies in the mouse model of hypertrophy derived from cardiac-specific Dicer deletion, showed that miR-1 also targets sorcin, which functions as a regulator of calcium signaling and excitation-contraction coupling.<xref rid="bib76" ref-type="bibr"> ⁷⁶ </xref> </p><p>With regards to the role of SERCA2a in the failing myocardium, functional screening of 875 miRNAs, identified miR-25 as a suppressor of SERCA2a expression and consequently a potent regulator of intracellular calcium handling. MiRNA-25 has also been found overexpressed in human and experimental HF. Moreover, experiments in cardiomyocyte-like HL-1 cells demonstrated that miR-25 delayed calcium uptake kinetics, whilst AAV9-mediated overexpression in a mouse model of HF led to loss of contractile function. Importantly, inhibition of miR-25 expression via antagomiRs in a mouse model of HF halted the established HF, and improved cardiac function and survival, thereby suggesting that miR-25 may be a novel therapeutic target for HF.<xref rid="bib180" ref-type="bibr"> ¹⁸⁰ </xref> These findings suggest that miRNAs, among their many mechanisms of contributing to HF, may also impair different aspects of calcium homeostasis in the cardiomyocytes.</p></sec><sec><title>miRNAs impact on mitochondrial dysfunction underlying HF</title><p>Interestingly, recent studies suggest that miRNAs may be implicated in HF development via impairing mitochondrial function. In specific, <italic>in vivo</italic> systemic administration of cloned miR-181c packaged in lipid-based nanoparticles in rats led to reduced exercise capacity and development of HF symptoms. Mir-181c acts via targeting mitochondrial COX1 (cytochrome c oxidase subunit 1), and miR-181c treatment selectively affected the expression of mitochondrial complex IV genes in the heart. Importantly, following miR-181c administration several mitochondrial functions were impaired, such as O₂ consumption, ROS production, matrix calcium levels as well as mitochondrial membrane potential.<xref rid="bib181" ref-type="bibr"> ¹⁸¹ </xref> Another group sought to investigate the role of miR-30c in the heart, due to the implication of miR-30 family in several aspects of CMC function and heart pathophysiology. To this aim they generated transgenic mice specifically overexpressing miR-30c in CMCs. They observed that these animals develop severe DCM after 6 weeks of age, and expression analysis of the transgenic hearts prior to phenotype onset revealed changes indicating mitochondrial function impairment. In addition to these findings, mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV were downregulated at the protein level. These observations indicate that miR-30c triggered mitochondrial dysfunction may account for the DCM phenotype of the miR-30c transgenic mice, thus uncovering a specific role of miR-30c in cardiac physiology.<xref rid="bib182" ref-type="bibr"> ¹⁸² </xref> Overall, these findings indicate a role of miRNAs in mitochondrial expression modifications that may underlie cardiac dysfunction.</p></sec></sec></sec><sec><title>MiRNAs in the diagnosis and prognosis of HF</title><p>Since HF is a highly heterogeneous disease, both in terms of etiology, clinical manifestation and outcome, early diagnostic and/or prognostic markers could considerably contribute towards the timely detection and more effective management of the disease.<xref rid="bib128" ref-type="bibr"> ¹²⁸ </xref> Towards this direction, significant ongoing efforts are aiming to depict miRNAs that could fulfill this role. A plethora of studies refer to observed changes in miRNA expression as potentially relevant in the diagnostic or prognostic setting. However, a very limited number of studies have been designed to address the <italic>per se</italic> diagnostic classification and/or prognostic value of miRNA markers. These studies have assessed cardiac tissue biopsies derived during surgery and peripheral blood.</p><sec><title>Diagnosis</title><sec><title>Cardiac tissue miRNAs</title><p>A recent study by Leptidis et al performed next generation sequencing in human failing left ventricles of end-stage HF patients of HCM and DCM etiology and reported over 250 significantly changed miRNAs in HF.<xref rid="bib33" ref-type="bibr"> ³³ </xref> Interestingly, the miRNA signatures differed significantly based on the pathology preceding HF (DCM or HCM),<xref rid="bib33" ref-type="bibr"> ³³ </xref> a finding consistent with other studies of distinct miRNA profiles in different HF diagnostic groups (e.g. DCM, ICM, AS). Importantly, the differences reported by the latter study appear to suffice for accurate patient classification.<xref rid="bib69" ref-type="bibr"> ⁶⁹ </xref> In specific, Ikeda et al used 67 microRNA signatures of control, ICM, DCM and AS heart tissue in order to develop a microRNA-based classifier. A diagnostic group label was assigned to each of the samples, which interestingly, corresponded to the clinical diagnosis 69% of the time (P&#x000a0;&#x0003c;&#x000a0;0.001).<xref rid="bib69" ref-type="bibr"> ⁶⁹ </xref> Nevertheless, cardiac tissue miRNA signatures would have a limited diagnostic value, due to the requirement of a cardiac biopsy. However, if cardiac miRNA signatures prove to correlate with circulating miRNA signatures, they could be easily translated to clinical practice, facilitating patient classification, and potentially prognosis and treatment.</p></sec><sec><title>Circulating blood miRNAs</title><p>A number of studies have focused on the miRNA expression in HF patient peripheral blood. Among them, several have pointed to an increase in miR-423-5p, often combined with a number of other miRNAs. For example, it has been proposed that increased serum levels of miR-423-5p, along with miR-320a, -22, and miR-92 can be used to identify patients with systolic HF and correlate with clinical prognostic parameters such as elevated serum natriuretic peptide levels, a wide QRS (Q, R, S waves of an electrocardiogram) and dilatation of the left ventricle and left atrium.<xref rid="bib129" ref-type="bibr"> ¹²⁹ </xref> Similarly, another group suggested that increased plasma levels of miR-423-5p can be a diagnostic biomarker of HF caused by DCM, while they correlated positively with N-terminal pro-brain natriuretic peptide (NT-proBNP) levels.<xref rid="bib130" ref-type="bibr"> ¹³⁰ </xref> However, it should be noted, that miR-423-5p has been investigated extensively in the context of multiple cardiac pathologies, with contradictory findings to date. Additional research is therefore needed, before final conclusions can be reached and findings are translated to the clinic.</p><p> <italic>Voellenkle et al</italic> investigated the miRNA expression pattern of peripheral blood mononuclear cells (PBMCs) in chronic HF patients suffering from ICM and nonischemic DCM.<xref rid="bib134" ref-type="bibr"> ¹³⁴ </xref> This group reported that three miRNAs (miR-107, -139, -142-5p) were decreased in both patient groups, while each group also featured additional altered miRNAs, and specifically decreased miR-125b, -497 in ICM, and increased miR-142-3p,-29b in nonischemic DCM.<xref rid="bib134" ref-type="bibr"> ¹³⁴ </xref> These findings suggest that chronic HF has a distinct miRNA expression profile in PBMCs, along with etiology-dependent changes that may allow patient classification, upon further validation of these results.</p></sec></sec><sec><title>Prognosis</title><sec><title>Circulating miRNAs as prognostic markers</title><p>In the context of identifying predictors of the development of ischemic HF in post AMI patients, the analysis of 377 miRNAs pointed to three p53-responsive microRNAs, namely miR-192, -194, and -34a, that were increased in the serum of patients who developed HF within one year of AMI onset.<xref rid="bib131" ref-type="bibr"> ¹³¹ </xref> Moreover, a significant correlation was observed between miR-194, -34a expression levels and left ventricular end-diastolic dimension.<xref rid="bib131" ref-type="bibr"> ¹³¹ </xref> These overall findings indicate that HF-related circulating miRNAs may serve as accurate biomarkers only for specific patient groups, and require further investigation in order to specify the conditions under which they could facilitate HF diagnosis and prognosis in the clinical setting.</p><p>He et al assessed the diagnostic and prognostic value of circulating miR-328 and -134 in AMI patients.To this aim, this group performed qPCR in plasma samples of 359 AMI patients and 30 healthy volunteers, and in parallel measured high-sensitivity cardiac troponin T (hs-cTnT) levels. Whilst miR-328 and -134 plasma levels were found to be significantly higher in patients in comparison to healthy controls, the diagnostic value of these miRs as determined by ROC curve analysis was significant, but inferior to (hs-cTnT) levels for AMI diagnosis. Interestingly though, the levels of these two circulating miRNAs were found to be associated with the risk of mortality and development of HF within 6 months after infarction (miR-328: OR 7.35, 95 % confidence interval 1.07-17.83, P&#x000a0;&#x0003c;&#x000a0;0.001, miR-134: OR 2.28, 95 % confidence interval 1.03-11.32 P&#x000a0;&#x0003c;&#x000a0;0.001).<xref rid="bib183" ref-type="bibr"> ¹⁸³ </xref> As such, miR-328 and 134 could be utilized as prognostic markers of post AMI clinical outcome.</p><p>Qiang et al investigated the miRNA expression profiles of endothelial progenitor cells (EPCs) isolated from venous blood of chronic HF patients with ICM or non-ischemic CM. This study identified sixteen miRNAs as differentially expressed between the two patient groups (miR-126, -508-5p, -34a, -210, -490-3p, -513-5p, -517c, -518e, -589, -220c, -200a*, -186*, -7i*, -200b*, -595, -662) and conducted a survival analysis using the patients' two-year follow up data. As a result, the levels of two of the differentially expressed miRNAs, miR-126 and -508-5p, were identified as independent prognostic factors of survival in both patient groups (P&#x000a0;=&#x000a0;0.003; HR (hazard ratio): 0.19; 95% CI (confidence intervals): 0.06-0.58, and P&#x000a0;=&#x000a0;0.002; HR: 2.292; 95% CI: 1.37-3.84 respectively).<xref rid="bib132" ref-type="bibr"> ¹³² </xref> This study brought to light two miRNAs that could be possibly used as prognostic markers of the clinical outcome of CHF.</p><p>In another study, the plasma concentrations of miR-126, -122 and -499 were measured in 33 congestive HF patients with ischemic heart disease and 17 asymptomtic controls. MiR-126 plasma levels were found to be decreased in HF patients, and negatively correlated with age, logBNP (B-type natriuretic peptide) and NYHA (New York Heart Association) class.<xref rid="bib135" ref-type="bibr"> ¹³⁵ </xref> Interestingly, miR-126 levels increased with improvement of the NYHA class from IV to III, in ten of the HF patients investigated. This finding is in line with a putative correlation of miR-126 with HF clinical outcome suggested by Qiang et al in 2013. However, miR-126 downregulation has also been related to coronary artery disease.<xref rid="bib136" ref-type="bibr"> ¹³⁶ </xref> Further investigation is required in order to assess if miR-126 downregulation is etiology-dependent or pertinent to HF development.</p><p>Goren et al aimed to identify circulating miRNAs that can be used as markers for atrial fibrillation (AF), given that AF is associated with poor prognosis in HF patients. They performed a genome wide screen for miRNAs in platelet and serum samples from 41 patients with systolic HF and 35 controls without cardiac disease. They observed that miR-150 levels were 3.2 fold lower in platelets of AF patients when compared to non AF HF patients, and 1.5 fold lower in the respective AF serum samples in comparison to non AF HF. Moreover, the serum levels of (cell-free) miR-150 in AF patients were found to be correlated with platelet levels of miR-150.<xref rid="bib184" ref-type="bibr"> ¹⁸⁴ </xref> Further investigation is required in order to assess if the markedly reduced miR-150 level in platelets and serum can be utilized as a prognostic marker for HF patients.</p><p>In agreement with HF patient studies, a recent analysis of hypertension-induced HF in rats detected significantly increased plasma levels of miR-423-5p, -16,- 20b, -93, -106b, and -223.<xref rid="bib133" ref-type="bibr"> ¹³³ </xref> The levels of these miRNAs were also measured during disease progression, at 2, 4, 6, 8 weeks after the high-salt diet onset in the salt-sensitive rats. Interestingly, some of the observed changes in miRNA expression paralleled disease progression. Specifically the levels of miR-106b and 93b showed significant upregulation at week 2 after diet onset, miR-20b at week 4, miR-19b at weeks 2 and 8, miR-423-5p at week 8 and miR-223 at weeks 6 and 8.<xref rid="bib133" ref-type="bibr"> ¹³³ </xref> The same study also assessed the levels of miR-16, -20b, -93, -106b, -223, and miR-423-5p after treatment with ACE inhibitors or mir-208 inhibition, and reported attenuation of their increase. These data suggest that the pattern of circulating miRNAs expression may be representative of distinct time points during HF progression, and as such they may be utilized in the prognostic setting. Additionally, early evidence indicates that circulating miRNAs could also be used to monitor response to HF treatment.<xref rid="bib133" ref-type="bibr"> ¹³³ </xref> </p></sec></sec></sec><sec><title>MiRNA and novel therapeutic approaches for HF</title><sec><title>RNA interference as a therapeutic approach</title><p>RNA interference (RNAi) technology has emerged as an effective method to manipulate gene expression.<xref rid="bib137" ref-type="bibr"> ¹³⁷ </xref> Importantly, RNAi has been recently proposed as a novel therapeutic strategy for manipulating dysregulated gene expression in human disease, and the first clinical trials using RNAi therapeutics are highly promising.<xref rid="bib138" ref-type="bibr"> ^{138&#x02013;140} </xref> The basic principle of RNAi is triggering gene expression silencing by an 18&#x02013;27 nucleotide long small RNA that identifies the target mRNA(s) via base pairing, with the most important classes of small RNAs utilized being miRNA and small interfering RNA (siRNA).<xref rid="bib141" ref-type="bibr"> ¹⁴¹ </xref> These two types of small RNAs have a similar course of action, but different biogenesis. MiRNAs originate from hairpin molecules containing ssRNAs (described in Section 1), whereas siRNAs originate from dsRNA which is in turn processed by Dicer and then directed to the target mRNA in the same manner as miRNA. In mammalian cells, two approaches are followed in order to achieve RNAi mediated gene silencing: the RNA- and the DNA-based approach. In the first approach, 21 base siRNA duplexes are chemically synthesized and delivered directly to the target cells, resulting in a transient gene silencing effect due to the short half-life of siRNA.<xref rid="bib142" ref-type="bibr"> ¹⁴² </xref> In the DNA-based approach, short hairpin RNA (shRNA) are delivered into the cell via viral vectors, and consequently shRNAs are synthesized in the nucleus and exported to the cytoplasm through the miRNA machinery, to be processed by Dicer and become siRNA effectors, thus achieving long term gene suppression.<xref rid="bib143" ref-type="bibr"> ^143,144,145 </xref> </p><p>Being an effective tool for gene silencing, siRNA emerges as a potential therapeutic agent for CVD and HF, according to <italic>in vitro</italic> and <italic>in vivo</italic> studies. A representative example of the therapeutic applications of siRNA in HF is the knock down of phospholamban (PLN) via RNAi in the TAC rat model of HF.<xref rid="bib146" ref-type="bibr"> ¹⁴⁶ </xref> PLN is a muscle-specific protein acting as an inhibitor of SERCA2A, but upon its phosphorylation triggered by &#x003b2;-adrenergic stimulation, it fails to inhibit SERCA2A, thus leading to increased heart contractility.<xref rid="bib147" ref-type="bibr"> ¹⁴⁷ </xref> Notably, mutations in PLN gene underlie an inherited form of DCM that presents with severe CHF in humans,<xref rid="bib148" ref-type="bibr"> ¹⁴⁸ </xref> whilst suppression of Pln has been engaged aiming to preserve Serca2 activity and prevent HF in animal models of HF.<xref rid="bib149" ref-type="bibr"> ^149,150 </xref> Suckau et al developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB), which was administered intravenously to TAC rats, in order to suppress Pln expression in the heart via RNAi.<xref rid="bib146" ref-type="bibr"> ¹⁴⁶ </xref> Interestingly, cardiac Pln protein levels were reduced to 25% and the observed suppression of Serca2 was restored in TAC rats, ultimately resulting in the attenuation of TAC- induced cardiac dilation, hypertrophy and fibrosis. These findings have been confirmed and expanded by other groups.<xref rid="bib151" ref-type="bibr"> ^{151&#x02013;156} </xref> Overall, it emerges that suppression of PLN or PP1 by RNAi could provide novel therapeutic strategies to fight HF.</p><p>Although the mechanism of RNAi and its therapeutic efficacy are not yet fully elucidated, RNAi emerges as a promising therapeutic strategy. It has been demonstrated that RNAi techniques have great sensitivity and specificity for the target gene, whilst its cooperation with the cell's own miRNA machinery may allow the transcriptional suppression of virtually any gene of interest. However, the therapeutic use of RNAi in humans has yet to overcome a number of obstacles, such as effective <italic>in vivo</italic> delivery method to specific tissue or cells, specific siRNAs designed for each mRNA target with diminished off-target effects, and avoidance of innate immunity activation by siRNAs.<xref rid="bib157" ref-type="bibr"> ^{157&#x02013;160} </xref> </p><p>Interestingly, these concerns may soon subside as recent studies showed that intravenous administration of nanoparticle-enclosed siRNAs is safe, and capable of triggering target-specific suppression of gene expression via an RNAi mechanism of action in cancer patients.<xref rid="bib161" ref-type="bibr"> ^161,162 </xref> Importantly, in a phase I trial, researchers showed that intravenous administration of the siRNA ALN-PCS -targeting the circulating protein PSCK9, in order to lower LDL plasma levels- resulted in significant plasma level reduction of PSCK9 (70%), and led to reduction of LDL (40%).<xref rid="bib140" ref-type="bibr"> ¹⁴⁰ </xref> To date, the latter study is the first to show that a therapeutic agent based on RNAi technology can be safely administered and affect a clinically validated endpoint, thus indicating that novel and effective RNAi-based drugs may soon be an alternative option to existing therapeutics.</p></sec><sec><title>MiRNAs as therapeutic targets: AntagomiRs and miRNA mimics</title><p>As aforementioned, several dysregulated miRNAs have been associated with HF pathogenesis and HF related pathologies, thus the targeted modulation of miRNA expression and activity may be a promising therapeutic approach to improve HF clinical management.</p><p>The targeted regulation of miRNA pathways could be facilitated by a variety of molecular tools, divided into two main categories: anti-miRNAs (antagomiRs) and miRNA mimics. AntagomiRs are modified antisense nucleotides that can trigger downregulation of the intracellular levels of selected miRNAs. AntagomiRs may intervene at multiple levels on the cellular miRNA machinery, including i) binding to mature miRNA within RISC and serving as a competitive inhibitor, ii) binding to pre-miRNA and inhibiting their processing and incorporation to the RISC complex, and iii) inhibiting the processing or the exit of pre-miRNA or pri-miRNA from the nucleus.<xref rid="bib163" ref-type="bibr"> ^163,164 </xref> Importantly, in all cases, antagomiRs activity ultimately results in increased intracellular levels of the corresponding mRNA targets. Conversely, miRNA mimics are synthetic RNA duplexes that mimic the function of endogenous mature miRNAs, and aim to decrease the levels of selected mRNA targets.<xref rid="bib165" ref-type="bibr"> ^165,166 </xref> </p><p>Interestingly, several investigating groups have engaged antagomirs in an attempt to inverse the pathological phenotype that was seemingly triggered by specific miRNAs in HF. For example, Montgomery et al administered antagomiR-208a to Dahl rats with hypertension-induced HF, and prevented the pathological &#x0201c;myosin switch&#x0201d; and cardiac remodeling, whereas cardiac function, overall health and survival were markedly improved.<xref rid="bib167" ref-type="bibr"> ¹⁶⁷ </xref> Ucar et al used antagomiRs to target the pro-hypertrophic miR-132 and miR-212, in mice with heart specific overexpression of these miRNAs presenting with cardiac hypertrophy and HF. Accordingly, injection of antagomiR-132 rescued cardiac hypertrophy and HF in vivo, whereas transgenic mice lacking both miR-212 and -132 were protected from TAC-induced hypertrophy, and were partially protected from TAC- induced cardiac fibrosis, dilatation and impaired left ventricular function. These data indicate a causal role of miR-132 and a contributing role of miR-212 in the development of hypertrophy and HF in vivo, whilst suppression of miR-132 via antagomiRs emerges as a possible therapeutic approach for HF.<xref rid="bib103" ref-type="bibr">¹⁰³</xref></p><p>In contrast, inhibition of endogenous miR-21 or miR-18b was shown to increase hypertrophic growth in cultured CMCs.<xref rid="bib99" ref-type="bibr"> ⁹⁹ </xref> However, the latter study is in contrast with the findings of other groups regarding the role of miR-21 in hypertrophy. Cheng et al performed antisense-mediated depletion of miR-21 in hypertrophic CMCs which overexpressed miR-21, and reported significantly decreased CMC hypertrophy.<xref rid="bib98" ref-type="bibr"> ⁹⁸ </xref> Moreover, Thum et al used antagomirs to inhibit mir-21 in TAC mice, and as a result the TAC-induced cardiac hypertrophy was attenuated.<xref rid="bib84" ref-type="bibr"> ⁸⁴ </xref> Interestingly though, Patrick et al claimed that genetic deletion or inhibition of miR-21 in mice did not altered the hypertrophy they displayed in response to cardiac stressing stimuli (TAC, MI, chronic calcineurin activation, infusion of Ang II), implying that mir-21 is not essential for the development of pathological cardiac hypertrophy.<xref rid="bib168" ref-type="bibr"> ¹⁶⁸ </xref> This discrepancy was subject to further discussion, and the different length of the anti- mir-21 oligonucleotides used by the groups of Thum and Patrick (22-mer vs 8-mer) were suggested as the cause of this inconsistence, whilst the difference in the phenotype of the mir-21 deficient mice has yet to be explained.<xref rid="bib169" ref-type="bibr"> ^169,170 </xref> </p><p>MiR-21, being mainly expresses by CFs, has also emerged as a regulator of cardiac fibrosis, and as such Thum and Patrick also investigated the effect of miR-21 inhibition in this subpathhology. According to Thum et al, miR-21 inhibition protected TAC mice against cardiac fibrosis, but Patrick et al has called into question the role of mir-21 in cardiac fibrosis, as well as hypertrophy. The latter reported that genetic deletion or inhibition of miR-21 in mice did not have an effect on the fibrosis the developed in response to a variety of cardiac stressing stimuli (TAC, MI, chronic calcineurin activation, infusion of Ang II). This inconsistence may be due to a technicality (antagomir length), but leaves open the possibility of a contributing role of miR-21 in the development of cardiac fibrosis.</p><p>MiRNA mimics have been utilized in the experimental setting in order to normalize the expression of miR-9 which is observed downregulated during cardiac hypertrophy. Wang et al administered a double-stranded RNA miRNA mimic for miR-9 in rats with isoproterenol-induced cardiac hypertrophy, and successfully reduced the levels of miR-9 target myocardin, ultimately leading to attenuation of cardiac hypertrophy and improvement of cardiac function.<xref rid="bib109" ref-type="bibr"> ¹⁰⁹ </xref> Although the efficiency of miRNA mimics is subject to a number of limitations, regarding <italic>in vivo</italic> delivery methods, cellular uptake and off-target effects, this study provides a paradigm of a possible therapeutic approach, where exogenous supplementation of miRNA mimics could be used to replenish endogenous miRNAs that are reduced during cardiac disease. Overall, it is important to note that mimics (other than viral delivery) have thus far not proven to be a viable option <italic>in vivo</italic>. In fact, it is thought that <italic>in vivo</italic> methods, other than viral delivery of mimics, actually result in an miR inhibitory effect, rather than a mimic effect.</p><p>According to the aforementioned studies, miRNA modulation appears to be a promising tool for the development of novel therapeutic strategies against cardiac disease and HF. However, further investigation is still required in order to fully reveal the functions of antagomiRs and miRNA mimics, as well as their exact pharmacodynamic and pharmacokinetic characteristics, and the potential adverse reactions or side effects. In addition, comparisons of the specificity and efficacy of the different miRNA antagonism and mimicking chemistries will need to be made, and the most effective and safe modes of <italic>in vivo</italic> deliver are yet to be determined.</p></sec><sec><title>miRNAs in cardiac regeneration: a novel therapeutic approach</title><p>In the failing myocardium, systolic dysfunction may occur as a result of many subpathologies, with MI-induced cardiac injury being one of the most common.<xref rid="bib1" ref-type="bibr"> ¹ </xref> The loss of functional CMCs due to MI or HF may deteriorate cardiac function, and the adult heart has a very limited ability to repair damaged tissue through myocardial regeneration.<xref rid="bib171" ref-type="bibr"> ^{171&#x02013;175} </xref> CMCs have a very low proliferative rate during postnatal development, but recent evidence supports the increased CMC proliferation at the border zone of the heart post-MI in adult mice.<xref rid="bib176" ref-type="bibr"> ¹⁷⁶ </xref> Interestingly, several lines of evidence indicate that miRNAs are potent regulators of CMC cell cycle (see Section &#x0201c;miRNAs play a central role in cardiac development&#x0201d;), and could be manipulated to trigger CMC proliferation in order to achieve myocardial regeneration. For example, knock down of miR-195 increases mitotic CMCs, and inhibition of miR-29a induces cell proliferation, accelerates G1/S and G2/M transition, and enhances cell cycle gene expression, acting at least in part through cyclin D2.<xref rid="bib56" ref-type="bibr"> ^56,177 </xref> </p><p>Furthermore, exogenous administration of hsa-miR-590 and -199a, stimulates cardiac regeneration and reduces infarct size in animal models of MI. More importantly, miRNA-induced cardiac regeneration was accompanied by almost complete recovery of cardiac functional parameters (e.g. left ventricular ejection fraction LVEF, left ventricular fractional shortening LVFS).<xref rid="bib178" ref-type="bibr"> ¹⁷⁸ </xref> Similarly, the miR-17-92 cluster appears to be a potent stimulator of CMC proliferation in embryonic, postnatal and adult murine hearts.<xref rid="bib179" ref-type="bibr"> ¹⁷⁹ </xref> Overall, these findings point to miRNAs as a novel, promising approach for treating HF related CMC loss.</p></sec></sec><sec><title>Conclusion</title><p>The continuously expanding field of miRNA research has revealed the significant contribution of these small molecules in a broad range of physiological and pathological mechanisms (<xref ref-type="fig" rid="fig3">Figure 3</xref>). In the context of heart biology, miRNAs prove to be potent regulators of gene expression during cardiac development and are directly implicated in the onset and progression of heart failure, amongst other pathological conditions. These valuable new insights change our perception of disease pathogenesis, and unveil promising new diagnostic and prognostic markers. Importantly, miRNAs open the way to a new line of therapeutic approaches that could play a significant role in the future of the cardiology clinics.</p></sec>

The discovery of pulmonary circulation: From Imhotep to William Harvey

<p>In his quest to comprehend his existence, Man has long been exploring his outer world (macro-cosmos), as well as his inner world (micro-cosmos). In modern times, monmental advances in the fields of physics, chemistry, and other natural sciences have reflected on how we understand the anatomy and physiology of the human body and circulation. Yet, humanity took a long and winding road to reach what we acknowledge today as solid facts of cardiovascular physiology. In this article, we will review some of the milestones along this road.</p>

<contrib contrib-type="author"><name><surname>ElMaghawry</surname><given-names>Mohamed</given-names></name><xref ref-type="aff" rid="aff1 aff2"> ^1,2 </xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>Zanatta</surname><given-names>Alberto</given-names></name><xref ref-type="aff" rid="aff2"> ² </xref></contrib><contrib contrib-type="author"><name><surname>Zampieri</surname><given-names>Fabio</given-names></name><xref ref-type="aff" rid="aff2"> ² </xref></contrib>

Global Cardiology Science &#x00026; Practice

<p> <disp-quote><p>&#x0201c;The history of the pulmonary circulation provides a measure of Man's thinking about himself and his place in the Universe.&#x0201d;<xref rid="bib1" ref-type="bibr"> ¹ </xref> <italic>Alfred P. Fishman (1918&#x02013;1990), president of American Physiological Society.</italic> </p></disp-quote> </p><sec><title>The heart in ancient Egyptian medicine</title><p>The ancient Egyptians considered the heart as the central organ of the body, both physiologically and spiritually. The earliest hieroglyphic depiction of the heart was as an organ with eight vessels attached to it (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). After the third Dynasty, the heart was modified to a simpler jar-shape (<xref ref-type="fig" rid="fig1">Figure 1B</xref>)<xref rid="bib2" ref-type="bibr"> ² </xref>.</p><p>The Smith papyrus (ca. 1600 BC) is the oldest known surgical treatise on trauma. It was named after Edwin Smith, the American Egyptologist who purchased the scroll in Luxor in 1862. Many historians believe that the text of the Smith papyrus was copied from a much older document originally written by Imhotep, the prominent high priest and physician of the Old Kingdom (ca.3000-2500 BC). The first phrases of the Smith papyrus (ca. 1600 BC) demonstrated that ancient Egyptians directly associated the pulse with the heart: <italic>&#x0201c;The counting of anything with the fingers (is done) to recognize the way the heart goes. There are vessels in it leading to every part of the body. When a Sekhmet priest, any sinw doctor&#x02026; puts his fingers to the head to the two hands, to the place of the heart&#x02026; it speaks in every vessel, every part of the body&#x0201d;</italic> <xref rid="bib2" ref-type="bibr"> ^2,3 </xref>.</p><p>In the Ebers medical Papyrus (ca. 1555 BC), the heart is again described as the centre of a system of vessels supplying the body. Going beyond underlining the importance of pulse examination, the text also alludes to cardiac rhythm disturbances and heart failure: <italic>&#x0201c;From the heart arise the vessels which go to the whole body&#x02026; if the physician lay his finger on the head, on the neck, on the hand, on the epigastrium, on the arm or the leg, everywhere the motion of the heart touches him, coursing through the vessels to all the members&#x02026;. When the heart is diseased, its work is imperfectly performed; the vessels proceeding from the heart become inactive so that you cannot feel them&#x02026; If the heart trembles, has little power and sinks, the disease is advancing.&#x0201d;</italic> <xref rid="bib2" ref-type="bibr"> ² </xref> </p><p>The Egyptian cardiovascular medicine cannot be entirely separated from spirituality and mysticism, as the heart played a pivotal role in the ancient Egyptian theology. However, the early Egyptian medicine, with its advanced clinical examination and diagnosis, paved the way to the scientific foundations of Greek and Roman medicine.</p></sec><sec><title>Hippocrates and the four humours</title><p>Hippocrates of Cos (460-377 BC) is recognized by most scientific historians as the Father of Medicine. He revolutionized the views of medicine and disease, mainly by recognizing that disease occurred naturally and was not due to divine punishment. The Hippocratic Corpus is a collection of around seventy medical works from Alexandrian Greece. The Corpus was most probably not written by a single person, as it had different writing styles and variable subjects. It can be attributed, by consequence, to the &#x0201c;Hippocratic School&#x0201d;, a group of disciplines sharing similar views and methods<xref rid="bib2" ref-type="bibr"> ² </xref>.</p><p>In the Corpus, Hippocrates and his contemporaries theorized that health is a state of balanced humours while disease was a state of imbalanced humours. These humours are blood, black bile, yellow bile, and phlegm. The four humours correspond to the four elements of nature (earth, wind, fire, and water) that reflect the four primary physical qualities (hot, cold, dry, and wet). Each humour was characterized by one of the four elements and a couple of the four qualities: blood, for instance, corresponded to the &#x0201c;fire&#x0201d; and was &#x0201c;hot&#x0201d; and &#x0201c;wet&#x0201d;. The behaviour and effects on the body of each humour was strictly related, by analogy, with these physical characteristics. Thus, the blood, being hot and wet, was related to the &#x0201c;natural heat&#x0201d; of the body and its eventual prevalence could cause fever. The concept of the four humours would influence the medical paradigms, including those regarding the cardiovascular system for long centuries to come (<xref ref-type="fig" rid="fig2">Figure 2</xref>)<xref rid="bib4" ref-type="bibr"> ^4,5 </xref>.</p></sec><sec><title>The School of Alexandria</title><p>Around 300 years before Christ, Alexandria boasted a remarkable cultural and intellectual advancement. The Alexandria School of Medicine was mainly founded on the teachings of Hippocrates. In this era, three eminent figures shaped the views of their contemporaries on the cardiovascular system: Praxagoras, Herophilus, and Erasistartus.</p><p>Praxagoras of Cos (340 BC) was a renowned anatomist in the early history of the Alexandrian medicine. He was the first to identify anatomical differences between arteries and veins. He theorized that arteries begin in the heart and carry pneuma, while veins originate in the liver and carry blood. On semeiotics, he was of the very first to recognize the diagnostic values of the pulse.</p><p>Herophilus of Chalcedon (355-260 BC), was a scholar of Praxagoras. He produced a large volume of anatomical writings on central nervous, gastrointestinal, and reproductive systems. Regarding cardiovascular system, Herophilus recognized that arteries are thicker than veins; he also noticed the exception of this rule at the lung vessels.</p><p>Erasistratus of Iulis on Ceos (315-240 BC), working initially with Herophilus, considered the heart to be the source of both arteries and veins. He postulated an open-air system in which veins distribute blood through the body, while arteries contain air alone. However, he did observe that arteries &#x02013; when punctured &#x02013; do bleed. To explain the paradox of bleeding arteries, he suggested that blood moves from veins to arteries via invisible channels after the arteries empty their content of air to the body<xref rid="bib3" ref-type="bibr"> ³ </xref> (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p></sec><sec><title>Galen of Pergamenon</title><p>Claudius Galenus, the prominent physician, surgeon and philosopher, was born in Pergamum (currently located near the city of Bergama in Turkey) around 129 AD (<xref ref-type="fig" rid="fig4">Figure 4</xref>). He studied medicine in Pergamum, Smyrna, Corinth, and Alexandria. He later resided in Rome and became the physician of the Roman emperors: Marcus Aurelius, Commodus, and Septus Severus. By the time of his death (between 207 and 216 AD), Galen had left an almost unsurpassed legacy of medical and philosophical writings. Galen's theories would impact medical sciences for long centuries, influencing Roman, Islamic and Renaissance scholars.</p><p>Galen's medical theories were founded on the Greek anatomy and physiology background and on Aristotle's deductive logic. Galen adopted the four humours paradigm, emphasizing the importance of the role of nutrition in human physiology. However, most of Galen's anatomical studies were based on observations made on living or dead animals, particularly apes and oxen, as he was not allowed access to human bodies.</p><p>According to Galen, the liver was the source of all veins and the principle organ for blood production. Nutrients were concocted in the gut to form chyle which was then transformed into blood by the liver. The blood moved from the hepatic vein to the vena cava and supplied all parts of the body above and below the liver. In contrast to Erasistratus theory, Galen believed that arteries were filled with blood, which was infused with the vital spirits by a mixture of air from the lungs through the pulmonary vein and heat from the heart. The blood passed from the right ventricle to the left ventricle through invisible pores present in the inter-ventricular septum. The heart itself was not a muscle and did not have a pumping function; blood simply passed through it. Like Erasistratus, Galen adopted that blood was not recycled, but rather evaporated or consumed by the organs through a single-pass open system<xref rid="bib6" ref-type="bibr"> ⁶ </xref> (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p><p>Galen's theory remained dominant until the fall of the Roman Empire. Also during the medieval ages in the Latin West, there was no interest to challenge ancient Greek and Roman science. In fact, Galen's work was fitted to Christian doctrine and thus rendered immune to questioning. This dogmatic view on Galen's teachings would last till the rise of the Renaissance in Europe.</p></sec><sec><title>The Renaissance</title><sec><title>Leonardo da Vinci</title><p>The Renaissance polymath Leonardo da Vinci (1452-1512) was one of the first Westerns to oppose the anatomical dogmas of Galen. In contrast to Galen, da Vinci described the heart as a muscle and considered the atria as cardiac chambers. His anatomical drawings of the heart and its valves were highly accurate (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Of interest, da Vinci also provided first descriptions of atherosclerotic coronaries. However, da Vinci was influenced by the Galenic anatomy. For instance, he drew the four chambers of the heart clearly depicting the invisible pores for the passage of blood from the right to the left ventricle (<xref ref-type="fig" rid="fig5">Figure 5B</xref>)<xref rid="bib6" ref-type="bibr"> ⁶ </xref>.</p></sec><sec><title>Servetus</title><p>Michael Serveto (1511-1553), was a Spanish philosopher and theologian (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). He published a theological treatise entitled &#x0201c;<italic>Christianismo restituti&#x0201d;</italic> where he challenged the views of Galen and proposed that blood must pass from the right ventricle to the lungs, where it is mixed with air and then back to the left ventricle (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). The exact details of how a theologian, who never systematically conducted a systematic anatomical research, reached these conclusions are still unknown. Servetus' views on religion and non-trinitarian Christology were condemned by both Catholics and Protestants. Michael Servetus was eventually denounced by John Calvin and was burnt, with most of his books, at the stake as a heretic by the city council of Geneva<xref rid="bib7" ref-type="bibr"> ⁷ </xref>.</p></sec></sec><sec><title>The School of Padua</title><p>The University of Padua is one of the oldest universities in the world. It was founded in 1222 by a group of scholars from University of Bologna seeking more academic freedom. During the Renaissance and under the influence of the Republic of Venice, Padua University medical school witnessed its golden age. Because of its academic autonomy and independence of political or religious influences, Padua was the destination of Europe's best scientists of the time<xref rid="bib8" ref-type="bibr"> ⁸ </xref>.</p><p>Andreas Vesalius (1514&#x02013;64) was born in Brabant (modern-day Belgium). He was a professor of anatomy at the University of Padua and considered by many as the founder of modern anatomy (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). By the age of 29, Vesalius had reshaped the study of human anatomy through his seven-book masterpiece &#x0201c;<italic>De humani corporis fabrica</italic>&#x0201d;, published in 1543 (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). Unlike Galen, Vesalius carried out human corpse dissections systematically and challenged many of Galen's anatomical views. In the sixth book of the <italic>fabrica</italic>, focusing on the heart and associated organs, Vesalius rectified Galen's notion that the great blood vessels originate from the liver. Moreover, in the second 1555 edition, he questioned the existence of the inter-ventricular pores<xref rid="bib9" ref-type="bibr"> ⁹ </xref>.</p><p>Realdo Colombo (1516&#x02013;1559), was an Italian anatomist and a scholar of Vesalius at the University of Padua (<xref ref-type="fig" rid="fig8">Figure 8</xref>). Colombo could not prove the presence of the inter-ventricular pores described by Galen. He theorized the pulmonary transit of blood instead of its passing through the invisible pores<xref rid="bib10" ref-type="bibr"> ¹⁰ </xref>. Interestingly, Colombo was a contemporary of Servetus. However, he made no reference to Servetus. The question whether Servetus was influenced by Colombo, or the other way around, or they produced their work independent of each other, was never resolved.</p></sec><sec><title>Girolamo Fabrizio d'Aquapendente</title><p>Fabrizio d'Aquapendente (1537-1619), also known as Fabricius, was a pioneer in embryology, anatomy, and surgery (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). During his professorship in Anatomy in Padua, the first stable anatomical theatre in the world was built. This anatomical theatre is still present at Palazzo Del Bo at the University of Padua (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). His anatomical studies included a description of the valves present in large veins which render the backward flow of venous blood improbable<xref rid="bib11" ref-type="bibr"> ¹¹ </xref>. Fabricius was the anatomy and surgery professor by the time William Harvey was studying medicine in Padua.</p></sec><sec><title>Andrea Cesalpino's Circulation</title><p>Andrea Cesalpino (1519-1603), was the director of the botanical garden in Pisa (<xref ref-type="fig" rid="fig10">Figure 10</xref>). He had limited studies in physiology. He theorized the pulmonary circulation without knowing the work of Realdo Colombo. Cesalpino formally coined the term &#x0201c;Circulation&#x0201d; to describe the physiology of blood. However, his concepts on circulation were chemical rather than physical, involving the continuous evaporation and condensation of blood. He was also one of the first to draw attention towards the swelling of the vein which takes below and never above the ligation, in contrast to Galen's teachings<xref rid="bib6" ref-type="bibr"> ⁶ </xref>.</p></sec><sec><title>William Harvey</title><p>William Harvey (1578-1657) was born in Kent, England (<xref ref-type="fig" rid="fig11">Figure 11A</xref>). In 1597, he finished his degree in arts at Gonville and Caius College, Cambridge. He later studied medicine in Padua, the greatest medical school of the time. In Padua, he was directly influenced by Fabricius and Galileo. In 1628, Harvey published his groundbreaking theory on blood circulation in a modest 72-page book written in Latin, entitled <italic>&#x0201c;Exercitatio anatomica de motu cordis et sanguinis in animalibus&#x0201d;</italic>. Harvey's work was met with much scepticism at the time of its publication as it challenged the existing dogmas of the time<xref rid="bib6" ref-type="bibr"> ⁶ </xref>.</p><p>In his seminal <italic>&#x0201c;de motu cordis et sanguinis&#x0201d;</italic>, Harvey laid the foundation of the modern concepts of blood circulation. He postulated that the main organ responsible for circulation was the heart and not the liver. He disagreed with the notion that the right ventricle only serves to nourish the lungs, and that blood passes from the right ventricle to the left ventricle through invisible inter-ventricular pores. He approved Colombo's views that blood must pass from the right side through a pulmonary transit to the left side of the heart. He also theorized that the intrinsic motion of the heart originate is the systole and not the diastole, and that arterial pulsations were due to impulses of the blood from the left ventricle. By estimating the cardiac output in about 12 kilos (3.000 &#x0201c;drachmas&#x0201d;) of blood every 30 minutes, Harvey argued that the quantity of blood is too great to be explained by a single-pass, open system and that blood must move in a circulatory pattern. Using his classical tourniquet experiment, Harvey demonstrated that blood moved into the limbs through the arteries and returned from it through the veins (<xref ref-type="fig" rid="fig11">Figure 11B</xref>). He also endorsed Fabricius'teachings that backward flow in the veins was not possible because of the venous valves. Harvey opposed the Galenic tradition that blood evaporated through skin breathing. Instead, he proposed that blood passed from the arterial side to the venous side through pores in the tissue<xref rid="bib6" ref-type="bibr"> ^6,12 </xref>.</p></sec><sec><title>Marcello Malpighi</title><p>In the <italic>de motu cordis</italic>, Harvey alluded to the possible presence of pulmonary capillaries and called them &#x0201c;pulmonum caecas porositates et vasorum eorum oscilla&#x0201d;, that is &#x0201c;the invisible porosity of the lungs and the minute cavities of their vessels&#x0201d;. Marcello Malpighi (1628&#x02013;1694) was an Italian physician, working in Pisa and Bologna, and one of the early pioneers of microscopical anatomy and histology (<xref ref-type="fig" rid="fig12">Figure 12A</xref>). With the help of the newly invented microscope, Malpighi solidified Harvey's concepts and was the first man ever to describe the pulmonary capillaries and alveoli<xref rid="bib13" ref-type="bibr"> ¹³ </xref> (<xref ref-type="fig" rid="fig12">Figure 12B</xref>).</p></sec><sec><title>The role of Ibn Al-Nafis</title><p>Three centuries before the works of Servetus, Colombo, Harvey, and Malpighi, the eminent thirteenth century Syrian physician Ibn Al-Nafis described the pulmonary circulation, alluding also to the presence of the pulmonary capillaries<xref rid="bib14" ref-type="bibr"> ¹⁴ </xref>. In a document entitled <italic>&#x0201c;Commentary on Anatomy in Avicenna's Canon&#x0201d;</italic>, the 29-year-old Ibn Al-Nafis challenged the classical anatomical teachings of Avicenna (<xref ref-type="fig" rid="fig12">Figure 12</xref>).</p><p>Avicenna (Ibn Sina in Arabic) (980&#x02013;1037) was a Persian physician and polymath. He was the most authoritarian figure in medicine during the Islamic Golden Era, that he was dubbed the title (ElSheikh AlRayees), or the &#x0201c;President Sheikh/ Grand Master&#x0201d;. His works, such as &#x0201c;The book of healing&#x0201d; and &#x0201c;the Canon in medicine&#x0201d;, were used as the fundamental textbooks in medical schools all over the world for as late as mid seventeenth century (<xref ref-type="fig" rid="fig13">Figure 13</xref>). Avicenna's medicine was markedly influenced by the Hippocratic and Galenic humourism and he adopted the Galenic concepts on cardiovascular medicine. The commentary written by Ibn Al-Nafis was only rediscovered in 1924 by an Egyptian PhD student &#x0201c;Muhyo AlDeen El-Tatawi&#x0201d;, at the Prussian State Library in Berlin. El-Tatawi later sent the document to Max Meyerhof, an experienced medical orientalist in Cairo. Meyerhof authenticated the document and subsequently translated the manuscript to German, French, and English<xref rid="bib14" ref-type="bibr"> ^14,15 </xref>.</p><p>In the <italic>Commentary</italic>, Ibn Al-Nafis denied the presence of the inter-ventricular pores that allowed the passage of blood from the right to the left ventricle. He emphasized this point more than once in the script<italic>: &#x0201c;&#x02026; but there is no passage between these two cavities [right and left ventricles]; for the substance of the heart is solid in this region and has neither a visible passage, as was thought by some persons, nor an invisible one which could have permitted the transmission of blood, as was alleged by Galen. The pores of the heart there are closed and its substance is thick.&#x0201d;</italic> and <italic>&#x0201c;There is no passage at all between these two ventricles; if there were the blood would penetrate to the place of the spirit [left ventricle] and spoil its substance. Anatomy refutes the contentions [of former authors]; on the contrary, the septum between the two ventricles is of thicker substance than other parts to prevent the passage of blood or spirits which might be harmful. Therefore the contention of some persons to say that this place is porous, is erroneous; it is based on the preconceived idea that the blood from the right ventricle had to pass through this porosity&#x02013;and they are wrong.&#x0201d;</italic> </p><p>Ibn Al-nafis argued that since there was no communication between the right and left ventricles through the inter-ventricular septum, then the output of the right ventricle could only reach the left ventricle via the pulmonary circulation: <italic>&#x0201c;the blood after it has been refined in this cavity [right ventricle], must be transmitted to the left cavity where the [vital] spirit is generated.&#x0201d;. &#x0201c;For the penetration of the blood into the left ventricle is from the lung, after it has been heated within the right ventricle and risen from it, as we stated before.&#x0201d;</italic> </p><p>Moreover, in an inspired prediction to Malpighi's descriptions 400 years later on the pulmonary capillaries and alveoli, Ibn Al-Nafis stated that there must be small communications between the pulmonary artery and the pulmonary vein: <italic>&#x0201c;And for the same reason there exists perceptible passages (or pores, manafidh) between the two [blood vessels, namely pulmonary artery and pulmonary vein].&#x0201d;. Also, he wrote:</italic> <italic>&#x0201c;The lungs are composed of three parts, one of which is the bronchi, the second the branches of the arteria venosa and the third the branches of the vena arteriosa, all of them connected by loose porous flesh.&#x0201d;</italic> </p><p>Finally, Ibn Al-Nafis also described accurately the coronary circulation: <italic>&#x0201c;His (Avicenna's) statement that the blood that is in the right side is to nourish the heart is not true at all, for the nourishment to the heart is from the blood that goes through the vessels that permeate the body of the heart&#x0201d;</italic>.<xref rid="bib15" ref-type="bibr"> ^15,16 </xref> </p></sec><sec><title>Ibn Al-Nafis, the Man</title><p>The full name of Ibn Al-Nafis was Abu Al-Hassan Alaa Al-Deen Ali ibn Abi-Hazm al-Qarshi al-Dimashqi<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref>. He was born in Qarsh, Syria, in 1213. Ibn Al-Nafis had his early medical education and practice in Damascus Medical College Hospital (Bimaristan Al-Noori) (<xref ref-type="fig" rid="fig14">Figure 14</xref>). Apart from medicine, he also learned jurisprudence, literature and theology. In 1236, moved to Cairo, Egypt where he initially worked at the Nur Al-Din Bimaristan. Subsequently he became chief of physicians at Bimaristan Al Mansouri. Other than his ground-breaking Commentary on Anatomy in Avicienna's Canon, Ibn Al-Nafis worked on his massive medical text book <italic>&#x0201c;El-Shamil&#x0201d;</italic> or the <italic>&#x0201c;The Comprehensive Book on the art of Medicine&#x0201d;. El-Shamil</italic> was an enormous body of work; some consider it one of the largest medical texts written by a single person, made of almost 300 books including most of medical sciences known at the time of Ibn Nafis. Unlike Avicenna's <italic>Canon</italic> and Rhazes' <italic>Comprehensive book of medicine</italic>, ElShamil was largely lost and only few scripts remain in the hands of historians today. Ibn Al-Nafis also wrote commentaries on Hippocrates, Avicenna, and Galen.</p><p>Ibn Al-Nafis was not simply a physician but an exemplary polymath. Regarding philosophy and arts, Ibn Al-Nafis had works on the logic of Avicenna <italic>&#x0201c;The Signs&#x0201d;</italic> and <italic>&#x0201c;The Guidance&#x0201d;,</italic> and on Aristotle's <italic>Organon</italic>. Most importantly, Ibn Al-Nafis wrote &#x0201c;<italic>Theologus Autodidactus&#x0201d;,</italic> the first theological novel known in Arabic literature and an early example of science fiction and &#x0201c;coming-of-age&#x0201d; genres. In addition, Ibn Al-Nafis produced works on Arabic language <italic>&#x0201c;Road to Eloquence&#x0201d;</italic> and on Islamic studies <italic>&#x0201c;A short account on the methodology of Hadith&#x0201d;</italic>. Ibn Al-Nafis based his scientific approach on experimentation and direct observation. He was critical to past knowledge and challenged scientific dogmas. He also used the Aristotelian logic for scientific deduction. He wrote extensively on the importance of categorization and classification of sciences and diseases<xref rid="bib17" ref-type="bibr"> ¹⁷ </xref>.</p></sec><sec><title>The unresolved questions</title><p>A few intriguing questions remain: did Servetus and Realdo Colombo read about Ibn Al-Nafis' work on pulmonary circulation? Servetus was a revolutionist theologian and had explored many texts by Islamic philosophers like Averros. While researching the Arabic philosophy, did he come across Ibn Al-Nafis' <italic>commentary</italic>? Realdo Colombo was a Professor in the progressive University of Padua, which was under the influence of the Republic of Venice at his time. Venice was always the window of medieval Europe to the Orient. Moreover, many Venetians were passionate book and manuscript collectors. Did Colombo have access to Ibn Al-Nafis' work by then? Colombo and Servetus lived in the same time, and Padua was known to be the most autonomous scientific institute of that time. Did Servetus communicate with Colombo to share with him his findings, knowing that Padua may give him scientific refuge?</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>The cardiovascular anatomy and physiology remained elusive to the human knowledge for long years. Humanity took a long and winding road to comprehend the nature of blood circulation (<xref ref-type="fig" rid="fig15">Figure 15</xref>). All the aforementioned scientists were visionaries. Each persisted on searching for the Truth to the best of his capabilities. Each strived to pass on his scientific message to the future generations and Humanity after him.<disp-quote><p>&#x0201c;<italic>When hearing something unusual, do not preemptively reject it, for that would be folly. Indeed, unconventional things may be true, and familiar and praised things may prove to be lies. Truth is truth unto itself, not because many people say it is.</italic>&#x0201d;<xref rid="bib17" ref-type="bibr"> ^17,18 </xref> (<xref ref-type="fig" rid="fig16">Figure 16</xref>)</p><p>Ibn Al-Nafis, <italic>&#x0201c;Commentary on Anatomy in Avicenna's Canon&#x0201d;</italic> </p></disp-quote> </p></sec>

Double outlet right atrium with three atrioventricular valves

<p>The atrioventricular (AV) valves are complex anatomical structures which perform sophisticated functions.<xref rid="bib1" ref-type="bibr"> ¹ </xref> A wide spectrum of malformations of these valves can occur in patients with AV septal defects. We here describe the anatomic and functional abnormalities of a rare form of the disease, where two valves connected the right atrium to both the right and left ventricles, in addition to a third valve that connected the left atrium to the left ventricle, with no evidence of regurgitation or cyanosis in spite of the relatively large communication between the right atrium and the left ventricle. In addition, the patient had severe subaortic stenosis. The pathophysiology, hemodynamics and method of repair of the condition are discussed.</p>

<contrib contrib-type="author"><name><surname>Hosny</surname><given-names>Hatem</given-names></name></contrib><contrib contrib-type="author"><name><surname>AbdelSalam</surname><given-names>Sherien</given-names></name></contrib><contrib contrib-type="author"><name><surname>Wally</surname><given-names>Hossam</given-names></name></contrib><contrib contrib-type="author"><name><surname>Yacoub</surname><given-names>Magdi</given-names></name><xref ref-type="corresp" rid="caf1">*</xref></contrib>

Global Cardiology Science &#x00026; Practice

<sec><title>Patient and methods</title><p>A 12-year-old female patient, presented with dyspnea on moderate effort. She had no history of cyanosis. Her resting oxygen saturation was 98%. Echocardiography revealed the diagnosis of atrioventricular septal defect (AVSD) with two adequate ventricles, closed atrial component and almost closed ventricular component, with what seemed to be aneurysmal tissue that had a tiny leak (<xref ref-type="fig" rid="fig1">Figures 1</xref>, <xref ref-type="fig" rid="fig2">2</xref>). A cord could be seen attached to that aneurysmal tissue (<xref ref-type="fig" rid="fig3">Figure 3</xref>). There was mild regurgitation through &#x0201c;cleft&#x0201d; left atrioventricular (AV) valve. The patient also had a subaortic membrane with a peak gradient of 70&#x000a0;mmHg across the left ventricular outflow tract, mild aortic regurgitation and left ventricular hypertrophy. There was no right or left ventricular dilatation.</p><p>The patient was referred to surgery for resection of the subaortic membrane and repair of the left AV valve. In surgery, the right atrium was opened for trans-septal access of the left AV valve. On opening the right atrium, two AV valves were found: a bigger AV valve opening to the right ventricle, directly attached to the muscular interventricular septum with no ventricular septal defect or aneurysmal tissue; and another small orifice opening to the left ventricle (<xref ref-type="fig" rid="fig4">Figure 4</xref>). There was no atrial septal defect. So the atrial septum was incised at the fossa ovalis, through which the left AV valve was seen opening to the left ventricle with &#x0201c;a cleft&#x0201d; (zone of apposition between the bridging leaflets).</p><p>The fossa ovalis was excised, creating good communication and the &#x0201c;cleft&#x0201d; in the left AV valve was closed. Then a fresh autologous pericardial patch was used to separate the two right AV valve orifices (<xref ref-type="fig" rid="fig5">Figure 5</xref>). The patch was then used to separate the right and left atria, leaving the small orifice connected to the left atrium (<xref ref-type="fig" rid="fig6">Figures 6</xref>, <xref ref-type="fig" rid="fig7">7</xref>). The coronary sinus was kept in the left atrium to avoid making a &#x0201c;waist&#x0201d; between the left AV valve and the small orifice. The subaortic membrane was resected with a limited myectomy.</p><p>The patient had a smooth post-operative course and was discharged from hospital after 5 days. The post-operative echo showed trivial right and left AV valve regurgitation. The mean diastolic gradient across the left AV valve was 4&#x000a0;mmHg. There was no significant gradient across the left ventricular outflow tract. The patient remained symptom-free and with the same echo findings in the routine follow-up after 2 months.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>This report illustrates several features of a rare variant of AV septal defect, with three AV valve orifices. The patient had AVSD with totally closed atrial component dividing the common AV valve into a right and left components. Misalignment between the atrial septum and muscular ventricular septum caused overriding and straddling of the right component of the common AV valve. The ventricular component of the AVSD has also totally closed causing the crest of the ventricular septum to divide the right AV valve into a valve connecting the right atrium to the right ventricle and another smaller valve connecting the right atrium to the left ventricle. Thus although the atrioventricular connections were seemingly concordant, at the same time there was double outlet right atrium and double inlet left ventricle with two balanced ventricles (<xref ref-type="fig" rid="fig8">Figure 8</xref>).</p><p>The tiny systolic flow, detected pre-operatively, that was thought to be a leak in the aneurysmal tissue closing the ventricular septal defect, was actually trivial regurgitation in the small orifice of the right AV valve connecting the right atrium to the left ventricle (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>Although there was a communication between the right atrium and the left ventricle, the patient did not give history of cyanosis and her resting oxygen saturation was 98%. This can be explained by the fact that the blood preferentially flowed from the right atrium to the right ventricle because the AV valve connecting them was significantly bigger than the other small orifice opening to the left ventricle, in addition the left ventricular compliance was much less than that of the right ventricle, which is secondary to the coexisting left ventricular Hypertrophy due to the subaortic membrane. Due to the difference in compliance between the right and left ventricles, which is increased further in our patient by the presence of left ventricular hypertrophy, due to the co existing subaortic stenosis. This is clearly evident by color Doppler in the pre-operative echo that shows almost no forward flow (from the right atrium to the left ventricle) across the small orifice (<xref ref-type="fig" rid="fig1">Figure 1</xref>), in comparison to the post-operative echo, that shows good flow across both orifices (<xref ref-type="fig" rid="fig7">Figure 7</xref>).</p><p>We chose to connect the small orifice to the left atrium rather than just closing it, because the left AV valve alone would have been small for the patient's body size, especially after closing the &#x0201c;cleft&#x0201d;.</p><p>AVSD can rarely occur without inter-atrial or inter-ventricular communications.<xref rid="bib2" ref-type="bibr"> ² </xref> The hallmark of diagnosis would then be the presence of common AV junction with trileaflet left AV valve.</p><p>Double orifice left AV valve occurs in AVSD when a tongue of tissue extends between the mural leaflet and one of the LV components of the bridging leaflets.<xref rid="bib3" ref-type="bibr"> ³ </xref> It occurs in about five percent of patients with partial AVSD.<xref rid="bib4" ref-type="bibr"> ⁴ </xref> This can also rarely occur with the right AV valve. Surgical repair of the left AV valve involves closure of the cleft in the main orifice leaving the accessory orifice intact, and the bridging tissue should not be divided as it is crucial for valve function.<xref rid="bib5" ref-type="bibr"> ⁵ </xref> </p><p>Double orifice left AV valve occurs when the two left valve orifices drain to the same ventricle. But if each orifice drains to a different ventricle, this is called double outlet atrium.<xref rid="bib6" ref-type="bibr"> ^6,7 </xref> Double outlet atrium is a quite rare condition. It can be double outlet right atrium or double outlet left atrium, and is generally caused by misaligned atrial or ventricular septae.<xref rid="bib8" ref-type="bibr"> ⁸ </xref> In some situations, as in our case, this can result in the presence of three AV valves.<xref rid="bib8" ref-type="bibr"> ^8&#x02013;10 </xref> If one AV connection is absent with straddling of the solitary AV valve, the condition will represent uni-atrial but bi-ventricular connection.<xref rid="bib11" ref-type="bibr"> ¹¹ </xref> </p><p>In conclusion, this was a rare case of AVSD with intact and misaligned atrial and ventricular septae and overriding and straddling of the right AV valve resulting in double outlet right atrium and double inlet left ventricle; in addition to subaortic membrane.</p></sec>

Correction: Can Tweets Predict Citations? Metrics of Social Impact Based on Twitter and Correlation with Traditional Metrics of Scientific Impact

Could not extract abstract

<contrib contrib-type="editor"><name><surname>Eysenbach</surname><given-names>Gunther</given-names></name></contrib>

Journal of Medical Internet Research

<p>A minor error in the references section in the originally published version of the editorial by Eysenbach (J Med Internet Res 2011;13[4]:e123) on the relationship between citations and tweetations has been corrected; in addition, references being part of the dataset are no longer cited as &#x0201c;references&#x0201d;. The now corrected problem with the references was a &#x0201c;formatting/presentation&#x0201d; problem only and had no impact on the study findings. The originally published article stated correctly that all 55 articles published between issue 3/2009 and 2/2010 were included, but the cited references erroneously contained 12 additional references from issue 2/2009, which were not part of the analysis, for the reasons described in the article (sparse tweetations pre-issue 3/2009). In the corrected version we have not only removed these extra 12 references (31-42), but we also took the opportunity to move all other references of included articles (43-97) into a new Multimedia Appendix 2, no longer citing them in the &#x0201c;References&#x0201d; section. We now refer to them in the paper by article ID (last 4-digits of the DOI), where we previously used in-text citations (Table 2 and Discussion). While there was nothing wrong with the way the articles were cited previously, and while we think that citing the JMIR articles whose impact we discuss in the paper is proper and necessary, we want to avoid any potential impression that this editorial artificially skews JMIR's future impact factor. One way to avoid this is to move the references to a separate file. The original decision to cite them as references was made for the sake of convenience for our readers, to prevent them from having to look up the references in a separate file or by DOI. JMIR has no space limitations and generally prefers to cite references in the article rather than in an Appendix; for readers downloading a PDF file it is more convenient to have all references in a single file rather than having to download a separate Appendix. The decision to now move these references into a Multimedia Appendix was made after a reader and publishing colleague pointed out that citing these articles may increase JMIR&#x02019;s impact factor. Although none of the two peer-reviewers, both experts in scientometrics, were originally concerned about citing the included articles as references, and even though any potential additional impact factor points after the decimal point caused by the original editorial would probably have been neglible (after all, these articles are already highly cited: altogether, 638 times, according to Google Scholar), and even though Thomson Reuters also publishes a journal impact factor that excludes journal self-citations, we wish to avoid any potential debate or uncertainty on what proportion of future JMIR impact factors were caused by this editorial, and have therefore decided to pre-emptively move these references into a separate file (Multimedia Appendix 2). The article correction was made on January 4, 2012, before submission to PubMed Central, Swets and other content aggregators and databases, and before indexing by Thomson Reuters. Having to remove references from a manuscript to preserve the validity of a journal-level impact metric is somewhat troubling, but if anything, then this perhaps illustrates the limitations and tyranny of the impact factor, and why we should consider additional metrics.</p>

Correction: Improving the Quality of Web Surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES)

Could not extract abstract

<contrib contrib-type="editor"><name><surname>Eysenbach</surname><given-names>Gunther</given-names></name></contrib>

Journal of Medical Internet Research

<p>An error in the CHERRIES statement has been corrected (J Med Internet Res 2004;6[3]:e34). In the original paper, in table 1, under the recommendations on how response rates (view rate, participation rate, and completion rate) should be calculated, denominators and numerators were flipped. The view rate should be the ratio of unique survey visitors divided by unique site visitors. The participation rate should be the ratio of those who agreed to participate divided by unique first survey page visitors. The completion rate is the ratio of the number of people who finished the survey divided by those who agreed to participate. The corrections have been made in the table in both columns. A corrected version has been submitted to PubMed Central, but incorrect versions may exist on other sites.</p>

Differential Susceptibility of Bacteria to Mouse Paneth Cell &#x003b1;-Defensins under Anaerobic Conditions

<p>Small intestinal Paneth cells secrete &#x003b1;-defensin peptides, termed cryptdins (Crps) in mice, into the intestinal lumen, where they confer immunity to oral infections and define the composition of the ileal microbiota. In these studies, facultative bacteria maintained under aerobic or anaerobic conditions displayed differential sensitivities to mouse &#x003b1;-defensins under <italic>in vitro</italic> assay conditions. Regardless of oxygenation, Crps 2 and 3 had robust and similar bactericidal activities against <italic>S. typhimurium</italic> and <italic>S. flexneri</italic>, but Crp4 activity against <italic>S. flexneri</italic> was attenuated in the absence of oxygen. Anaerobic bacteria varied in their susceptibility to Crps 2-4, with Crp4 showing less activity than Crps 2 and 3 against <italic>Enterococcus faecalis</italic>, and <italic>Bacteroides fragilis</italic> in anaerobic assays, but <italic>Fusobacterium necrophorum</italic> was killed only by Crp4 and not by Crps 2 and 3. The influence of anaerobiosis in modulating Crp bactericidal activities <italic>in vitro</italic> suggests that &#x003b1;-defensin effects on the enteric microbiota may be subject to regulation by local oxygen tension.</p>

<contrib contrib-type="author"><name><surname>Mastroianni</surname><given-names>Jennifer R.</given-names></name><xref ref-type="aff" rid="af1-antibiotics-03-00493">1</xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Wuyuan</given-names></name><xref ref-type="aff" rid="af2-antibiotics-03-00493">2</xref></contrib><contrib contrib-type="author"><name><surname>Selsted</surname><given-names>Michael E.</given-names></name><xref ref-type="aff" rid="af1-antibiotics-03-00493">1</xref></contrib><contrib contrib-type="author"><name><surname>Ouellette</surname><given-names>Andr&#x000e9; J.</given-names></name><xref ref-type="aff" rid="af1-antibiotics-03-00493">1</xref><xref rid="c1-antibiotics-03-00493" ref-type="corresp">*</xref></contrib>

Antibiotics

<sec><title>1. Introduction</title><p>Defensins are broad-spectrum microbicides with activities against diverse microbes and certain viruses [<xref rid="B1-antibiotics-03-00493" ref-type="bibr">1</xref>]. The &#x003b1;-defensin subfamily consists of amphipathic ~4.5 kDa peptides that share a common triple-stranded &#x003b2;-sheet topology. &#x003b1;-Defensins also have nine conserved residue positions, including Arg and Glu positions that form a salt bridge, a conserved Gly residue on a beta turn, and six Cys residues that form the characteristic tridisulfide array [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>]. Although these canonical features are highly conserved, the remaining 22&#x02013;25 residue positions may be occupied by varied amino acids, creating diverse &#x003b1;-defensin molecules with often differing target cell specificities [<xref rid="B1-antibiotics-03-00493" ref-type="bibr">1</xref>,<xref rid="B3-antibiotics-03-00493" ref-type="bibr">3</xref>]. Members of the &#x003b1;-defensin subfamily kill bacteria <italic>in vitro</italic> by electrostatic attractions between the cationic peptides and the electronegative bacterial cell envelope and subsequent hydrophobic interactions with phospholipid acyl chains that induce membrane disruption [<xref rid="B4-antibiotics-03-00493" ref-type="bibr">4</xref>,<xref rid="B5-antibiotics-03-00493" ref-type="bibr">5</xref>,<xref rid="B6-antibiotics-03-00493" ref-type="bibr">6</xref>]. In addition, certain &#x003b1;-defensins bind and inactivate lipid II thereby inhibiting cell wall biogenesis [<xref rid="B7-antibiotics-03-00493" ref-type="bibr">7</xref>,<xref rid="B8-antibiotics-03-00493" ref-type="bibr">8</xref>].</p><p>Enteric &#x003b1;-defensins are released apically by Paneth cells into the lumen of small intestinal crypts [<xref rid="B9-antibiotics-03-00493" ref-type="bibr">9</xref>,<xref rid="B10-antibiotics-03-00493" ref-type="bibr">10</xref>,<xref rid="B11-antibiotics-03-00493" ref-type="bibr">11</xref>]. In the small bowel, secreted Paneth cell &#x003b1;-defensins and additional host defense molecules confer immunity against certain pathogens, and they determine the composition of the ileal microbiota [<xref rid="B12-antibiotics-03-00493" ref-type="bibr">12</xref>,<xref rid="B13-antibiotics-03-00493" ref-type="bibr">13</xref>,<xref rid="B14-antibiotics-03-00493" ref-type="bibr">14</xref>]. &#x003b1;-Defensins constitute the majority of bactericidal peptide activity released by Paneth cells, and mice that are defective in Paneth cell homeostasis are subject to dysbiosis and blooms of select bacterial species [<xref rid="B15-antibiotics-03-00493" ref-type="bibr">15</xref>,<xref rid="B16-antibiotics-03-00493" ref-type="bibr">16</xref>,<xref rid="B17-antibiotics-03-00493" ref-type="bibr">17</xref>,<xref rid="B18-antibiotics-03-00493" ref-type="bibr">18</xref>]. In addition, Paneth cell &#x003b1;-defensins persist in mouse colonic lumen, although their role in colonic innate immunity is uncertain, given the 10¹² to 10¹⁴ bacteria per gram of tissue luminal contents in that environment [<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>,<xref rid="B20-antibiotics-03-00493" ref-type="bibr">20</xref>]. </p><p>The gastrointestinal tract is colonized by complex microbial consortia, which are critical in mucosal protection, immunological development, nutrition and metabolism [<xref rid="B20-antibiotics-03-00493" ref-type="bibr">20</xref>,<xref rid="B21-antibiotics-03-00493" ref-type="bibr">21</xref>,<xref rid="B22-antibiotics-03-00493" ref-type="bibr">22</xref>]. It is estimated that 99% of intestinal microbiota are strict anaerobes, predominantly members of the phyla Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria [<xref rid="B20-antibiotics-03-00493" ref-type="bibr">20</xref>,<xref rid="B23-antibiotics-03-00493" ref-type="bibr">23</xref>], and they constitute a potential infectious challenge if homeostasis of the intestinal epithelium is disrupted. In mouse ileum, the composition of the microbiota is determined by Paneth cell &#x003b1;-defensins, perhaps by selection of peptide-tolerant bacterial species [<xref rid="B14-antibiotics-03-00493" ref-type="bibr">14</xref>]. For example, the relative numbers of Firmicutes and Bacterioidetes in ileum of mice expressing a human DEFA5 transgene (<italic>DEFA5</italic> (+/+)) and congenic FVB mice are markedly different [<xref rid="B14-antibiotics-03-00493" ref-type="bibr">14</xref>,<xref rid="B20-antibiotics-03-00493" ref-type="bibr">20</xref>]. These findings illustrate how Paneth cell secretion of a single additional &#x003b1;-defensin can influence the commensal population, and they provide rationale for characterizing the effects of enteric &#x003b1;-defensins on anaerobic bacteria.</p><p>Although antimicrobial activities of &#x003b1;-defensins have been studied extensively in the presence of oxygen [<xref rid="B1-antibiotics-03-00493" ref-type="bibr">1</xref>,<xref rid="B24-antibiotics-03-00493" ref-type="bibr">24</xref>,<xref rid="B25-antibiotics-03-00493" ref-type="bibr">25</xref>,<xref rid="B26-antibiotics-03-00493" ref-type="bibr">26</xref>], their microbicidal effects against anaerobes of the gastrointestinal microbiota have remained mostly unknown. Against facultative periodontal bacteria, antibacterial activities of HNPs 1&#x02013;3 under aerobic and anaerobic conditions varied with the microbial target [<xref rid="B27-antibiotics-03-00493" ref-type="bibr">27</xref>,<xref rid="B28-antibiotics-03-00493" ref-type="bibr">28</xref>]. Under both conditions, the more electropositive rabbit NP-1 &#x003b1;-defensin peptide proved more potent than human neutrophil &#x003b1;-defensins (HNPs), suggesting that mouse &#x003b1;-defensins, also strongly cationic, may be particularly bactericidal under anaerobic conditions. Human &#x003b1;-defensin HD5 and human &#x003b2;-defensins (hBDs) 1&#x02013;3 also showed variable antimicrobial activities against anaerobes in assays that measured membrane potential as an index of bacterial viability [<xref rid="B29-antibiotics-03-00493" ref-type="bibr">29</xref>], and HD5 was active against facultatives but had low activity against strict anaerobes <italic>in vitro</italic>. Among mouse &#x003b1;-defensins, Crp4 has shown selective bactericidal activities against certain, but not all, anaerobic bacterial species [<xref rid="B30-antibiotics-03-00493" ref-type="bibr">30</xref>]. Here, we report on the relative bactericidal activities of mouse Paneth cell &#x003b1;-defensins against anaerobic and facultative bacteria under strict anaerobic conditions. Under these conditions, the activity of individual mouse Paneth cell &#x003b1;-defensins was highly variable against facultatives as a function of anaerobiosis. Anaerobic bacterial species, including <italic>Bacteroides fragilis</italic> (Bacteroidetes) and <italic>Clostridium difficile</italic> (Firmicutes), phyla whose numbers <italic>in vivo</italic> are affected by Paneth cell &#x003b1;-defensins [<xref rid="B31-antibiotics-03-00493" ref-type="bibr">31</xref>], also displayed variable sensitivity to these &#x003b1;-defensins.</p></sec><sec><title>2. Results and Discussion</title><sec><title>2.1. &#x003b1;-Defensin Activities against Facultative Bacteria under Aerobic and Anaerobic Conditions</title><p>To test whether mouse &#x003b1;-defensins assayed in an anaerobic environment against facultative and strict anaerobic bacterial species retain structural integrity, we assessed peptide homogeneity and molecular masses by AU-PAGE (<xref ref-type="fig" rid="antibiotics-03-00493-f001">Figure 1</xref>A) and MALDI-TOF MS and showed that peptides maintained their disulfide arrays under anaerobic conditions. Samples of proCrp4, Crp2, Crp3, and Crp4 dissolved in 0.01% acetic acid, 10 mM PIPES, and 1% (v/v) Brucella broth to replicate assay conditions were incubated aerobically or anaerobically and tested for spontaneous disulfide bond reduction in the absence of oxygen. After 2 h under anaerobic conditions, the four peptides (<xref ref-type="fig" rid="antibiotics-03-00493-f001">Figure 1</xref>B) had atomic masses equal to native, oxidized peptides, showing that anaerobic conditions did not reduce disulfide bonds to free thiols. Also, peptide mobilities in AU-PAGE, a gel system that separates &#x003b1;-defensin disulfide bond variants or foldamers at high resolution [<xref rid="B32-antibiotics-03-00493" ref-type="bibr">32</xref>], were those of the native peptides (<xref ref-type="fig" rid="antibiotics-03-00493-f001">Figure 1</xref>A). Thus, the tridisulfide arrays of these &#x003b1;-defensins were unaffected by anaerobic assay conditions. </p><p>The bactericidal activities of mouse Crps 2&#x02013;4 were compared against facultative bacterial species under aerobic and anaerobic conditions. <italic>In vitro</italic>, Crps 2&#x02013;4 kill 99.9% of most bacteria at &#x02264;3 &#x003bc;M peptide levels when assayed under normoxia [<xref rid="B13-antibiotics-03-00493" ref-type="bibr">13</xref>,<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>,<xref rid="B24-antibiotics-03-00493" ref-type="bibr">24</xref>]. Here, we measured their relative activities in the presence or absence of air over 1 to 15 &#x003bc;M peptide levels. Under either condition, Crps 2&#x02013;4 were bactericidal against <italic>S. typhimurium</italic>, <italic>S. flexneri</italic>, and <italic>E. coli</italic> ML35 (<xref ref-type="fig" rid="antibiotics-03-00493-f002">Figure 2</xref>C&#x02013;H and <xref ref-type="fig" rid="antibiotics-03-00493-f003">Figure 3</xref>A,D,G), and Crps 2 and 3 had robust and similar bactericidal activities against <italic>S. typhimurium</italic> and <italic>S. flexneri</italic>, regardless of oxygenation. However, under anaerobiosis, Crp4 bactericidal activity was reduced markedly against <italic>S. flexneri</italic> and modestly so against <italic>S. typhimurium</italic>. Regardless of assay conditions, proCrp4 lacked activity (<xref ref-type="fig" rid="antibiotics-03-00493-f002">Figure 2</xref>A,B), consistent with its lack of activity prior to proteolytic activation by MMP-7 [<xref rid="B33-antibiotics-03-00493" ref-type="bibr">33</xref>]. As expected from previous determinations of <italic>in vitro</italic> bactericidal activities [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>,<xref rid="B24-antibiotics-03-00493" ref-type="bibr">24</xref>], Crp4 potency was greater than that of Crps 2 and 3 when assays were performed in air (<xref ref-type="table" rid="antibiotics-03-00493-t001">Table 1</xref>). However, under anaerobic conditions, the activity of 15 &#x003bc;M Crp4 against <italic>S. flexneri</italic> was significantly lower than Crps 2 and 3. Thus, direct cell killing of facultative bacteria by individual Paneth cell &#x003b1;-defensins varies as a function of the presence or absence of oxygen.</p></sec><sec><title>2.2. Sensitivity of E. coli to Mouse &#x003b1;-Defensins under Aerobic and Anaerobic Conditions</title><p>To examine whether pathogenic <italic>E. coli</italic> strains are as susceptible to mouse Paneth cell &#x003b1;-defensins as a laboratory-adapted strain under anaerobic conditions, three strains of <italic>E. coli</italic> were tested for defensin sensitivity. All <italic>E. coli</italic> strains were susceptible to Crps 2&#x02013;4, and Crp4 had the greatest activity against all strains, regardless of assay conditions. On the other hand, pathogenic <italic>E. coli</italic> ci and EPEC strains survived exposure to 1&#x02013;5 &#x003bc;M Crps 2 and 3 better under anaerobic conditions than in air (<xref ref-type="table" rid="antibiotics-03-00493-t001">Table 1</xref>, <xref ref-type="fig" rid="antibiotics-03-00493-f003">Figure 3</xref>B,C,E,F), and <italic>E. coli</italic> ci was less susceptible to Crp4 under anaerobic conditions (<xref ref-type="fig" rid="antibiotics-03-00493-f003">Figure 3</xref>H).</p><fig id="antibiotics-03-00493-f001" position="float"><label>Figure 1</label><caption><p>Acid-Urea PAGE of &#x003b1;-Defensins after Anaerobic Incubation. (<bold>A</bold>) cryptdin (Crp)2, Crp3, Crp4, and proCrp4 were incubated under anaerobic conditions (see Experimental) for 2 h and analyzed by AU-PAGE. (1) Aerobic control peptides dissolved in in 0.01% acetic acid, (2) Peptides incubated under anaerobic conditions in 0.01% acetic acid, (3) Peptides incubated in 0.01% acetic acid, 1% Brucella broth (BRU, see Experimental) broth under anaerobic conditions as described in the Experimental section; (<bold>B</bold>) ProCrp4, Crp2, Crp3, Crp4 primary structures are shown for reference with disulfide pairings shown below the Crp4 sequence. Arrow at right indicates the final cleavage event in proCrp processing by matrix metalloproteinase-7, the activating convertase. Dashes were introduced to maintain the cysteine spacing in proCrp4 and Crp4 for alignment with Crp2 and Crp3.</p></caption><graphic xlink:href="antibiotics-03-00493-g001"/></fig><fig id="antibiotics-03-00493-f002" position="float"><label>Figure 2</label><caption><p>Bactericidal Activities of Mouse &#x003b1;-Defensins against Facultative Organisms under Aerobic and Anaerobic Conditions. <italic>S. typhimurium</italic> 14028s (A, C, E, G) and <italic>Shigella flexneri</italic> BS497 (B, D, F, H) were Target Organisms for 0, 1, 5, or 15 &#x003bc;M proCrp4 (A, B), Crp2 (C, D), Crp3 (E, F), and Crp4 (G, H) under Both Aerobic (open symbols) and Anaerobic (Closed Symbols) Conditions. Data from three independent experiments are expressed as the mean &#x000b1; standard deviations.</p></caption><graphic xlink:href="antibiotics-03-00493-g002"/></fig><fig id="antibiotics-03-00493-f003" position="float"><label>Figure 3</label><caption><p>Bactericidal Activities of Crps 2-4 against <italic>E. coli</italic> Strains under Aerobic and Anaerobic Conditions. At concentrations of 1, 5, or 15 &#x003bc;M Crp2 (A, B, C), Crp3 (D, E, F), and Crp4 (G, H, I) were assayed for bactericidal activity against <italic>E. coli</italic> ML35 (A, D, G), <italic>E. coli</italic> clinical isolate (B, E, H), and an EPEC strain (C, F, I) under aerobic (open symbols) and anaerobic (closed symbols) conditions. Data shown are from three independent experiments expressed as means &#x000b1; standard deviations.</p></caption><graphic xlink:href="antibiotics-03-00493-g003"/></fig><table-wrap id="antibiotics-03-00493-t001" position="float"><object-id pub-id-type="pii">antibiotics-03-00493-t001_Table 1</object-id><label>Table 1</label><caption><p>Minimum Bactericidal Concentrations (MBC) Values were Determined as the Lowest Peptide Concentrations that Reduced the Number of Viable Bacteria by 99.9%. Due to the large difference between tested concentrations, a range is given. </p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" rowspan="1" colspan="1">Bacteria</th><th align="center" valign="middle" rowspan="1" colspan="1">Oxygen Status</th><th align="center" valign="middle" rowspan="1" colspan="1">Crp2 MBC (&#x003bc;M)</th><th align="center" valign="middle" rowspan="1" colspan="1">Crp3 MBC (&#x003bc;M)</th><th align="center" valign="middle" rowspan="1" colspan="1">Crp4 MBC (&#x003bc;M)</th></tr></thead><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Shigella flexneri</italic> BS497</td><td align="left" valign="middle" rowspan="1" colspan="1">aerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">90% ^a</td><td align="center" valign="middle" rowspan="1" colspan="1">99% ^a</td><td align="center" valign="middle" rowspan="1" colspan="1">99% ^a</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Salmonella</italic> Typhimurium WT</td><td align="left" valign="middle" rowspan="1" colspan="1">aerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">99% ^a</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>E. coli</italic> ML35</td><td align="left" valign="middle" rowspan="1" colspan="1">aerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>E. coli</italic> clinical isolate</td><td align="left" valign="middle" rowspan="1" colspan="1">aerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">1</td><td align="center" valign="middle" rowspan="1" colspan="1">1</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">EPEC</td><td align="left" valign="middle" rowspan="1" colspan="1">aerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Clostridium difficile</italic> </td><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Bacteroides fragilis</italic> </td><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">0%^a</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Fusobacterium necrophorum</italic> </td><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">0% ^a</td><td align="center" valign="middle" rowspan="1" colspan="1">60% ^a</td><td align="center" valign="middle" rowspan="1" colspan="1">1&#x02013;5</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>Enterococcus faecalis</italic> </td><td align="left" valign="middle" rowspan="1" colspan="1">anaerobic</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">5&#x02013;15</td><td align="center" valign="middle" rowspan="1" colspan="1">90% ^a</td></tr></tbody></table><table-wrap-foot><fn><p>If 99.9% killing was not reached, then the percentage of bacteria killed at the highest tested concentration, 15 &#x003bc;M, is shown.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>2.3. Differential Effects of Crps 2&#x02013;4 against Anaerobic Bacteria</title><p>The susceptibility of anaerobic bacteria to &#x003b1;-defensins were determined for Crps 2&#x02013;4, showing that the four species tested responded differently to specific peptides. <italic>C. difficile</italic> (Firmicutes), <italic>B. fragilis</italic> (Bacteroidetes) and the facultative anaerobe <italic>E. faecalis</italic> (Firmicutes) were selected for study, because the ratio of Firmicutes to Bacteroidetes of the ileal microbiota change markedly as a function of qualitative differences in Paneth cell &#x003b1;-defensins [<xref rid="B31-antibiotics-03-00493" ref-type="bibr">31</xref>]. Also, we chose the veterinary and human pathogen <italic>F. necrophorum</italic>, because Fusobacteria have been implicated in exacerbating inflammatory bowel disease and in colon cancer [<xref rid="B34-antibiotics-03-00493" ref-type="bibr">34</xref>,<xref rid="B35-antibiotics-03-00493" ref-type="bibr">35</xref>]. Under anaerobic conditions, Crps 2 and 3 showed greater bactericidal activity against <italic>E. faecalis</italic>, and <italic>B. fragilis</italic> than Crp4 (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>B, C), greater activity against <italic>C. difficile</italic> at &#x02264;5 &#x003bc;M but the same activity as Crp4 at 20 &#x003bc;M peptide levels (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>A). However, Crps 2 and 3 both lacked activity against <italic>F. necrophorum</italic> in contrast to Crp4, which was highly bactericidal against <italic>F. necrophorum</italic> (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>D). Although Crp4 was the most bactericidal of the known mouse &#x003b1;-defensins in previous <italic>in vitro</italic> assays performed in room air [<xref rid="B24-antibiotics-03-00493" ref-type="bibr">24</xref>], the results of the current study are consistent with the low Crp4 activity reported against <italic>B. fragilis</italic> [<xref rid="B30-antibiotics-03-00493" ref-type="bibr">30</xref>], which is sensitive to Crps 2 and 3 (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>). Therefore, anaerobic bacteria vary in susceptibility to mouse Paneth cell &#x003b1;-defensins, and individual &#x003b1;-defensins differ in their activities against anaerobic bacterial species.</p><fig id="antibiotics-03-00493-f004" position="float"><label>Figure 4</label><caption><p>Bactericidal Activities of Crps 2-4 against Anaerobic Bacteria. <italic>C. difficile</italic> ATCC 9689 (A), <italic>E. faecalis</italic> ATCC 29214 (B), <italic>B. fragilis</italic> ATCC 25285 (C), and <italic>F. necrophorum</italic> ATCC 25286 (D) were incubated with 1, 5, or 15 &#x003bc;M Crp 2 (-&#x025a0;-), Crp3 (-&#x02666;-), and Crp4 (-&#x025bc;-) under anaerobic conditions in three independent experiments. Data are expressed as the means &#x000b1; standard deviations.</p></caption><graphic xlink:href="antibiotics-03-00493-g004"/></fig></sec><sec><title>2.4. Comparative Bactericidal Activities of Mouse &#x003b1;-Defensin Mixtures</title><p>Inbred strains of mice, C57BL/6 mice in particular, are polymorphic with respect to Paneth cell expression of &#x003b1;-defensins [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>]. Specifically, most inbred strains, including BALB/c, FVB, C3H/HeJ/N, and 129/SvJ strains as well as outbred Swiss mice, produce high levels of Crps 1&#x02013;6 [<xref rid="B36-antibiotics-03-00493" ref-type="bibr">36</xref>]. However, C57BL/6 mice lack the <italic>Defa1</italic>, <italic>2</italic>, <italic>4</italic> and <italic>6</italic> genes for Crps 1, 2, 4, and 6, expressing instead high levels of Crps 20, 21, 23, and 27 [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>]. Because the peptides tested in this study are absent from C57BL/6 mice, the most frequent background for genetic modification, we tested the bactericidal activity of &#x003b1;-defensin mixtures from outbred Swiss and C57BL/6 small intestine [<xref rid="B24-antibiotics-03-00493" ref-type="bibr">24</xref>,<xref rid="B36-antibiotics-03-00493" ref-type="bibr">36</xref>]. </p><p>&#x003b1;-Defensin mixtures were purified from Swiss and C57BL/6 mouse small intestinal protein extracts by sequential gel permeation and cation exchange chromatography [<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>,<xref rid="B37-antibiotics-03-00493" ref-type="bibr">37</xref>]. Because they exhibited variable peptide-specific sensitivities to Crps 2&#x02013;4 (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>), <italic>F. necrophorum</italic> and <italic>C. difficile</italic> were selected as target organisms for comparisons of the native &#x003b1;-defensin mixtures. Despite qualitative differences in &#x003b1;-defensin content of the two mixtures [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>,<xref rid="B33-antibiotics-03-00493" ref-type="bibr">33</xref>], the bactericidal activities of the mixed &#x003b1;-defensin preparations were the same against either anaerobic species (<xref ref-type="fig" rid="antibiotics-03-00493-f005">Figure 5</xref>).</p><fig id="antibiotics-03-00493-f005" position="float"><label>Figure 5</label><caption><p>Bactericidal Activities of Swiss and C57BL/6 Native &#x003b1;-Defensin Mixtures against <italic>C. difficile</italic> and <italic>F. necrophorum</italic>. <italic>C. difficile</italic> ATCC 9689 (A) and <italic>F. necrophorum</italic> ATCC 25286 (B) were test anaerobes for bactericidal assays using preparations of &#x003b1;-defensins from complete ileum of mice that have qualitatively different &#x003b1;-defensins [<xref rid="B2-antibiotics-03-00493" ref-type="bibr">2</xref>]. Bacteria were incubated with 5, 20, or 40 &#x003bc;g/mL of the following: Crp4 (-&#x025bc;-), outbred Swiss Crps (-&#x025a0;-), or C57BL/6 Crps (-&#x02666;-). Surviving bacteria were counted with data expressed as the percent of bacterial survival in each sample relative to control samples lacking peptides (<xref ref-type="sec" rid="sec3-antibiotics-03-00493">Experimental Section</xref>). Data from three independent experiments are expressed as means &#x000b1; standard deviations.</p></caption><graphic xlink:href="antibiotics-03-00493-g005"/></fig></sec><sec><title>2.5. Discussion</title><p>To simulate the anaerobic conditions under which &#x003b1;-defensin-microbial interactions are believed to occur in the ileum and colonic lumen, we have tested the effect of anaerobiosis on mouse &#x003b1;-defensin bactericidal activity <italic>in vitro</italic>. Unexpectedly, the activity of Crp4, a consistently potent microbicide when assays are performed in air, was attenuated against the facultative pathogen <italic>S. flexneri</italic> under anaerobic conditions (<xref ref-type="fig" rid="antibiotics-03-00493-f002">Figure 2</xref>). In addition, differential Crp2-4 activities were evident against <italic>E. coli</italic> strains, with Crps 2 and 3 exhibiting attenuated activities against the two pathogenic <italic>E. coli</italic> strains tested under anaerobic conditions (<xref ref-type="fig" rid="antibiotics-03-00493-f003">Figure 3</xref>). Moreover, <italic>E. faecalis</italic>, and <italic>B. fragilis</italic> were sensitive to Crps 2 and 3 at low peptide levels, with Crp4 again showing markedly lower activity at 15 &#x003bc;M peptide levels when assayed under anaerobiosis (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>). In direct contrast to these results, only Crp4 was bactericidal against <italic>F. necrophorum</italic> (<xref ref-type="fig" rid="antibiotics-03-00493-f004">Figure 4</xref>), revealing extensive variability of &#x003b1;-defensin-mediated killing as a function of anoxia. Given the variability of individual Crp activities and the differential effects of oxygen on them, we compared native &#x003b1;-defensin mixtures from C57BL/6 and outbred Swiss mice against <italic>F. necrophorum</italic> and <italic>C. difficile</italic>. Against these strict anaerobes, the combined bactericidal activities of the different Crp mixtures were equivalent (<xref ref-type="fig" rid="antibiotics-03-00493-f005">Figure 5</xref>). Thus, although the anaerobic environment alters the activities of individual Crp peptides against specific bacterial species, mixtures of distinctly different Crp peptides in molar ratios reflecting those in Paneth cell secretions, are similar in their overall bactericidal effects against the anaerobes tested.</p><p>Anatomically restricted to the small bowel, Paneth cells secrete dense core granules rich in &#x003b1;-defensins and host defense molecules apically into the lumen [<xref rid="B38-antibiotics-03-00493" ref-type="bibr">38</xref>,<xref rid="B39-antibiotics-03-00493" ref-type="bibr">39</xref>,<xref rid="B40-antibiotics-03-00493" ref-type="bibr">40</xref>]. Luminal &#x003b1;-defensins confer immunity to oral infection and define the composition of the ileal microbiota [<xref rid="B14-antibiotics-03-00493" ref-type="bibr">14</xref>,<xref rid="B41-antibiotics-03-00493" ref-type="bibr">41</xref>], and defects in delivery of activated Paneth cell &#x003b1;-defensins to the lumen affect enteric innate immunity adversely [<xref rid="B40-antibiotics-03-00493" ref-type="bibr">40</xref>,<xref rid="B42-antibiotics-03-00493" ref-type="bibr">42</xref>]. Although Paneth cell &#x003b1;-defensins are secreted only in the small bowel, the peptides can be recovered from distal colonic lumen [<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>,<xref rid="B37-antibiotics-03-00493" ref-type="bibr">37</xref>]. Because the colonic microbial burden is orders of magnitude greater than in small bowel and colonic &#x003b1;-defensins are less abundant than in small intestine, &#x003b1;-defensins may not affect the colonic microbiota by direct peptide-mediated cell killing. In mouse studies where Paneth cell &#x003b1;-defensins were shown to alter the composition of the ileal microbiota, the composition of the cecal and colonic microbiota were unaffected by Paneth cell &#x003b1;-defensins [<xref rid="B14-antibiotics-03-00493" ref-type="bibr">14</xref>]. Also, peptides that are not bactericidal when assayed under in anaerobic conditions may have bacteriostatic affects under those conditions, and that possibility has not been tested in the studies presented here. </p><p>The differential susceptibilities to cryptdins of bacteria grown anaerobically may result from alterations in membrane composition and metabolism. The &#x003b1;-defensins studied here cause direct bactericidal action by peptide-mediated membrane disruption at the concentrations tested [<xref rid="B43-antibiotics-03-00493" ref-type="bibr">43</xref>,<xref rid="B44-antibiotics-03-00493" ref-type="bibr">44</xref>,<xref rid="B45-antibiotics-03-00493" ref-type="bibr">45</xref>]. In response to environmental changes or chronic peptide exposure, bacteria may acquire resistance to antimicrobial peptides by altering membrane fluidity and by decreasing the net electronegative charge of the bacterial cell surface [<xref rid="B46-antibiotics-03-00493" ref-type="bibr">46</xref>,<xref rid="B47-antibiotics-03-00493" ref-type="bibr">47</xref>,<xref rid="B48-antibiotics-03-00493" ref-type="bibr">48</xref>]. For example, <italic>S. typhimurium</italic> modifies its membrane via lipid A acylation [<xref rid="B47-antibiotics-03-00493" ref-type="bibr">47</xref>], and bacteria regulate lipid A modification in response to Mg²⁺ concentrations [<xref rid="B46-antibiotics-03-00493" ref-type="bibr">46</xref>]. As anaerobiosis influences virulence and pathogenesis by processes such as iron acquisition and sequestration [<xref rid="B49-antibiotics-03-00493" ref-type="bibr">49</xref>,<xref rid="B50-antibiotics-03-00493" ref-type="bibr">50</xref>,<xref rid="B51-antibiotics-03-00493" ref-type="bibr">51</xref>,<xref rid="B52-antibiotics-03-00493" ref-type="bibr">52</xref>], oxygen tension, much like iron concentration, may signal changes that alter susceptibility to &#x003b1;-defensins by inducing lipid A or lipoteichoic acid modification. Alternatively, anaerobiosis may alter membrane energetics or bacterial growth, increasing susceptibility to specific &#x003b1;-defensins under anaerobic conditions as was observed for <italic>S. flexneri</italic> with Crp4.</p><p>Variations in the microbiota such as those associated with changes to Paneth cell &#x003b1;-defensin composition have implications in host health and disease. Bacteria can modulate the composition of the microbiota by competing for growth-limiting resources or by production of direct microbicides such as bacteriocins and lantibiotics [<xref rid="B53-antibiotics-03-00493" ref-type="bibr">53</xref>,<xref rid="B54-antibiotics-03-00493" ref-type="bibr">54</xref>]. In addition, proteases secreted by <italic>E. faecalis</italic>, for example, convert proCrp4 to mature Crp4 <italic>in vitro</italic> [<xref rid="B37-antibiotics-03-00493" ref-type="bibr">37</xref>], suggesting that bacteria may regulate the microbiota by activating or degrading AMPs in the lower gastrointestinal tract. In humans, changes in the microbiota and &#x003b1;-defensin expression levels are two of among many factors associated with ileal Crohn&#x02019;s disease [<xref rid="B42-antibiotics-03-00493" ref-type="bibr">42</xref>,<xref rid="B55-antibiotics-03-00493" ref-type="bibr">55</xref>,<xref rid="B56-antibiotics-03-00493" ref-type="bibr">56</xref>], and changes to the microbiota are linked to chronic inflammatory disorders and systemic diseases, including ulcerative colitis, obesity, cancer, diabetes, allergic reactions, and cardiovascular disease [<xref rid="B57-antibiotics-03-00493" ref-type="bibr">57</xref>,<xref rid="B58-antibiotics-03-00493" ref-type="bibr">58</xref>,<xref rid="B59-antibiotics-03-00493" ref-type="bibr">59</xref>,<xref rid="B60-antibiotics-03-00493" ref-type="bibr">60</xref>]. Because of the role of anaerobic commensal organisms in overall health, understanding the interplay between host defense peptides, including &#x003b1;-defensins, and the mainly anaerobic intestinal flora may have consequences in innate immunity and in disease.</p></sec></sec><sec id="sec3-antibiotics-03-00493"><title>3. Experimental Section</title><sec><title>3.1. Preparation of Recombinant and Synthetic Peptides</title><p>Crp4 and proCrp4 were produced by recombinant methods and purified to homogeneity as described [<xref rid="B61-antibiotics-03-00493" ref-type="bibr">61</xref>,<xref rid="B62-antibiotics-03-00493" ref-type="bibr">62</xref>]. Briefly, recombinant peptides were expressed in <italic>Escherichia coli</italic> as N-terminal His6-tagged fusion proteins using the pET-28a expression vector (Novagen Inc., Madison, WI) and isolated by affinity purification. Affinity purified recombinant fusion proteins were cleaved with CNBr to separate the vector coded fusion protein from the expressed &#x003b1;-defensin, diluted, lyophilized, and recombinant peptides were purified to homogeneity by C18 RP-HPLC as before [<xref rid="B33-antibiotics-03-00493" ref-type="bibr">33</xref>,<xref rid="B61-antibiotics-03-00493" ref-type="bibr">61</xref>,<xref rid="B63-antibiotics-03-00493" ref-type="bibr">63</xref>]. Peptide homogeneity was assessed by analytical RP-HPLC and acid-urea (AU)-PAGE [<xref rid="B32-antibiotics-03-00493" ref-type="bibr">32</xref>], and peptide masses were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a Microflex LRF mass spectrometer in linear mode (Bruker Daltonics, Maynard, MA, USA).</p><p>Homogeneous preparations of Crp2 and Crp3 were synthesized using Boc chemistry for solid phase peptide synthesis as described for human alpha-defensins [<xref rid="B64-antibiotics-03-00493" ref-type="bibr">64</xref>,<xref rid="B65-antibiotics-03-00493" ref-type="bibr">65</xref>,<xref rid="B66-antibiotics-03-00493" ref-type="bibr">66</xref>]. The molecular masses of resultant Crp2 and Crp3 in their reduced forms were verified by electrospray ionization mass spectrometry to be within experimental error of their calculated theoretical values. After HPLC purification, the synthetic polypeptides were oxidatively folded at 0.25 mg/mL in 50 mM Tris/HCl buffer containing 1 M guanidinium HCl, 3 mM reduced glutathione, and 0.3 mM oxidized glutathione, pH 8.3, followed by HPLC purification to homogeneity (<xref ref-type="fig" rid="antibiotics-03-00493-f001">Figure 1</xref>A). The formation of three intra-molecular disulfides was ascertained by the loss of 6 mass units upon folding. </p></sec><sec><title>3.2. Purification of Tissue-Derived Mouse Enteric &#x003b1;-Defensins</title><p>&#x003b1;-Defensins were isolated from complete mouse ileum, consisting of organ plus luminal contents, as described [<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>,<xref rid="B36-antibiotics-03-00493" ref-type="bibr">36</xref>]. Briefly, segments of complete ileum excised from outbred Swiss or C57BL/6 mice immediately after euthanasia were homogenized in 100 mL ice-cold 60% acetonitrile, 1% trifluoroacetic acid (TFA), incubated at 4 &#x000b0;C overnight, clarified by centrifugation, and lyophilized. Extract proteins dissolved in 5 mL of 5% acetic acid were chromatographed on 10 &#x000d7; 120 cm Bio-Gel P-60 columns (Bio-Rad), and &#x003b1;-defensins were identified as rapidly migrating peptides in P-60 fractions by MALDI-TOF MS and AU-PAGE analysis [<xref rid="B32-antibiotics-03-00493" ref-type="bibr">32</xref>,<xref rid="B36-antibiotics-03-00493" ref-type="bibr">36</xref>]. &#x003b1;-Defensins were purified further by cation exchange chromatography of pooled &#x003b1;-defensin-containing fractions and peptide quantities were determined using the Pierce Protein Assay [<xref rid="B19-antibiotics-03-00493" ref-type="bibr">19</xref>].</p></sec><sec><title>3.3. Bacterial Species and Culture Conditions</title><p>Anaerobic manipulations were performed in a Bactron II anaerobic chamber (Sheldon Manufacturing, Cornelius, OR, USA). <italic>Bacteroides fragilis</italic> ATCC 25285, <italic>Clostridium difficile</italic> ATCC 9689, <italic>Enterococcus faecalis</italic> ATCC 29214, and <italic>Fusobacterium necrophorum</italic> ATCC 25286 were cultured anaerobically on pre-reduced anaerobically sterilized (PRAS) Brucella blood agar plates and in PRAS Brucella broth (Anaerobe Systems, Morgan Hill, CA, USA). Wild-type <italic>Salmonella enterica</italic> serovar Typhimurium 14028s, <italic>Shigella flexneri</italic> BS497 [<xref rid="B67-antibiotics-03-00493" ref-type="bibr">67</xref>], <italic>Escherichia coli</italic> ML35 ATCC 43827, <italic>E. coli</italic> clinical isolate (<italic>E. coli</italic> ci) obtained from The University of California Irvine Medical Center (Orange, CA, USA), and enteropathogenic <italic>E. coli</italic> E2348/69 (EPEC) [<xref rid="B68-antibiotics-03-00493" ref-type="bibr">68</xref>] were cultured on trypticase soy agar (TSA) and in trypticase soy broth (TSB) under both aerobic and anaerobic conditions (<xref ref-type="table" rid="antibiotics-03-00493-t001">Table 1</xref>). We thank Drs. Mike Cox and Jeremy McDonald (Anaerobe Systems, Inc., Morgan Hill, CA, USA) for <italic>B. fragilis</italic> ATCC 25285, <italic>C. difficile</italic> ATCC 9689, <italic>E. faecalis</italic> ATCC 29214, and <italic>F. necrophorum</italic> ATCC 25286 and for advice for culturing anaerobic bacteria, Dr. Philippe Sansonetti (Pasteur Institute, Paris, France) for <italic>Shigella flexneri</italic> BS497, and Dr. Gail Hecht (University of Illinois, Chicago, IL, USA) for EPEC E2348/69. Media and solutions were degassed and with all plasticware were equilibrated inside the anaerobic chamber for a minimum of 18 h before use. PRAS supplies were manufactured and packed under anaerobic conditions to avoid exposure to oxygen and oxygen damage. Cultures were grown at 37 &#x000b0;C with (aerobes) or without (anaerobes) agitation. </p></sec><sec><title>3.4. Bactericidal Assay</title><p>The sensitivity of bacteria to &#x003b1;-defensins was assayed in 96-well polypropylene round-bottom microtiter plates (Corning). Samples of proCrp4, Crp2, Crp3, Crp4, and tissue-derived Paneth cell &#x003b1;-defensin peptide mixtures in 5 &#x003bc;L 0.01% acetic acid were mixed with 35 &#x003bc;L 10 mM PIPES plus 1% (v/v) of appropriate medium (PIPES-media). Purified Crps were assayed in triplicate dilution series at 1, 5, or 15 &#x003bc;M, and tissue-derived peptide mixtures at 5, 20, and 40 &#x003bc;g/mL. Exponential phase bacterial cells were deposited by centrifugation at 2000 <italic>g</italic> for 2 min, washed with 10 mM PIPES-media and resuspended in 10 mM PIPES-media. Bacterial cells were added to a final cell densities that ranged from 1 &#x000d7; 10⁵ to 2 &#x000d7; 10⁶ CFU/mL, incubated for 1 h at 37 &#x000b0;C with gentle, intermittent agitation, then diluted serially ten-fold from 1:10 to 1:10⁶ in TSB or BRU. Diluted cell suspensions were incubated 24&#x02013;48 h at 37 &#x000b0;C, and bactericidal activity was assessed by absence of growth, correlated to CFU/well, and survivors were enumerated based on the number of dilutions required to extinguish growth. In testing native &#x003b1;-defensin mixtures, results were expressed as percent of input bacteria that survived peptide exposure: % bacterial killing = [(CFU per well exposed to peptide/CFU per well not exposed to peptide) &#x000d7; 100]. Minimum bactericidal concentrations (MBC) of individual peptides were determined as the lowest peptide concentration that reduced bacterial cell survival by 1000-fold, or that reduced cell viability by 99.9%. </p></sec></sec><sec><title>4. Conclusions</title><p>To simulate the anaerobic conditions under which &#x003b1;-defensin-microbial interactions are thought to occur in the ileum and colonic lumen, we have tested the effect of anaerobiosis on mouse &#x003b1;-defensin bactericidal activity <italic>in vitro</italic>. Facultative bacteria maintained under aerobic or anaerobic conditions displayed differential sensitivities to mouse &#x003b1;-defensins, and anaerobic bacteria varied extensively in their susceptibility to Crps 2-4. Comparisons of native &#x003b1;-defensin mixtures from C57BL/6 and outbred Swiss mice against <italic>F. necrophorum</italic> and <italic>C. difficile</italic> showed that the combined bactericidal activities of the different &#x003b1;-defensin mixtures were similar. Thus, although the anaerobic environment alters the activities of individual Crp peptides against specific bacterial species, mixtures of distinctly different Crp peptides in molar ratios reflecting Paneth cell secretions, are similar in their overall bactericidal effects against the anaerobes tested. These findings suggest that the effects of Paneth cell &#x003b1;-defensins on the enteric microbiota may be subject to regulation by local oxygen tension.</p></sec>

Nutritional Assessment and Antioxidant Activities of Different Varieties of <italic>Vigna radiata</italic>

<p>Three cultivars of <italic>Vigna radiata</italic>, namely, NM-92, NM-98, and NM-06, were analyzed for their proximate composition. The samples were also tested by HPLC for amino acid content. The data showed that all the varieties had same moisture level. The maximum ash content (4.29%) was present in NM-92, and crude fat (2.26%) was highest in NM-98 while NM-06 contained maximum amount of crude protein. About eighteen types of amino acids were detected in each of the three varieties. Acidic amino acids, that is, aspartic and glutamic acids, were in considerable amount ranged from 13 to 23% followed by leucine, isoleucine, alanine, valine, lysine, phenyl alanine, serine, and arginine which fell in the range of 3&#x02013;8% of total protein. The maximum amount (13.00 and 22.80%) of aspartic and glutamic acids was present in NM-98. Similarly arginine (6.83%) and serine (5.45%) were also in highest amount in this variety. Leucine (7.46%) was maximum in NM-92 variety. NM-06 contained almost all the amino acids in lesser quantity except for few like threonine, proline, glycine, and alanine. It was concluded from the present study that varieties were of different nutritional value and HPLC was a sensitive method for amino acids determination. Antioxidant activities of all three varieties were also assayed and showed significant results.</p>

<contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">http://orcid.org/0000-0002-2860-467X</contrib-id><name><surname>Ullah</surname><given-names>Riaz</given-names></name><xref ref-type="aff" rid="I1"> ¹ </xref><xref ref-type="corresp" rid="cor1"> ^* </xref></contrib><contrib contrib-type="author"><name><surname>Ullah</surname><given-names>Zain</given-names></name><xref ref-type="aff" rid="I2"> ² </xref></contrib><contrib contrib-type="author"><name><surname>Al-Deyab</surname><given-names>Salem S.</given-names></name><xref ref-type="aff" rid="I3"> ³ </xref></contrib><contrib contrib-type="author"><name><surname>Adnan</surname><given-names>Muhammad</given-names></name><xref ref-type="aff" rid="I4"> ⁴ </xref></contrib><contrib contrib-type="author"><name><surname>Tariq</surname><given-names>Akash</given-names></name><xref ref-type="aff" rid="I4"> ⁴ </xref></contrib>

The Scientific World Journal

<sec id="sec1"><title>1. Introduction</title><p>Legumes are those species of family Leguminosae (Fabaceae) which are consumed directly by human beings, most commonly as mature dry seeds but occasionally as immature seeds enclosed in pods [<xref rid="B1" ref-type="bibr">1</xref>]. The leguminous grains, also known as pulses, are famous for their high quality protein content. Grain legumes are often considered as meat substitutes for people in less developed countries [<xref rid="B2" ref-type="bibr">2</xref>]. One member of legumes known as Mung bean (<italic>Vigna radiata</italic>), also called &#x0201c;green gram,&#x0201d; mainly grown in tropical regions of Asia, is an important summer-growing, annual crop [<xref rid="B3" ref-type="bibr">3</xref>].<italic> Vigna radiata</italic> has a short growing season and is relatively drought-tolerant. Mung bean is thought to be originated in South and Southeast Asian regions. It is widely grown in India, Pakistan, Bangladesh, Myanmar, Thailand, Philippines, China, and Indonesia. It is the second major pulse crop in Pakistan which has covered an area of 200 thousand acres with annual production of about 93 thousand tones equal to 446&#x02009;kg&#x02009;per&#x02009;ha [<xref rid="B4" ref-type="bibr">4</xref>]. In KPK various regions like Malakand, Kohat, Karak, Bannu, and Dera Ismail Khan districts are famous for its production; however total production in the province is 12.6 tones on 8.1 hectors, so we selected different varieties of Mung bean from different regions of Khyber Pakhtunkhwa (KPK), Pakistan [<xref rid="B3" ref-type="bibr">3</xref>, <xref rid="B5" ref-type="bibr">5</xref>].<italic> Vigna radiata</italic> fits well in our cropping system and it is grown as intercrop with other Kharif crops [<xref rid="B6" ref-type="bibr">6</xref>]. It contains isoflavoineds having estrogen and antioxidant activities, used as prevention of many diseases such as cancer; it also exhibits antimicrobial and insecticidal activities [<xref rid="B7" ref-type="bibr">7</xref>]. There are two main varieties, golden and green, the name denoting the color of dry seed. The climate and soil requirements of<italic> Vigna radiata</italic> are sandy loam soil with moderate moisture content. Mung bean is resistant to most of the diseases and insects pests of legume family.<italic> Vigna radiata</italic> is used as supplement for cereal based human diets due to its high lysine content. The grain has about 19.05 to 23.86% protein, 247.67 to 277.3&#x02009;mg/100&#x02009;g calcium, and 5.03 to 12.63&#x02009;mg/100&#x02009;g iron [<xref rid="B8" ref-type="bibr">8</xref>]. The nutritional quality of these grains may be impaired due to the presence of trypsin and other such antinutrients. This may cause low protein digestibility and minerals availability in legumes. In rural areas the immature green pods are also used as vegetables.<italic> Vigna radiata</italic> stalks, leaves, and husks constitute a significant proportion of livestock feed. The sprouted beans are consumed as cooked vegetable, usually in oriental dishes, or eaten raw in salads [<xref rid="B9" ref-type="bibr">9</xref>]. Protein of<italic> Vigna radiata</italic> is known as complete protein due to the presence of many of essential amino acids. Amino acids are required by man and other animals for their normal growth and health. Every cell of the body contains thousands of different proteins, each digested to carry specific function. And each protein is composed of a basic set of 20 amino acids. The acidic part (carboxylic group) transfers a proton to the basic part amino group (NH) that results in zwitterion. Amino acid in the zwitterion form is electrically neutral because the negative charge is balanced by the positive charge [<xref rid="B10" ref-type="bibr">10</xref>].</p><p>In the developing countries, like Pakistan, India, and Bangladesh, where mostly people are vegetarian for economic and religious reasons, they usually suffer from protein malnutrition problems as their diet is lacking lysine, so they are requested to eat food legumes because they are rich in lysine [<xref rid="B11" ref-type="bibr">11</xref>]. Keeping in view the importance of legumes, this work was an endeavor to highlight the nutritional importance by analyzing<italic> Vigna radiata</italic> for its certain chemical constituents, that is, proximate composition and amino acid profile.</p></sec><sec id="sec2"><title>2. Material and Methods</title><sec id="sec2.1"><title>2.1. Sample Collection</title><p>Three varieties of<italic> Vigna radiata,</italic> that is, NM-92, NM-98, and NM-06, were collected from National Institute for Food and Agriculture (NIFA) Research Centre, TARNAB, Peshawar. All the samples were collected in one-kilogram plastic bags and were stored at room temperature. Similarly all the three verities were crushed and dipped in methanol for one month. It was shaken throughout and finally methanol was evaporated through rotary evaporator. The crude extracts of methanol of all three varieties were studied for antioxidant activities.</p></sec><sec id="sec2.2"><title>2.2. Sample Preparation</title><p>Seeds of each variety were shifted from dirt and malformed kernels and then samples were stored at room temperature and one-third of the seeds were ground in the grinder and then further ground to fine particles by chopper in the laboratory for chemical analysis.</p></sec><sec id="sec2.3"><title>2.3. Proximate Composition</title><p>Samples of<italic> Vigna radiata</italic> varieties were analyzed for crude protein, crude fat, ash, and moisture by standard methods of AOAC [<xref rid="B12" ref-type="bibr">12</xref>].</p></sec><sec id="sec2.4"><title>2.4. Moisture Contents</title><p>Moisture was determined by oven drying method. 1&#x02009;g of each sample was accurately weighed in a Petri dish. The partially covered Petri dish was placed in an oven at 105&#x000b0;C for 6 hours. After cooling the Petri dish in the desiccators for 30&#x02009;minutes, it was reweighed. The percent moisture was calculated as <disp-formula id="eq1"><label>(1)</label><mml:math id="M1"><mml:mtable><mml:mtr><mml:mtd><mml:mtext>Moisture</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>content</mml:mtext><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>%</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>Weight</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>loss</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mi>on</mml:mi><mml:mo>&#x02061;</mml:mo><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>drying</mml:mtext><mml:mo>&#x000d7;</mml:mo><mml:mn mathvariant="normal">100</mml:mn></mml:mrow><mml:mrow><mml:mtext>weight</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>of</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>sample</mml:mtext></mml:mrow></mml:mfrac><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula> </p></sec><sec id="sec2.5"><title>2.5. Crude Fats</title><p>Crude fat was determined by ether extract method using Soxhlet apparatus. 1&#x02009;g of each moisture sample was wrapped in filter paper, placed in thimble, and then introduced into extraction tube. Receiving flasks were filled up to 1/3 with hexane and fitted into apparatus. After complete extraction, the hexane from the receiving flasks was evaporated on water bath, the ether extract was dried in an electric oven, and the flasks were reweighted. The percent crude fat was determined by using the following formula: <disp-formula id="eq2"><label>(2)</label><mml:math id="M2"><mml:mtable><mml:mtr><mml:mtd><mml:mi>%</mml:mi><mml:mtext>Crude</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>Fat</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mtext>Weight</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>of</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>ether</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>extract</mml:mtext><mml:mo>&#x000d7;</mml:mo><mml:mn mathvariant="normal">100</mml:mn></mml:mrow><mml:mrow><mml:mtext>weight</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>of</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>sample</mml:mtext></mml:mrow></mml:mfrac><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula> </p></sec><sec id="sec2.6"><title>2.6. Crude Proteins</title><p>One gram of each sample was taken in digestion flasks. 8&#x02009;g of digestion mixture, that is, K₂So₄&#x000b7;CuSo₄ (8&#x02009;:&#x02009;1), and 12&#x02009;mL of concentrated H₂So₄ were added. Some pieces of fumic stones were also added to avoid bumping of the solution. Digestion was carried out by heating the mixture until it becomes clear.</p><p>After cooling, the digest was transferred to 100&#x02009;mL volumetric flask and volume was made up to the mark with distilled water. Distillation was carried out in Markham Still Distillation apparatus. 10&#x02009;mL of digest was introduced in the distillation tube through funnel. Then 10&#x02009;mL of 40% NaOH was gradually added through the same way. Distillation was carried out for at least 10 minutes and NH₃ produced was collected as NH₄OH in a conical flask containing 20&#x02009;mL of 4% boric acid solution with few drops of modified methyl red indicator and during distillation yellowish color appears due to ammonium hydroxide.</p><p>The distillate was titrated against standard 0.1&#x02009;N&#x02009;HCL solutions till the appearance of pink color. A bland (experiment without sample) was also run through all the steps as above. Percent of crude proteins was calculated by multiplying the percent nitrogen with appropriate factor: <disp-formula id="eq3"><label>(3)</label><mml:math id="M3"><mml:mtable><mml:mtr><mml:mtd><mml:mfrac><mml:mrow><mml:mi>%</mml:mi><mml:mtext>N</mml:mtext><mml:mo>=</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mi>S</mml:mi><mml:mo>&#x02212;</mml:mo><mml:mi>B</mml:mi><mml:mo>)</mml:mo></mml:mrow><mml:mo>&#x000d7;</mml:mo><mml:mi>N</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mn mathvariant="normal">0.014</mml:mn><mml:mo>&#x000d7;</mml:mo><mml:mi>D</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mn mathvariant="normal">100</mml:mn></mml:mrow><mml:mrow><mml:mtext>Weight</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>of</mml:mtext><mml:mi>&#x02009;&#x02009;</mml:mi><mml:mtext>sample</mml:mtext><mml:mo>&#x000d7;</mml:mo><mml:mi>V</mml:mi></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula> where <italic>S</italic> = sample titration reading, <italic>B</italic> = blank titration reading, <italic>N</italic> = normality of HCL, <italic>D</italic> = dilution of sample after digestion, <italic>V</italic> = volume taken for titration, and 0.014 = Milliequivalent Wt. of Nitrogen Crude proteins (%) = %N &#x000d7; 6.25.</p></sec><sec id="sec2.7"><title>2.7. Analysis of Amino Acid by HPLC</title><p>Six test tubes (1.5 &#x000d7; 10&#x02009;cm) were washed and placed in oven at 100&#x000b0;C for 20 minutes for drying. Each dried test tube was put 40&#x02009;mg dried ground Mung bean sample, 10&#x02009;mL 6 N HCL and small portion of phenol was added. All the tubes were placed in vacuum desiccators and 700&#x02009;mm Hg vacuum was created in order to stop the breakdown of amino acids by oxidation reaction. Desiccators were then placed in oven at 100&#x000b0;C for three days. 25 microliters of each hydrolysate was transferred to sample tube (6 &#x000d7; 50&#x02009;mm) and was dried in vacuum and 50 microliters of dissolving reagent (methanol + distilled water + TEA; 2&#x02009;:&#x02009;2&#x02009;:&#x02009;1) was added to sample tube, mixed, and dried twice in vacuum. After this 50 derivatizing reagents containing methanol, TEA, water, and PITC (7&#x02009;:&#x02009;1&#x02009;:&#x02009;1&#x02009;:&#x02009;1) were added and incubated at room temperature for 20 minutes followed by drying in vacuum for 10 minutes. Methanol was then added to each sample tube and dried twice in vacuum. The residues were then dissolved in 200 microliters of solution containing disodium hydrogen phosphate and acetonitrile (95&#x02009;:&#x02009;5); 20 microliters was injected in HPLC and the data was recorded by SW 32 software system.</p></sec><sec id="sec2.8"><title>2.8. Antioxidant Bioassay</title><p>DPPH (diphenylpicrylhydrazyl) method was adopted for antioxidant activities. Test samples were allowed to react with stable free radical, 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) (from Sigma Aldrich), for half an hour at 37&#x000b0;C. The concentration of DPPH was kept as 300&#x02009;<italic>&#x003bc;</italic>M. The test samples methanol extracts of NM-92, NM-98, and NM-06 were dissolved in DMSO while the DPPH solution was prepared in ethanol. After incubation, decrease in absorption was measured at 515&#x02009;nm using multiplate reader (Spectra MAX-340). Percent radical scavenging activity by samples was determined in comparison with a DMSO treated control group [<xref rid="B12" ref-type="bibr">12</xref>]. % radical scavenging activity was calculated by using the following formula: %RSA = 100 &#x02212; {(OD&#x02009;&#x02009;test&#x02009;&#x02009;compound/OD&#x02009;&#x02009;control)} &#x000d7; 100 &#x02009;[<xref rid="B13" ref-type="bibr">13</xref>].</p></sec></sec><sec id="sec3"><title>3. Results and Discussion</title><p>Three varieties of<italic> Vigna radiata</italic> (NM-98 and NM-06) obtained from NIFA (Nuclear Institute of Food and Agriculture) were analyzed for their important proximate composition and amino acid contact. The data was presented in the form of table and graphs as shown below.</p><p>The data (<xref ref-type="table" rid="tab1">Table 1</xref>) showed that all the varieties had same moisture level. The maximum ash contact (4.29%) was present in NM-92. Crude fats (2.26%) were highest in NM-06 which contained maximum amount of crude protein. The present data was in good line with [<xref rid="B14" ref-type="bibr">14</xref>] while studying the effect of processing on different pulses containing Mung beans. The reported ash content was up to 3.34%, the crude fat was up to 1.34%, and the crude protein was up to 22.90%. The data was also in good comparison with [<xref rid="B1" ref-type="bibr">1</xref>, <xref rid="B3" ref-type="bibr">3</xref>] also reporting same kind of data while analyzing different Mung bean cultivars.</p><p>About eighteen amino acids were detected (<xref ref-type="table" rid="tab2">Table 2</xref>) in each of the three varieties. The data showed that acidic amino acids, that is, aspartic and glutamic acids, were in considerable amount ranging from 13 to 23%, followed by leucine, isoleucine, alanine, valine, lysine, phenyl, alanine, serine, and arginine which fall in the range of 3&#x02013;8% of total protein. The maximum amount (13.00 and 22.80%) of aspartic and glutamic acids was present in NM-98. Similarly arginine (6.38%) and serine (5.45%) were also in highest amount in this variety. Leucine (7.46%) was in NM-92 variety. NM-06 contained almost all the amino acids in lesser quantity except for few like threonine, proline, glycine, and alanine. The present data was in good line with [<xref rid="B15" ref-type="bibr">15</xref>] which studied the nutritional composition and antinutritional factors of<italic> Vigna radiata</italic> seeds as affected by home traditional processes. The data was also close to the results of [<xref rid="B16" ref-type="bibr">16</xref>] which analyzed various<italic> Vigna radiata</italic> cultivars for their nutrition.</p><p>Mung bean (<italic>Vigna radiata</italic>) belongs to family Leguminosae (Fabaceae) and is the second major pulse crop in Pakistan. It is used as a supplement for cereal based human diets due to its high lysine content. The grain has about 19.05&#x02013;23.86% protein, 247.67&#x02013;277.3&#x02009;mg/100&#x02009;gms calcium, and 5.30 to 12.63&#x02009;mg/100&#x02009;gms iron. The nutritional quality of these grains may be impaired due to the presence of trypsin and other such antinutrients. In the rural areas the immature green pods are also used as vegetables. Mung bean stalks, leaves, and husks constitute a significant proportion of livestock feed. The sprouted beans are consumed as cooked vegetables, usually in oriental dishes and are eaten raw in salads. Three cultivars of Mung beans, NM-92 and NM-98, were analyzed for their proximate composition. The samples were also tested by HPLC for amino acid content. The data showed that all the varieties had same moisture level. The maximum ashes content (4.29%) was present in MN-92. Crude fats (2.26%)&#x02009; were highest in MN-98 while MN-06 contained maximum amount of crude proteins. About eighteen types of amino acids were detected in each of the three varieties. Acidic amino acids, that is, aspartic and glutamic acid, were in considerable amount ranged from 13 to 23% followed by leucine, isoleucine aniline, valine, lysine phenyl alanine, serine, and arginine which fell in the range of 3&#x02013;8% of total protein; the maximum amount (13.00 and 22.80%) of aspartic and glutamic acid is present in MN-98. Similarly arginine (6.83%) and serine (5.45%) were also in highest amount in this variety. Leucine (7.46%) was maximum in MN-92 variety. MN-06 contained almost all the amino acids in lesser quantity except for few like threonine, praline, glycine, and alanine.</p><p>It was concluded from the present study that different varieties have different nutritional values and HPLC was a sensitive method for amino acid determination.</p><sec id="sec3.1"><title>3.1. Antioxidant Activities</title><p>Free radicals are responsible for many diseases like cancer and AIDS. Antioxidants due to their scavenging activity are useful for the management of those diseases. DPPH stable free radical method is a sensitive way to determine the antioxidant activity of plant extracts [<xref rid="B13" ref-type="bibr">13</xref>]. Keeping in mind the importance of antioxidant activity, the methanolic crude extracts of all the three varieties of<italic> Vigna radiata</italic>, namely, NM-92, NM-98, and NM-06, were screened for antioxidant activity. Results obtained are given in <xref ref-type="table" rid="tab3">Table 3</xref>. From <xref ref-type="table" rid="tab3">Table 3</xref>, it is clear that NM-92 showed the highest activity 66.08%.&#x000b1;0.01 followed by NM-98, showing 64.15%.&#x000b1;0.01. It is comparable with standard showing result of 95.12% &#x000b1; 0.01.</p></sec></sec><sec id="sec4"><title>4. Conclusion</title><p>It was concluded that<italic> Vigna radiata</italic> contained various nutrients in appreciable quantities. All the cultivars contained crude protein in maximum amount which were comparable to those of animals sources. The HPLC method was used for amino acids determination was accurate regularly be utilized for the same purpose. It also showed significant antioxidant activities. It is recommended that the<italic> Vigna radiata</italic> due to high nutritional value and antioxidant potential could be used in daily human diet which would be helpful in fulfilling the protein need of the body. It is also recommended that utmost precautions should be taken while hydrolyzing samples for HPLC recommendation of amino acid, because high risks of oxidation contamination with other proteins are present.</p></sec>

Consequences of tropical land use for multitrophic biodiversity and ecosystem functioning

<p>Our knowledge about land-use impacts on biodiversity and ecosystem functioning is mostly limited to single trophic levels, leaving us uncertain about whole-community biodiversity-ecosystem functioning relationships. We analyse consequences of the globally important land-use transformation from tropical forests to oil palm plantations. Species diversity, density and biomass of invertebrate communities suffer at least 45% decreases from rainforest to oil palm. Combining metabolic and food-web theory, we calculate annual energy fluxes to model impacts of land-use intensification on multitrophic ecosystem functioning. We demonstrate a 51% reduction in energy fluxes from forest to oil palm communities. Species loss clearly explains variation in energy fluxes; however, this relationship depends on land-use systems and functional feeding guilds, whereby predators are the most heavily affected. Biodiversity decline from forest to oil palm is thus accompanied by even stronger reductions in functionality, threatening to severely limit the functional resilience of communities to cope with future global changes.</p>

<contrib contrib-type="author"><name><surname>Barnes</surname><given-names>Andrew D.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Jochum</surname><given-names>Malte</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Mumme</surname><given-names>Steffen</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Haneda</surname><given-names>Noor Farikhah</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Farajallah</surname><given-names>Achmad</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Widarto</surname><given-names>Tri Heru</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Brose</surname><given-names>Ulrich</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Systemic Conservation Biology, J.F. Blumenbach Institute of Zoology and Anthropology, University of G&#x000f6;ttingen</institution>, Berliner Strasse 28, 37073 G&#x000f6;ttingen, <country>Germany</country></aff><aff id="a2"><label>2</label><institution>Department of Silviculture, Faculty of Forestry, Bogor Agricultural University</institution>, Darmaga Campus, Bogor 16680, <country>Indonesia</country></aff><aff id="a3"><label>3</label><institution>Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University</institution>, Darmaga Campus, Bogor 16680, <country>Indonesia</country></aff>

Nature Communications

<p>The transformation from natural ecosystems to agricultural land use and its continued intensification has led to extensive losses in biodiversity and ecosystem services<xref ref-type="bibr" rid="b1">1</xref> resulting in the degradation of human well being<xref ref-type="bibr" rid="b2">2</xref>. The transformation of lowland tropical rainforest to oil palm (<italic>Elaeis guineensis</italic> Jacq.) plantations has gained more recent attention as an especially severe threat to tropical biodiversity<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>. In the last 25 years the total plantation area of oil palm has tripled, with current global estimates of over 15 million hectares<xref ref-type="bibr" rid="b3">3</xref>, making this crop one of the world&#x02019;s most rapidly expanding forms of agriculture<xref ref-type="bibr" rid="b5">5</xref>. It is now clear that the expansion of oil palm agriculture is one of the greatest causes of deforestation<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>, and this threat appears to be increasing without respite as Indonesia, one of the world&#x02019;s leaders in oil palm, makes plans to double production by 2020 (ref. <xref ref-type="bibr" rid="b8">8</xref>). The rapid expansion of such large-scale land-use transformation raises questions about the impending implications for biodiversity and ecosystem functioning in the tropics.</p><p>Despite a broad consensus that biodiversity is positively correlated with ecosystem functioning in controlled experiments<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, there are few real-world examples of such biodiversity&#x02013;ecosystem functioning relationships<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>. In fact, until now there have been no studies that explore the relationship between biodiversity and ecosystem functioning in ecosystems undergoing agricultural land-use transformation to oil palm. Thus, our knowledge of this globally important land-use conversion is strongly limited. Furthermore, over the past decade there have been important advances towards multitrophic approaches in research investigating biodiversity&#x02013;ecosystem functioning relationships<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>. Despite these advances, however, we are still substantially limited by the lack of clear approaches to quantify single measures of ecosystem functioning that can be compared among any combination of trophic levels. This has resulted in our inability to directly look at whole-community relationships between entire species assemblages and the respective functional processes carried out in these communities.</p><p>Here we use the total energy flux between functional feeding guilds as a measure of multitrophic ecosystem functioning, as many studies have suggested process rates, such as energy fluxes, to be important proxies for ecosystem functioning<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b17">17</xref>. Depending on the resource pool that the energy flux comes from, these fluxes can be directly related to ecosystem services such as decomposition<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref>, plant biomass production<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref> or biocontrol through predation<xref ref-type="bibr" rid="b22">22</xref>. These energy flux calculations are based on metabolic scaling theory<xref ref-type="bibr" rid="b23">23</xref> and principles of food-web energy dynamics<xref ref-type="bibr" rid="b18">18</xref>. Using individual metabolic rates that are dependent on body mass, environmental temperature and phylogenetic grouping<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b24">24</xref>, combined with resource-specific assimilation efficiencies<xref ref-type="bibr" rid="b25">25</xref> and energy loss to predation<xref ref-type="bibr" rid="b18">18</xref>, we present this energy flux calculation as a unified measure of multitrophic ecosystem functioning (<xref ref-type="fig" rid="f1">Fig. 1</xref>). Studies that incorporate diversity across trophic levels to test the relationship between biodiversity and ecosystem functioning have predominantly used only biomass as the measure of ecosystem function<xref ref-type="bibr" rid="b26">26</xref>. However, the metabolic activity and thus the energy-processing rates of these biomass pools can vary substantially. Integrating over body mass, phylogeny and temperature with their constraints on metabolic rates, and additionally taking into account assimilation efficiencies and loss to predation, our measure of whole-community energy flux inherently incorporates not only biomass but also other important ecosystem attributes enabling the quantification of emergent functional properties of ecosystems that would otherwise remain undetected. As such, our measure of energy flux provides a comprehensive and robust measure of multitrophic ecosystem functioning that can be utilized for modelling biodiversity&#x02013;ecosystem functioning relationships for any assemblage of taxonomic groups, while incorporating multiple ecological functions.</p><p>In the tropical lowland rainforests of Sumatra, Indonesia, which have been undergoing vast land-use transformation to oil palm<xref ref-type="bibr" rid="b7">7</xref>, we quantify the impacts of this transformation ranging from tropical secondary rainforest, jungle rubber and intensively managed rubber, to oil palm. We utilize data gathered from 32 sites in Sumatra, Indonesia, comprising 2,415 populations of 871 species. First, we investigate the biodiversity value of jungle rubber, conventional rubber and secondary forest compared with oil palm agriculture by comparing observed species richness, density and biomass of litter-associated macroinvertebrate communities across these systems. Second, as a multitrophic measure of the rate of ecosystem processes carried out by these communities, we calculate total solid fresh mass energy flux in a system by incorporating community metabolism<xref ref-type="bibr" rid="b27">27</xref>, resource-specific assimilation efficiencies and biomass loss to predation<xref ref-type="bibr" rid="b18">18</xref> into whole-community energy flux equations (<xref ref-type="fig" rid="f1">Fig. 1</xref>). This provides a quantitative measure of multitrophic ecosystem functioning, defined here as the total flux of energy from any resource pool to consumer trophic levels. In addition, this measure can be attributed to specific functional feeding guilds within communities to look for patterns in ecosystem functioning at different trophic levels. Using the energy-mass flow conversion<xref ref-type="bibr" rid="b28">28</xref>, we express energy flux as kilograms per hectare, per year and explore the relationship between total species diversity and energy flux, distinguishing among four transformation systems to test for land-use-dependent biodiversity&#x02013;ecosystem functioning relationships. Our results demonstrate strong losses in species diversity that, in turn, predict reductions in whole-community energy fluxes. However, these reductions are strongest in oil palm systems, suggesting that land-use conversion from forest to oil palm causes disproportionally strong losses in multitrophic ecosystem functioning.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Transformation to oil palm leads to biodiversity loss</title><p>Using generalized linear mixed effects models, we show that transformation of tropical rainforest to oil palm plantations leads to severe losses in species richness (45% decline), animal density (48% decline) and biomass (52% decline; <xref ref-type="fig" rid="f2">Fig. 2a&#x02013;c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>), supporting previous studies suggesting that land-use transformation to oil palm poses one of the greatest threats to global biodiversity<xref ref-type="bibr" rid="b3">3</xref>. Beyond mere diversity effects, land-use transformation altered animal densities and biomass, threatening to not only drive species extinctions but also to eliminate vital ecological functions. The effects of land-use transformation on species richness and animal densities were additionally dependent on functional feeding guilds, with predators decreasing in species richness and density most rapidly (<xref ref-type="fig" rid="f2">Fig. 2a&#x02013;c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>) as could be expected for higher trophic level feeding guilds<xref ref-type="bibr" rid="b29">29</xref>. Such alteration of higher trophic levels is likely to have severe indirect functional impacts on other functional guilds within the trophic network<xref ref-type="bibr" rid="b30">30</xref>.</p></sec><sec disp-level="2"><title>Community metabolism</title><p>Summing up individual metabolic rates, we demonstrate that transformation of forest to oil palm yields a 51% decrease in community metabolism, with jungle rubber and rubber only 16% and 10% below forest levels of community metabolism, respectively. However, all systems yielded significantly higher community metabolism than oil palm (<xref ref-type="fig" rid="f2">Fig. 2d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>). As such, we show that ecosystem energy processing is critically reduced in oil palm plantations. Interestingly, biomass responses to land-use transformation among feeding guilds were not clearly comparable to responses in community metabolism (<xref ref-type="fig" rid="f2">Fig. 2c,d</xref>). This suggests that systematic changes in species composition, body-mass distributions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>) and biomass exhibited a complex interaction in determining the functional consequences of land-use transformation.</p></sec><sec disp-level="2"><title>Whole-community energy fluxes and ecosystem functioning</title><p>Aiming to visualize the complex interplay between community biomass dynamics and energy flux, we constructed energy networks for the four transformation systems (<xref ref-type="fig" rid="f3">Fig. 3</xref>) based on total energy fluxes as a promising way to quantify multitrophic ecosystem functioning (<xref ref-type="fig" rid="f1">Fig. 1</xref>). In addition to the general decreases in biomass (node sizes in <xref ref-type="fig" rid="f3">Fig. 3</xref>) and energy-processing rates (arrow widths in <xref ref-type="fig" rid="f3">Fig. 3</xref>), we also found a systematic shift from predator to omnivore dominance when comparing forest and oil palm systems. Specifically, we found that predator biomass in oil palm yielded only 25% of their biomass in forest (0.424 and 1.664&#x02009;kg&#x02009;ha^&#x02212;1, respectively), while the predator-driven energy flux was reduced to 46% of the energy flux driven by predators in forest (30.697 and 66.816&#x02009;kg&#x02009;ha^&#x02212;1 per year, respectively). In contrast, omnivore biomass in oil palm was 22% higher than in the forest (0.767 compared with 0.629&#x02009;kg&#x02009;ha^&#x02212;1), while omnivore-driven energy flux in the oil palm was 47% lower than in forest communities (32.531 compared with 61.900&#x02009;kg&#x02009;ha^&#x02212;1 per year; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>), suggesting a considerable mismatch of biomass and energy flux, partly dependent on the trophic group in question. In our analyses, this disparity finds its explanation in varying body-mass distributions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>) and assimilation efficiencies that strongly modify how biomass translates into total resource assimilation rates (<xref ref-type="fig" rid="f1">Fig. 1</xref>). These results suggest that biomass, alone, may be an unsuitable proxy for general ecosystem functioning in animal communities.</p></sec><sec disp-level="2"><title>Multitrophic biodiversity-ecosystem function relationships</title><p>Until now, most studies investigating biodiversity&#x02013;ecosystem function relationships have focused on single trophic levels<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref>. We present a new approach to easily quantify multitrophic ecosystem functioning, requiring only information on body mass, phylogeny, temperature and assimilation efficiencies to overcome previous limitations in biodiversity&#x02013;ecosystem functioning research. Utilizing this approach, we also investigated the relationship between species richness and ecosystem functioning, identifying a clear linear positive effect of diversity on total energy flux (<xref ref-type="fig" rid="f4">Fig. 4a</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). The relationship between diversity and energy flux was dependent on land-use transformation system, whereby oil palm and jungle rubber showed the strongest decrease in energy flux per unit loss in species richness (<xref ref-type="fig" rid="f4">Fig. 4a</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). Our results suggest that each loss of species in oil palm and jungle rubber therefore would be followed by proportionately higher losses in energy flux, compared with equal species losses in forest and rubber. We found the same pattern as in the overall trend for the predator group, which showed transformation system-dependent relationships between species richness and energy flux (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). However, for omnivores, detritivores and herbivores there was a linear effect of diversity on energy flux driven by these groups; however, this effect was independent of transformation system (<xref ref-type="fig" rid="f4">Fig. 4b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). This implies that studies focusing on single trophic levels, or even specific species, may fail to detect the alteration of ecosystem processes resulting from land-use transformation. These results call for a wider application of multitrophic approaches that not only measure one ecosystem property, such as total productivity or decomposition, but that also aim to assess whole-community ecosystem processes such as total energy flux.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Our study reflects previous findings that the transformation of forest systems to oil palm has severe impacts not only on single animal populations but also on communities as a whole. In particular, species richness and animal biomass are most significantly affected. Furthermore, jungle rubber and rubber appear to represent intermediate steps in land-use intensification. Their higher levels of biodiversity and ecosystem functioning indicate that they potentially provide higher ecological value than oil palm. As such, these rubber land-use systems could present economically viable, lower intensity land-use alternatives.</p><p>By taking a multitrophic ecosystem functioning approach we demonstrate that, at the community level, species loss leads to a direct linear decrease in ecosystem functioning. This means that any species loss will be followed by a proportionate loss in function, and this relationship becomes proportionately stronger in more intensive transformation systems such as oil palm plantations. Thus, every one of the few species in high-intensity land-use systems is functionally more important than species in low-intensity systems where functional redundancy is likely to be higher<xref ref-type="bibr" rid="b33">33</xref>. Without explicit consideration of multiple trophic levels, such emergent properties are likely to be overlooked. Our study demonstrates the crucial implications of tropical land-use intensification for biodiversity and ecosystem functioning across multiple trophic levels, suggesting that these globally important impacts will likely resonate beyond previously explored trophic boundaries.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Study site and sampling design</title><p>Sampling took place in the Jambi province of Sumatra, Indonesia, a region known as a hotspot for biodiversity, but that has also already undergone extensive deforestation<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b34">34</xref>. In the second half of the last century, Sumatra&#x02019;s forests have experienced vast transformation to rubber and oil palm monocultures<xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>. This large-scale land-use conversion has left Sumatra with a very limited area of natural forest mainly restricted to national parks and even here, where logging has been reduced, it has not come to a complete halt<xref ref-type="bibr" rid="b37">37</xref>. This severe and extensive land-use transformation, that has progressed already further than in most other tropical landscapes, makes Sumatra a unique and ideal example system for studying the impacts of land-use conversion on biodiversity and ecosystem functioning.</p><p>We sampled secondary rainforest, jungle rubber, rubber and oil palm systems, replicated eight times across two landscapes (<italic>n</italic>=32; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). Sites were selected by first looking for landscapes in the Jambi province that still contained secondary rainforest. Second, we identified all lowland areas with little or no slope and then randomly selected two landscapes with 16 sites each. Among all of the 32 sampling sites, we maintained a minimum distance of 120&#x02009;m to insure independence of the epigaeic invertebrate communities sampled. The secondary-forest regions lie within two protected areas, Bukit Duabelas National Park and Harapan Rainforest, and represent the least influenced land-use system. Jungle rubber&#x02014;forest stands with a high percentage of rubber trees that are still regularly harvested&#x02014;represents a low-impact agroforestry system<xref ref-type="bibr" rid="b38">38</xref>. Rubber and oil palm plantations serve as locally common<xref ref-type="bibr" rid="b36">36</xref> high-impact monocultures. The 32 sites were carefully selected so that they were all of a similar age and from equal elevations close to the sea level. All agricultural systems (jungle rubber, rubber and oil palm) were treated and harvested by their owners with intensities typical for the respective transformation system.</p></sec><sec disp-level="2"><title>Animal sampling and calculation of response variables</title><p>Animal sampling took place between early October and early November 2012. All organisms were collected based on Permit No. 51/KKH-5/TRP/2014 issued by the Indonesian Institute of Sciences and the Ministry of Forestry. In all 32 of the 50 &#x000d7; 50-m sites, we sampled once in each of three 5 &#x000d7; 5-m subplots by sieving the leaf litter from 1&#x02009;m² through a coarse sieve of 2-cm-width mesh. In all, 7,472 macroinvertebrates were hand-collected from the sieving samples and stored in 65% ethanol. Specimens were identified to morphospecies and assigned to one of four feeding guilds: omnivores, detritivores, predators and herbivores, based on morphology and literature.</p><p>As biodiversity studies always suffer from undersampling and correlation of sample size with species richness, we compared observed species richness to both extrapolated and rarefied species richness, calculated in the &#x02018;vegan&#x02019; package in R<xref ref-type="bibr" rid="b39">39</xref>, to assess the accuracy of our species-sampling effort. To extrapolate sampled species richness, we used the nonparametric second-order jacknife estimator<xref ref-type="bibr" rid="b40">40</xref> to calculate extrapolated species richness from the three 1-m² subsamples at each of the 32 sites, revealing an estimated mean sampling coverage of 56% (s.d. of&#x000b1;2.393%) making the second-order jacknife estimator the most accurate extrapolation method<xref ref-type="bibr" rid="b40">40</xref>. In addition, we calculated sample-based rarefaction, whereby rarefaction curves were calculated for each of the 32 sampled sites and then cut off at the sample size of the smallest sample (40 individuals). Because of the very high attrition of data during the rarefaction procedure (a total of 6,192 out of 7,472 individuals, or 83%, were removed), the rarefied species richness yielded very little resemblance to observed species richness when comparing across transformation systems, resulting in almost no pattern of rarefied richness among transformation systems (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). The jacknife2-extrapolated species richness, however, was extremely closely correlated with observed species richness (Pearson&#x02019;s <italic>&#x003c1;</italic>=0.993) patterns among transformation systems (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>), suggesting that our observed species richness did in fact accurately capture realistic patterns in total species diversity across the land-use transformation systems.</p><p>For each of the 7,472 animals collected, we measured individual body length to an accuracy of 0.1&#x02009;mm using stage micrometres. We then converted all measured individual body lengths to fresh body mass using length-mass regressions and, where necessary, dry mass-fresh mass relationships from the literature (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>), yielding an estimated fresh mass in milligrams for every collected individual. Where family-specific relationships were not available or animal body lengths in our collection fell outside of the size ranges of published regressions, we then used regressions from higher-order taxonomic groupings. For heavily damaged individuals that could not be measured for body length, we assigned these individuals a fresh body mass from the median body mass of all animals from the same species or order where only one individual of that species was collected. We then calculated community biomass (mg fresh mass m^&#x02212;2) for each of the 32 communities by summing together all individual body masses calculated from length-mass regressions as derived from the individually measured body lengths.</p><p>We calculated individual metabolic rates for all 7,472 animals using body masses, temperature and phylogeny<xref ref-type="bibr" rid="b24">24</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>). Temperature was measured over a period of at least 2.5 months at 30-cm depth below the soil surface in each site and averaged for each transformation system in each of the two landscapes. From this, community metabolism was calculated by summing together all individual metabolic rates within each of the 32 sites, providing the total metabolic demand for each of the 32 communities. Using diet-specific assimilation efficiencies<xref ref-type="bibr" rid="b25">25</xref>, energy loss to predation and community metabolism, we analytically calculated energy fluxes for each of these communities<xref ref-type="bibr" rid="b18">18</xref> using the formula</p><p><disp-formula id="eq1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e432" xlink:href="ncomms6351-m1.jpg"/></disp-formula></p><p>where <italic>F</italic> is the total energy flux into the network node of a feeding guild, <italic>e</italic>_{<italic>a</italic>} is the diet-specific assimilation efficiency, <italic>X</italic> is the metabolic demand of the feeding guild and <italic>L</italic> is the loss to predation that the feeding guild is subjected to (<xref ref-type="fig" rid="f1">Fig. 1</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Methods</xref>). In order to calculate the fluxes between the functional feeding guilds, we constructed a general network of feeding relationships (link structure in <xref ref-type="fig" rid="f3">Fig. 3</xref>) that represents a null model for an energy network structure where no active preferences are assumed. We assumed that, of our four functional feeding guilds, energy fluxes to predators were split up equally into the three animal guilds below them. Energy fluxes to detritivores and herbivores were assumed to come from only detritus and plant material, respectively. Omnivores were assumed to receive energy in equal 25% proportions from the other three functional feeding groups (predators, detritivores and herbivores, making 75%) and the remaining 25% from both plant and detritus material combined (<xref ref-type="supplementary-material" rid="S1">Supplementary Methods</xref>).</p><p>To assess how these assumptions of feeding preferences might affect the calculations of total energy fluxes, we reconstructed the energy networks so that omnivores were assumed to only consume plant and detritus material (50% derived from each) but with no energy derived from animal material. We then recalculated total energy fluxes and found an overall decrease of up to 54%, which appeared to be highly consistent among the different land-use transformation systems. This consistency between models was especially evident after calculating the loss of energy flux in the three agriculturally used systems compared with the forest system, demonstrating a maximum of only 3% disparity between the two models (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). This sensitivity analysis indicated that our presented method is highly robust in calculating differences in energy fluxes among different systems. Accordingly, the null model was accepted as the simplest model with the least diet preferences assumed. However, we still suggest that studies adopting this method of energy flux calculation should assign feeding preferences with caution, or employ other techniques such as stable isotope analysis to estimate feeding preferences.</p></sec><sec disp-level="2"><title>Statistical analyses</title><p>Using mixed effects models (GLMM&#x02019;s), we tested the effects of &#x02018;transformation system&#x02019; and its interaction with functional feeding guild on community responses, with &#x02018;landscape&#x02019; as a random effect. &#x02018;Density&#x02019;, &#x02018;biomass&#x02019; and &#x02018;community metabolism&#x02019; were log₁₀-transformed to meet assumptions of normality and &#x02018;species richness&#x02019; (overdispersed poisson-distributed data) was modelled on a negative binomial distribution. We additionally explored biodiversity&#x02013;ecosystem functioning relationships by first testing for linearity of relationships using untransformed data. Once linearity was established, we then tested for the effects of log₁₀-transformed &#x02018;species richness&#x02019; and its interaction with &#x02018;transformation system&#x02019; on &#x02018;energy flux&#x02019; for overall data and repeated again for data from separate feeding guilds. In addition, because we suspected that our analyses could be affected by spatial autocorrelation, we calculated Moran&#x02019;s <italic>I</italic> values for each model&#x02019;s residuals and tested for spatial autocorrelation using the Moran&#x02019;s <italic>I</italic> standard deviate<xref ref-type="bibr" rid="b41">41</xref> in the &#x02018;spdep&#x02019; package in R 3.0.2 (ref. <xref ref-type="bibr" rid="b39">39</xref>). Results from these tests provided no support for the spatial autocorrelation of variation in any of the response variables tested (all Moran&#x02019;s <italic>I</italic> test results yielded <italic>P</italic>&#x0003e;0.4).</p><p>For all GLMM&#x02019;s, we applied a backward stepwise selection procedure to obtain the model of best fit, based on the Akaike Information Criterion (AIC). In this procedure, we constructed full models that contained all possible predictors and their interactions (&#x02018;transformation system&#x02019; and &#x02018;feeding guild&#x02019; for general community response models; &#x02018;species richness&#x02019; and &#x02018;transformation system&#x02019; for biodiversity&#x02013;ecosystem functioning models) and compared these full models and the model of the backward selection procedure to a null, intercept-only model. The model that yielded the lowest AIC score, with a minimum &#x00394;AIC of 2 units, was selected as the model of best fit. All analyses were conducted with the &#x02018;nlme&#x02019; and &#x02018;lme4&#x02019; packages in R 3.0.2 (ref. <xref ref-type="bibr" rid="b39">39</xref>).</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>A.D.B., M.J. and U.B. designed the study; A.D.B., M.J. and S.M. carried out the field and laboratory work; A.D.B. and M.J. prepared and analysed the data; all authors interpreted the results and wrote the paper.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Barnes, A. D. <italic>et al.</italic> Consequences of tropical land use for multitrophic biodiversity and ecosystem functioning. <italic>Nat. Commun.</italic> 5:5351 doi: 10.1038/ncomms6351 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-4, Supplementary Tables 1-5, Supplementary Methods and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6351-s1.pdf"/></supplementary-material></sec>

Brillouin light scattering from surface acoustic waves in a subwavelength-diameter optical fibre

<p>Brillouin scattering in optical fibres is a fundamental interaction between light and sound with important implications ranging from optical sensors to slow and fast light. In usual optical fibres, light both excites and feels shear and longitudinal bulk elastic waves, giving rise to forward-guided acoustic wave Brillouin scattering and backward-stimulated Brillouin scattering. In a subwavelength-diameter optical fibre, the situation changes dramatically, as we here report with the first experimental observation of Brillouin light scattering from surface acoustic waves. These Rayleigh-type surface waves travel the wire surface at a specific velocity of 3,400&#x02009;m&#x02009;s^&#x02212;1 and backscatter the light with a Doppler shift of about 6&#x02009;GHz. As these acoustic resonances are sensitive to surface defects or features, surface acoustic wave Brillouin scattering opens new opportunities for various sensing applications, but also in other domains such as microwave photonics and nonlinear plasmonics.</p>

<contrib contrib-type="author"><name><surname>Beugnot</surname><given-names>Jean-Charles</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lebrun</surname><given-names>Sylvie</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Pauliat</surname><given-names>Gilles</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Maillotte</surname><given-names>Herv&#x000e9;</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Laude</surname><given-names>Vincent</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sylvestre</surname><given-names>Thibaut</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Institut FEMTO-ST, Universit&#x000e9; de Franche-Comt&#x000e9;, CNRS</institution>, Besan&#x000e7;on 25030, <country>France</country></aff><aff id="a2"><label>2</label><institution>Laboratoire Charles Fabry, Institut d&#x02019;Optique, Universit&#x000e9; Paris-Sud, CNRS</institution>, Palaiseau 91127, <country>France</country></aff>

Nature Communications

<p>The complex and intriguing dynamics of light and sound interactions in tiny optical waveguides have recently witnessed a renewed interest because of their experimental realization in emerging key areas of modern physics<xref ref-type="bibr" rid="b1">1</xref>. For instance, the micro and nanostructuring of photonic crystal fibres (PCFs) allows for a tight confinement of both photons and phonons, giving rise to new characteristics for Brillouin scattering fundamentally different from those of standard optical fibres. These include the generation of multiple high-frequency hybrid transverse and longitudinal acoustic waves (LAWs) trapped within the small core of microstructured optical fibres<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. Strong photon&#x02013;phonon coupling has also recently been reported in optical microcavities and new concepts have been introduced such as cavity or surface optomechanics<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>, Brillouin cooling<xref ref-type="bibr" rid="b9">9</xref>, on-chip Brillouin scattering<xref ref-type="bibr" rid="b10">10</xref> and microcavity lasers<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>. Moreover, the recent demonstration of simultaneous photonic and phononic bandgaps in nanostructured materials has led to the development of innovative opto-acoustic devices called phoxonic crystals<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. In yet another example, tailorable Brillouin scattering was recently reported in nanoscale silicon waveguides<xref ref-type="bibr" rid="b15">15</xref>.</p><p>Among other microdevices, photonic silica microwires are the tiny and as-yet underutilized cousins of optical fibres<xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. These hair-like slivers of silica glass, fabricated by tapering optical fibres, enable enhanced nonlinear optical effects and applications not currently possible with comparatively bulky optical fibres. Although microfibres have helped greatly to enhance the optical Kerr effect and stimulated Raman scattering for supercontinuum generation<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref>, Brillouin scattering (BS) in these tiny waveguides has not been explored yet. Until now, only transverse acoustic resonances have been reported in long optical fibre tapers with a large diameter of a few micrometres<xref ref-type="bibr" rid="b20">20</xref>.</p><p>In this work, we present what is to our knowledge the first complete measurement and numerical modelling of Brillouin scattering in a subwavelength-diameter optical fibre, revealing the full elastic wave distribution of such tiny optical waveguides. More specifically, we demonstrate the all-optical generation of a new class of surface acoustic waves (SAWs) and report the observation of SAW Brillouin scattering (SAWBS) in the backward direction. In addition to surface waves, our experimental and theoretical investigations also show that silica microwires also exhibit several widely spaced Brillouin frequencies involving hybrid shear and LAWs, as previously demonstrated in small-core PCFs<xref ref-type="bibr" rid="b2">2</xref>. Our experimental measurements are checked against numerical simulations of the equations of elastodynamics including the electrostrictive stress.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Principle</title><p>Let us first describe the opto-mechanical interaction under consideration. When coherent laser light is coupled and guided into a long uniform optical microwire as that shown schematically in <xref ref-type="fig" rid="f1">Fig. 1</xref>, the light both excites and feels several types of elastic waves with similar micrometre-scale wavelengths. In standard optical fibres, the light is guided into the core and is therefore solely sensitive to both shear and longitudinal bulk acoustic waves, leading to well-known physical effects such as guided acoustic wave Brillouin scattering<xref ref-type="bibr" rid="b21">21</xref> and stimulated Brillouin scattering<xref ref-type="bibr" rid="b22">22</xref> (SBS), respectively. In contrast, in subwavelength-diameter optical fibres, the guided light and particularly the evanescent field interact with the outer surface. Light can thus shake the wire and generate SAWs. The associated mechanical ripples will lead to small periodic changes of the effective refractive index along the optical microwire. When passing through this moving refractive index grating, light undergoes Bragg scattering in the backward direction according to the phase-matching condition, as in fibre-based Brillouin scattering from LAWs. The backscattered Brillouin signal also undergoes a slight shift of its carrier frequency due to the Doppler effect according to the photon&#x02013;phonon energy conservation law <italic>&#x003bd;</italic>_B=2<italic>n</italic>_eff<italic>V</italic>/<italic>&#x003bb;</italic>, with <italic>n</italic>_eff, the effective refractive index of the microwire; <italic>&#x003bb;</italic>, the optical wavelength in vacuum and <italic>V,</italic> the acoustic phase velocity. The acoustic velocity however significantly differs for surface, shear and longitudinal waves. Surface waves travel at a velocity between 0.87 and 0.95 of a shear wave (for fused silica, <italic>V</italic>_S=3,400&#x02009;m&#x02009;s^&#x02212;1). This gives rise to new optical sidebands down-shifted from 6&#x02009;GHz in the light spectrum (<xref ref-type="fig" rid="f1">Fig. 1c</xref>). On top of surface waves, there are also bulk hybrid acoustic waves (HAW) involving both shear and longitudinal components because of the mechanical boundary conditions<xref ref-type="bibr" rid="b2">2</xref> (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). HAWs propagate at an intermediate speed between shear and longitudinal waves with acoustic frequencies ~9&#x02009;GHz. Furthermore, the sound energy density of surface waves is confined at the air&#x02013;silica interface of the microwire, leading to small mechanical ripples of a few picometre, whereas, the HAW energy density remains trapped within the core without altering the wire shape (<xref ref-type="fig" rid="f1">Fig. 1c</xref>).</p></sec><sec disp-level="2"><title>Experimental setup</title><p>In our experiment, we investigated several subwavelength-diameter silica optical fibres, as those shown schematically in <xref ref-type="fig" rid="f2">Fig. 2a</xref> and in the image of <xref ref-type="fig" rid="f2">Fig. 2c</xref>. They were drawn from a commercial single-mode fibre (SMF) using the heat-brush technique<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref> (for details, see Fabrication method). They have a waist diameter ~1&#x02009;&#x003bc;m, a length of 8&#x02009;cm and the input/output tapered fibre sections are 15-mm long. <xref ref-type="fig" rid="f2">Figure 2b</xref> shows the experimental setup to detect Brillouin backscattering<xref ref-type="bibr" rid="b25">25</xref>. As a pump laser, we used a narrow-linewidth continuous-wave distributed-feedback laser running at a wavelength of 1,550&#x02009;nm. The laser output was split into two beams using a fibre coupler. One beam was amplified and injected in the optical microwire through an optical circulator, while the other beam served for detection. We then implemented a heterodyne detection in which the backscattered light from the microwire was mixed with the input coming from a second fibre coupler. The resulting beat note was then detected using a fast photodiode and averaged Brillouin spectra recorded using an electrical spectrum analyser.</p></sec><sec disp-level="2"><title>Experimental results</title><p><xref ref-type="fig" rid="f3">Figure 3a</xref> shows the experimental Brillouin spectrum for an input power of 100&#x02009;mW and for a wire diameter of 1&#x02009;&#x003bc;m. We see the clear emergence of several frequency peaks with different weights and linewidths in a radio-frequency range from 6 to 11&#x02009;GHz. First, the high frequency at 10.86&#x02009;GHz originates from standard Brillouin scattering in the 2-m-long untapered fibre sections and can be disregarded. More importantly, three other peaks appear at 8.33, 9.3&#x02009;GHz, and slightly &#x0003e;10&#x02009;GHz, respectively. The two first peaks exhibit a linewidth ~25&#x02009;MHz, in good agreement with the acoustic amplitude lifetime in fused silica (~10&#x02009;ns). On the basis of numerical simulations described below, we identified them as resulting from Brillouin scattering from hybrid waves whereas the two other resonances ~6&#x02009;GHz are clearly the signature of SAWs.</p></sec><sec disp-level="2"><title>Numerical simulations</title><p>To better understand the nature of these acoustic modes, we performed numerical simulations on the basis of the equations of elastodynamics extended to account for electrostriction, a key physical effect in Brillouin scattering whereby matter become compressed under the effect of an electric field<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref> (for details, see Numerical method section). With respect to standard three-wave mixing SBS theory<xref ref-type="bibr" rid="b28">28</xref>, this new modelling provides an excellent estimate of the theoretical Brillouin gain spectrum by computing the energy density of acoustic phonons generated by light. We considered the silica microwire as a rod-type cylinder and we took into account all elastic and optical parameters of silica. For the sake of simplicity, we neglected the two conical tapered sections of the fibre taper. We must also stress that we did not include in our model the radiation pressure induced by light at the surface of the microwire due to Maxwell stress. Using the method described in ref. <xref ref-type="bibr" rid="b29">29</xref>, we have indeed theoretically estimated that the radiation pressure is &#x0003c;1% of the electrostrictive stress in silica wire of 1&#x02009;&#x003bc;m diameter. Radiation pressure should, however, be taken into account for nanowires whose diameter is &#x0003c;500&#x02009;nm. The results of our numerical simulations are shown in <xref ref-type="fig" rid="f3">Fig. 3b&#x02013;h</xref>. The elastic energy is plotted in <xref ref-type="fig" rid="f3">Fig. 3b</xref> versus acoustic frequency and for a wire diameter varying from 1 to 1.35&#x02009;&#x003bc;m. As can be seen, we retrieve most of the surface and hybrid acoustic resonances, as those observed experimentally in <xref ref-type="fig" rid="f3">Fig. 3a</xref>. There are, however, slight differences with experiment regarding the precise acoustic resonant frequencies. We attribute them to both the microwire uniformity and diameter uncertainty. Nevertheless, we can clearly identify two surface acoustic modes ~6&#x02009;GHz, and three hybrid acoustic modes ~9&#x02009;GHz. More information is provided by the modal distribution of the interacting waves. <xref ref-type="fig" rid="f3">Figure 3c</xref> shows the spatial distribution of the optical mode intensity in the waist region at a wavelength of 1,550&#x02009;nm. The black circle marks out the interface between silica and air. The optical mode is guided in the microwire with a rather long evanescent tail extending outside silica. The tight confinement of the optical field into the microwire induces an electrostrictive stress ~30 times larger than in usual optical fibre (up to 7&#x02009;kPa as compared with 250&#x02009;Pa under same incident optical power<xref ref-type="bibr" rid="b26">26</xref>). As a comparison, the radiation pressure is only 50&#x02009;Pa at the surface of microwire. The electrostrictive stress distribution, shown in <xref ref-type="fig" rid="f3">Fig. 3d</xref>, follows the optical mode distribution without extending out of the microwire. The sound energy density of the two SAWs at 5.382&#x02009;GHz and 5.772&#x02009;GHz are plotted in <xref ref-type="fig" rid="f3">Fig. 3e,f</xref> for a waist of 1.05&#x02009;&#x003bc;m. As can be seen, the elastic energy density is mainly localized below the wire surface. In contrast, the kinetic energy density of HAWs at 8.37 and 9.235&#x02009;GHz, shown in <xref ref-type="fig" rid="f3">Fig. 3g,h</xref>, are mostly confined within the core of the taper. The role of confining elastic energy is due to the elastic waveguide properties of such microwires, mostly its small diameter and hard mechanical boundary conditions.</p><p>To go further into details, we plot in <xref ref-type="fig" rid="f4">Fig. 4a</xref> the full elastic wave spectrum over a wider range of wire diameter till 3.6&#x02009;&#x003bc;m. For every diameter, the fundamental optical mode is recomputed and normalized. The refractive index <italic>n</italic>_eff increases smoothly with the diameter as the optical mode becomes more and more localized within silica rather than air. The two SAWs that we identified before exist for every diameter with a slight shift in frequency. Their acoustic energy is significant for small core but strongly reduces as the diameter increases beyond the optical wavelength (1.5&#x02009;&#x003bc;m). This observation is consistent with the simultaneous decrease of the overlap of SAW with the fundamental optical mode guided inside the core. <xref ref-type="fig" rid="f4">Figure 4a</xref> also shows that, for a given microwire diameter, multiple HAW with widely spaced frequencies can be simultaneously excited, as in small-core PCFs<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b5">5</xref>. This means that, in such small subwavelength waveguides, the light&#x02013;sound interaction is fundamentally different from standard optical fibres. Brillouin spectrum is not simply the signature of a single bulk longitudinal sound wave. In such tiny waveguides, instead, waveguide boundary conditions induce a strong coupling of shear and longitudinal displacements, resulting in a much richer dynamics of light interaction with hybrid and SAWs. We can thus see the emergence of several avoided crossings and acoustic resonance splitting in <xref ref-type="fig" rid="f4">Fig. 4a</xref>, due to the fact that HAWs are not orthogonal and thus strongly interact. This interaction is strong enough for certain acoustic frequencies to be forbidden at some anti-crossing points, as shown by the white arrows in <xref ref-type="fig" rid="f4">Fig. 4a</xref>. In contrast, the two SAW branches cross without interacting for a diameter ~0.85&#x02009;&#x003bc;m because they have orthogonal polarizations. <xref ref-type="fig" rid="f4">Figure 4b</xref> shows the associated transverse and axial displacements in the microwire for one of surface and hybrid modes, denoted A and B in <xref ref-type="fig" rid="f4">Fig. 4a</xref>. Transverse and axial displacements are simply defined as <inline-formula id="d33e362"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e363" xlink:href="ncomms6242-m1.jpg"/></inline-formula> and (|<italic>u</italic>_{<italic>z</italic>}|), respectively. <italic>R</italic> is defined as the ratio between spatial integrals of the shear and longitudinal displacements (<italic>R</italic>=1 for a pure shear wave). As can be seen, the surface acoustic mode A at 5.382&#x02009;GHz exhibits a transversal displacement of a few picometres and a weak axial displacement just below the wire surface. Clearly, this is the signature of a surface Rayleigh wave that combines both a longitudinal and transverse motion to create mechanical ripples with an elliptic orbit motion. In contrast, the displacements for the hybrid mode B are smaller (<italic>R</italic>=0.589) and mainly localized within the core of the wire.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>One may then wonder what is the scattering efficiency and if the threshold for stimulated regime is raised for SAWBS. To answer these important issues, we have estimated the Brillouin gain from the experimental spectrum in <xref ref-type="fig" rid="f3">Fig. 3a</xref>. Starting from the usual theory of Brillouin scattering<xref ref-type="bibr" rid="b30">30</xref>, the Brillouin gain <italic>g</italic>_B can be expressed as <inline-formula id="d33e396"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e397" xlink:href="ncomms6242-m2.jpg"/></inline-formula>, where <italic>n</italic>_eff is the effective refractive index of the microwire, <italic>P</italic>₁₂ is the elasto-optic constant and <italic>&#x003c1;</italic> is the mean density of fused silica (SiO₂), c is the speed of light in vacuum, <italic>&#x003bb;</italic> is the optical wavelength, <italic>&#x003bd;</italic>_{<italic>B</italic>} and &#x00394;<italic>&#x003bd;</italic>_{<italic>B</italic>} are the SAW frequency shift and FWHM width of the spectrum. From measurements, we get <italic>&#x003bd;</italic>_{<italic>B</italic>}=5.85&#x02009;GHz and &#x00394;<italic>&#x003bd;</italic>_{<italic>B</italic>}=20&#x02009;MHz for the first SAW peak, thus we found <italic>g</italic>_B=1.4.10^&#x02212;12&#x02009;m&#x02009;W^&#x02212;1, to be compared with <italic>g</italic>_B=3.10^&#x02212;11&#x02009;m&#x02009;W^&#x02212;1 for SMF fibres. If we now consider the effective mode area <italic>A</italic>_eff to get the Brillouin gain factor by unit length, we found that <italic>g</italic>_B/<italic>A</italic>_eff=8&#x02009;W^&#x02212;1&#x02009;m^&#x02212;1 for a 1-&#x003bc;m-diameter silica wire whereas it is only 0.4&#x02009;W^&#x02212;1&#x02009;m^&#x02212;1 for SMF fibres. That clearly means that the scattering efficiency is rather high. When examining carefully the spectrum of <xref ref-type="fig" rid="f3">Fig. 3a</xref>, one can indeed clearly see that the SAW efficiency in the microwire is just 2-dB below that of Brillouin scattering in the 2&#x02009;m-long fibre pigtails. However the threshold for stimulated regime is not raised over so short propagation distance. It can readily be derived from the Brillouin gain using the following standard formula<xref ref-type="bibr" rid="b30">30</xref> <italic>P</italic>_th=(21<italic>A</italic>_eff)/(<italic>Kg</italic>_B<italic>L</italic>_eff), where <italic>L</italic>_eff is the effective length that accounts for losses and <italic>K</italic> the polarization factor (<italic>K</italic>=1.5). Assuming 10&#x02009;dB of linear losses for the microwire from measurements, we found a high threshold power of <italic>P</italic>_th=60&#x02009;W in silica microwire which is well above the continuous-wave power used in the experiment (100&#x02009;mW). This high threshold power level could, however, be achieved using amplified long nanosecond optical pulses instead of continuous-wave power. There also could be improvement in microwire parameters such as optical loss. In addition, we can expect that the surface Brillouin threshold can be substantially reduced in optical microwire made of highly nonlinear chalcogenide glasses<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b31">31</xref>.</p></sec><sec disp-level="1" sec-type="conclusions"><title>Conclusion</title><p>In conclusion, we have reported a clear-cut evidence of a new type of Brillouin light scattering driven by SAWs by use of a subwavelength-diameter silica fibre. It has been further shown that the SAWs reported here travel the wire surface at a specific velocity of 3,400&#x02009;m&#x02009;s^&#x02212;1 and give rise to new useful optical sidebands around 6&#x02009;GHz in the scattering spectrum. This work thus may represent a landmark to foster the investigation of such surface waves in many different photonic platforms, such as PCFs and integrated optical devices (for example, chalcogenide and silicon chips). In addition, SAWBS can find strong potential applications for optical sensing and detection because these acoustic waves are inherently sensitive to surface features and defects and are already used in many sensing systems (for example, MEMS). They could be seen as a new class of Brillouin optical sensors that rely on the modulation of SAWs to sense a physical phenomenon (for example, temperature, stress, gas). Changes in amplitude and frequency of the Brillouin signal through the silica microwire could be advantageously used to measure the presence of the desired phenomenon. One can also imagine a strong interaction between surface waves and surface plasmons by depositing a thin layer of gold or silver<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>. These results thus show the potential of optical microwires and nanowires for surface Brillouin scattering<xref ref-type="bibr" rid="b34">34</xref>, optical sensing and nonlinear plasmonics<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>. Finally, this work contributes to the further understanding of the intriguing light&#x02013;sound interactions in subwavelength optics and in nanophotonics.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Fabrication method</title><p>The silica microwire has been tapered from a standard telecom fibre (SMF-28) using the heat-brush technique<xref ref-type="bibr" rid="b24">24</xref>. The fibre to be pulled is attached at two computer-controlled translation stages. The fibre is softened on its central part with a small butane flame. The gas flow is monitored and regulated by a mass-flow controller. The flame is kept fixed while the two translation stages elongate the fibre to create the microwire. The microwire shape is fully controlled by the trajectories of the two translation stages. The untapered parts, upstream and downstream the microwire, allow a very easy light injection and collection. During the pulling, a laser light is coupled inside the microwire and collected at the output end to control several parameters (transmitted power, mode shape and spectrum). With this home-made pulling platform, we routinely achieved light transmission larger than 90% over the full fibre including the tapered and untapered parts, even with microwire diameters of a few hundred nanometres and lengths up to several centimeters.</p></sec><sec disp-level="2"><title>Numerical method</title><p>Our experimental observations of surface and HAWs are modelled with the equations of elastodynamics including the electrostrictive stress induced by the optical field. Specifically, the displacements <italic>u</italic>_{<italic>i</italic>} in silica microwire are given by a simple partial differential equation which reads:</p><p><disp-formula id="eq3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e613" xlink:href="ncomms6242-m3.jpg"/></disp-formula></p><p>where <italic>c</italic>_{<italic>ijkl</italic>} is the rank-4 tensor of elastic constants. <inline-formula id="d33e622"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e623" xlink:href="ncomms6242-m4.jpg"/></inline-formula> is the eletrostrictive stress tensor, with the rank-4 susceptibility tensor &#x003c7;_{<italic>klij</italic>}=<italic>&#x003b5;</italic>_{<italic>km</italic>}<italic>&#x003b5;</italic>_{<italic>ln</italic>}<italic>p</italic>_{<italic>mnij</italic>} and <italic>p</italic>_{<italic>mnij</italic>} the elasto-optic tensor. &#x003b5;₀ is the permitivity of vacuum. The force term with detuning frequency <italic>&#x003c9;</italic>=<italic>&#x003c9;</italic>₁&#x02212;<italic>&#x003c9;</italic>₂ is proportional to <inline-formula id="d33e669"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e670" xlink:href="ncomms6242-m5.jpg"/></inline-formula> with <italic>k</italic>=<italic>k</italic>₁&#x02212;<italic>k</italic>₂. Here we assume that the total optical field results from the superposition of the pump and Brillouin Stokes waves with angular frequencies <italic>&#x003c9;</italic>_1,2 and axial wavevectors <italic>k</italic>_1,2. If the two optical waves are propagating in opposite directions (See <xref ref-type="fig" rid="f1">Fig. 1a,b</xref>) <italic>k</italic>&#x02248;2<italic>k</italic>₁ and we speak of backward SBS or SAWBS. We also consider in our model the phonon lifetime by taking into account the elastic losses assuming a complex tensor. This loss model is compatible with the usual assumption that the product of the quality factor Q and the acoustic frequency is a constant for a given material (for example, for silica, Q &#x000d7; f=5&#x02009;THz). Further applying Green&#x02019;s theorem to <xref ref-type="disp-formula" rid="eq3">equation (1)</xref>, we get</p><p><disp-formula id="eq6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e716" xlink:href="ncomms6242-m6.jpg"/></disp-formula></p><p>which accounts for the theorem of virtual work for the elestrostrictive stress. For numerical computations, we used the Galerkin nodal finite element method to transform the integral equation into the following linear matrix system</p><p><disp-formula id="eq7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e721" xlink:href="ncomms6242-m7.jpg"/></disp-formula></p><p>with mass matrix <italic>M</italic>, stiffness matrix <italic>K</italic>(<italic>k</italic>)=<italic>K</italic>₀+<italic>kK</italic>₁+<italic>k</italic>²<italic>K</italic>₂, and <italic>X</italic>(<italic>k</italic>)=<italic>X</italic>₀+<italic>kX</italic>₁. <italic>U</italic> is the vector norm of nodal displacements <italic>&#x0016b;</italic>_{<italic>i</italic>}. Solutions of <xref ref-type="disp-formula" rid="eq7">equation (3)</xref> as a function of frequency detuning yield rigorous distribution of displacements within microwire cross-section as shown in <xref ref-type="fig" rid="f3">Fig. 3</xref>. The associated sound energy density can be directly compared with experimental measurements of Brillouin scattering. Finally, the electrostriction stress tensor is defined by the optical modal distribution (<xref ref-type="fig" rid="f3">Fig. 3c,d</xref>), which is beforehand calculated using a finite element method (Comsol software).</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>J.-C.B. performed the main experimental observations and characterizations. T.S. wrote the main manuscript text and supervised the overall project. S.L. and G.P. designed and fabricated the optical microwires. V.L. and J.-C.B. developed the numerical model. H.M. launched the initial scientific project. All authors discussed the results and substantially contributed to the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Beugnot, J.-C. B. <italic>et al.</italic> Brillouin light scattering from surface acoustic waves in a subwavelength-diameter optical fibre. <italic>Nat. Commun.</italic> 5:5242 doi: 10.1038/ncomms6242 (2014).</p></sec>

Observation of strongly entangled photon pairs from a nanowire quantum dot

<p>A bright photon source that combines high-fidelity entanglement, on-demand generation, high extraction efficiency, directional and coherent emission, as well as position control at the nanoscale is required for implementing ambitious schemes in quantum information processing, such as that of a quantum repeater. Still, all of these properties have not yet been achieved in a single device. Semiconductor quantum dots embedded in nanowire waveguides potentially satisfy all of these requirements; however, although theoretically predicted, entanglement has not yet been demonstrated for a nanowire quantum dot. Here, we demonstrate a bright and coherent source of strongly entangled photon pairs from a position-controlled nanowire quantum dot with a fidelity as high as 0.859&#x000b1;0.006 and concurrence of 0.80&#x000b1;0.02. The two-photon quantum state is modified via the nanowire shape. Our new nanoscale entangled photon source can be integrated at desired positions in a quantum photonic circuit, single-electron devices and light-emitting diodes.</p>

<contrib contrib-type="author"><name><surname>Versteegh</surname><given-names>Marijn A. M.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Reimer</surname><given-names>Michael E.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>J&#x000f6;ns</surname><given-names>Klaus D.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Dalacu</surname><given-names>Dan</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Poole</surname><given-names>Philip J.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Gulinatti</surname><given-names>Angelo</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Giudice</surname><given-names>Andrea</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Zwiller</surname><given-names>Val</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Quantum Transport, Kavli Institute of Nanoscience, Delft University of Technology</institution>, Lorentzweg 1, 2628CJ Delft, <country>The Netherlands</country></aff><aff id="a2"><label>2</label><institution>National Research Council of Canada</institution>, Ottawa, Ontario, <country>Canada</country> K1A 0R6</aff><aff id="a3"><label>3</label><institution>Politecnico di Milano, Dipartimento di Elettronica Informazione e Bioingegneria</institution>, piazza Leonardo da Vinci 32, 20133 Milano, <country>Italy</country></aff><aff id="a4"><label>4</label><institution>Micro Photon Devices</institution>, Via Stradivari 4, 39100 Bolzano, <country>Italy</country></aff>

Nature Communications

<p>There are demanding requirements for an &#x02018;ideal&#x02019; entangled-photon source for implementing ambitious schemes in quantum information processing, such as that of a quantum repeater<xref ref-type="bibr" rid="b1">1</xref>. The source should meet the following criteria: high brightness combined with high-fidelity entanglement<xref ref-type="bibr" rid="b2">2</xref>, on-demand generation<xref ref-type="bibr" rid="b3">3</xref>, high extraction efficiency<xref ref-type="bibr" rid="b4">4</xref>, directional<xref ref-type="bibr" rid="b5">5</xref> and coherent emission<xref ref-type="bibr" rid="b6">6</xref>, as well as position control at the nanoscale<xref ref-type="bibr" rid="b7">7</xref>. It is extremely difficult to meet all of these requirements in a single device. Good candidates are semiconductor quantum dots embedded in nanowires.</p><p>The high refractive index of a nanowire waveguide around a quantum dot ensures that the emitted light is guided in the desired direction and a tapered end makes the light extraction very efficient<xref ref-type="bibr" rid="b4">4</xref>. With such a design, efficient single-photon generation has been demonstrated from a single nanowire quantum dot<xref ref-type="bibr" rid="b5">5</xref>. In addition, the emission mode-profile was shown to be directional and Gaussian<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>, a key requirement for efficient long-distance quantum communication in well-established telecommunication technology. Nanowires can be controllably positioned in uniform arrays<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref>, with the ability to independently control the dot size and waveguide shell around it<xref ref-type="bibr" rid="b12">12</xref>. Silicon segments and substrates can be included in the design<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref> and electrical contacts have been demonstrated on single nanowires for single-electron devices<xref ref-type="bibr" rid="b17">17</xref>, light-emitting diodes<xref ref-type="bibr" rid="b18">18</xref>, as well as single-photon avalanche photodiodes<xref ref-type="bibr" rid="b19">19</xref>. A significant advantage of using nanowire waveguides for efficient light extraction over other existing approaches, such as optical microcavities<xref ref-type="bibr" rid="b20">20</xref>, is the broad frequency bandwidth of operation<xref ref-type="bibr" rid="b21">21</xref>, which is needed for achieving bright entangled photon-pair generation via the biexciton&#x02013;exciton radiative cascade. This approach is especially advantageous for quantum dots emitting over a large spectral range and may also be implemented with II&#x02013;VI quantum dots where the biexciton binding energy is very large (&#x0003e;20&#x02009;meV)<xref ref-type="bibr" rid="b22">22</xref>.</p><p>A key feature of nanowires with embedded quantum dots grown in the [111]-direction is that the fine-structure splitting is expected to vanish<xref ref-type="bibr" rid="b23">23</xref>, which should result in excellent entangled photon emission via the biexciton&#x02013;exciton radiative cascade<xref ref-type="bibr" rid="b24">24</xref>. Our measurements realize this prediction and demonstrate the generation of strongly entangled photon pairs for the first time from a nanowire quantum dot. Our sources are ready to implement in advanced quantum information processing schemes without the need for any post-growth manipulation<xref ref-type="bibr" rid="b25">25</xref> or temporal post-selection<xref ref-type="bibr" rid="b26">26</xref>. Temporal post-selection can be a major source of photon losses and puts additional requirements on the measurement, thus limiting the scalability of quantum dot-based entangled photon sources. For practical applications it is therefore very useful that we can avoid temporal post-selection. Finally, due to the efficient waveguiding and the tapered end, which we created during the bottom-up growth of the nanowire, we measure a light extraction efficiency of 18&#x000b1;3% for the source. Importantly, due to a recent breakthrough in the nanowire growth<xref ref-type="bibr" rid="b12">12</xref>, this high efficiency is obtained while potentially meeting all of the criteria of an ideal entangled photon source.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Site-controlled quantum dots in tapered nanowire waveguides</title><p>The nanowires were grown by selective-area chemical beam epitaxy, which allows for control of the dot size and position, as well as enabling growth of the waveguide shell around the dot for efficient light extraction (see Methods). This technique has been demonstrated to yield defect-free, pure wurtzite nanowires, which is essential to obtain long single-photon coherence<xref ref-type="bibr" rid="b27">27</xref>. <xref ref-type="fig" rid="f1">Figure 1a</xref> shows a scanning electron microscopy (SEM) image of a tapered InP nanowire waveguide containing an InAsP segment, 200&#x02009;nm from the nanowire base, defining the optically active quantum dot that we study.</p><p>A spectrum taken under the excitation condition used for the quantum-state tomography measurements is depicted in <xref ref-type="fig" rid="f1">Fig. 1b</xref>. By performing cross-correlation measurements<xref ref-type="bibr" rid="b28">28</xref>, shown in the inset, and power-dependent measurements (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>), we identified the biexciton (XX) and exciton (X_A and X_B) transitions. The XX&#x02013;X_B cascade produces entangled photons. In contrast, a weak cascade is observed for XX&#x02013;X_A, which does not show entanglement. From these observations, X_A could be either a charged exciton or an exciton with a different hole state than X_B as is permitted by the wurtzite crystal structure. The transitions XX and X_B are resolution limited; single-photon interference measurements show excellent coherence of our entangled photons (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>). Autocorrelation measurements at saturation of the XX and X_B transitions show strong antibunching, indicative of nearly perfect single-photon pairs from the XX&#x02013;X_B cascade (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>).</p></sec><sec disp-level="2"><title>Light extraction efficiency</title><p>From the single-photon detector counts, 55 kilocounts per second for XX and 15 kilocounts per second for X_B, under pulsed excitation at 80&#x02009;MHz, we calculate a collection of 7.9 million XX and 2.0 million X_B photons per second into the first objective when taking our 0.7&#x000b1;0.1% setup efficiency at ~900&#x02009;nm into account. The X_A transition shows the highest intensity of 12.1 million photons per second and saturates our spectrometer&#x02019;s CCD camera with integration times as short as 1&#x02009;s under the excitation conditions used throughout our study (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). This radiative recombination pathway from XX competes strongly with the X_B emission, thus reducing the entangled photon-pair generation efficiency. Taking into account this competing recombination pathway for XX, we calculate a light extraction efficiency of 18&#x000b1;3% for the source. We expect that adding a gold mirror with thin dielectric layer below the nanowire will boost the efficiency nearly twofold<xref ref-type="bibr" rid="b21">21</xref>. Combining this mirror with further engineering of the nanowire shape promises extraction efficiencies exceeding 90% (ref. <xref ref-type="bibr" rid="b29">29</xref>).</p></sec><sec disp-level="2"><title>Low fine-structure-splitting system</title><p>Using polarization-dependent measurements, presented in <xref ref-type="fig" rid="f1">Fig. 1c</xref>, we obtain an estimation for the excitonic fine-structure splitting, <italic>S</italic>, by subtracting the XX transition from the X_B transition energy<xref ref-type="bibr" rid="b30">30</xref>. We obtain from the sine-function fit a fine-structure splitting of 1.2&#x02009;&#x003bc;eV. In the case of nanowires, the small fine-structure splitting is a result of growth on a [111]-oriented substrate and the symmetric hexagonal cross-section of the nanowire core, defining the quantum dot. This small value that we measure for the fine-structure splitting is crucial for the entanglement observation between XX and X_B photons without any temporal post-selection and is representative of the sample where, remarkably, a high percentage (&#x0003e;50%) of the measured quantum dots show a fine-structure splitting below 2&#x02009;&#x003bc;eV (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). The period of precession of the X_B spin can be estimated as <italic>h</italic>/<italic>S</italic>=3.5&#x02009;ns (ref. <xref ref-type="bibr" rid="b31">31</xref>), where <italic>h</italic> is Planck&#x02019;s constant. This period of precession is much longer than the X_B lifetime of 0.50&#x000b1;0.01&#x02009;ns as extracted from the XX&#x02013;X_B cross-correlation measurements without polarization selection (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>). Therefore, the X_B spin precession has only little influence on the correlations in polarization between the two photons.</p></sec><sec disp-level="2"><title>Polarization-entangled photon pairs</title><p>Twelve cross-correlation measurements in the rectilinear, diagonal and circular polarization bases are shown in <xref ref-type="fig" rid="f2">Fig. 2</xref>, where each histogram is composed of 64&#x02009;ps time bins. In the correlation measurements, the first letter stands for the measured polarization of the XX photon, whereas the second letter stands for the X_B photon. The strong correlations in <italic>HV</italic>, <italic>VH</italic>, <italic>DD</italic>, <italic>AA</italic>, <italic>RR</italic> and <italic>LL</italic>, together with the weak correlations in <italic>HH</italic>, <italic>VV</italic>, <italic>DA</italic>, <italic>AD</italic>, <italic>RL</italic> and <italic>LR</italic>, show that the two photons from the XX&#x02013;X_B cascade are entangled. Here, <italic>H</italic> and <italic>V</italic> are orthogonal linear polarizations (horizontal and vertical), <inline-formula id="d33e453"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e454" xlink:href="ncomms6298-m1.jpg"/></inline-formula> and <inline-formula id="d33e456"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e457" xlink:href="ncomms6298-m2.jpg"/></inline-formula> are diagonal and antidiagonal linear polarizations, whereas <inline-formula id="d33e459"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e460" xlink:href="ncomms6298-m3.jpg"/></inline-formula> and <inline-formula id="d33e462"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e463" xlink:href="ncomms6298-m4.jpg"/></inline-formula> are righthanded and lefthanded circular polarizations.</p><p>The quantum state we observe is different from the state that is measured for self-assembled quantum dots<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>. Typically, one measures for the XX&#x02013;X cascade bunching (positive correlations) in <italic>HH</italic> and <italic>RL</italic>, and antibunching (negative correlations) in <italic>HV</italic> and <italic>RR</italic>. However, we observe the opposite (<xref ref-type="fig" rid="f2">Fig. 2a,c</xref>). Only in the diagonal basis we see the usual correlations: bunching in <italic>DD</italic> and antibunching in <italic>DA</italic>. These results show that the two-photon quantum state is closer to <inline-formula id="d33e491"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e492" xlink:href="ncomms6298-m5.jpg"/></inline-formula> than to the commonly measured state <inline-formula id="d33e494"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e495" xlink:href="ncomms6298-m6.jpg"/></inline-formula> (refs <xref ref-type="bibr" rid="b31">7</xref>, <xref ref-type="bibr" rid="b32">25</xref>, <xref ref-type="bibr" rid="b33">32</xref>, <xref ref-type="bibr" rid="b34">33</xref>, <xref ref-type="bibr" rid="b35">34</xref>).</p></sec><sec disp-level="2"><title>Quantum-state tomography</title><p>We performed quantum-state tomography<xref ref-type="bibr" rid="b36">36</xref> to determine more precisely the quantum state of the photons and the degree of entanglement. The raw cross-correlation measurements needed to reconstruct the density matrix are shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>. The resulting density matrix is given in <xref ref-type="fig" rid="f3">Fig. 3a,b</xref>. The concurrence is 0.57&#x000b1;0.02. A positive value for the concurrence means that the correlations cannot be explained classically and that the photons are quantum entangled. In this calculation, all correlation counts in the full time window of 6.02&#x02009;ns are taken into account. The two-photon state has a fidelity of 0.762&#x000b1;0.002 to the maximally entangled state <inline-formula id="d33e526"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e527" xlink:href="ncomms6298-m7.jpg"/></inline-formula>, where <italic>J</italic>=<italic>He</italic>^{&#x02212;<italic>i&#x003b2;</italic>} cos<italic>&#x003b1;</italic>+<italic>Ve</italic>^{&#x02212;<italic>i&#x003b2;</italic>} sin<italic>&#x003b1;</italic> and <italic>W</italic>=&#x02212;<italic>He</italic>^{<italic>i&#x003b2;</italic>} sin<italic>&#x003b1;</italic>+<italic>Ve</italic>^{<italic>i&#x003b2;</italic>} cos<italic>&#x003b1;</italic> are two orthogonal elliptical polarizations. The angles <italic>&#x003b1;</italic> and <italic>&#x003b2;</italic> are specified in <xref ref-type="table" rid="t1">Table 1</xref>. The classical limit is 0.5, so this result shows a strong degree of entanglement, even without temporal selection.</p><p>Selection of a narrower time window yields higher values for the concurrence and the fidelity (<xref ref-type="table" rid="t1">Table 1</xref>). For example, for a time window of 0.13&#x02009;ns we calculate a concurrence of 0.80&#x000b1;0.02 and a fidelity of 0.854&#x000b1;0.006. The density matrix for this time window is presented in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>. Temporal selection yields stronger entanglement, because within a narrow time window the effects of spin precession and dephasing processes are smaller<xref ref-type="bibr" rid="b31">31</xref>. When we do not restrict our analysis to states of the form <inline-formula id="d33e595"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e596" xlink:href="ncomms6298-m8.jpg"/></inline-formula>, but instead calculate the fidelity to a general maximally entangled two-photon state, we find only slightly higher values (<xref ref-type="table" rid="t1">Table 1</xref>). The maximally entangled states to which the fidelity is maximal are very close to states of the form <inline-formula id="d33e602"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e603" xlink:href="ncomms6298-m9.jpg"/></inline-formula>.</p></sec><sec disp-level="2"><title>Two-photon quantum state modified by birefringence</title><p>Why do we measure <inline-formula id="d33e610"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e611" xlink:href="ncomms6298-m10.jpg"/></inline-formula> and not the usual two-photon state that is measured for quantum dots, namely <inline-formula id="d33e613"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e614" xlink:href="ncomms6298-m11.jpg"/></inline-formula>? The most probable reason is that the nanowire has a small anisotropy: it could have a slightly elongated cross-section. An extreme case is shown in the SEM image of <xref ref-type="fig" rid="f4">Fig. 4b</xref>. Such an anisotropy may be formed during the growth of the cladding around the core, and would then be unrelated to the shape of the quantum dot (for details of the growth, see the Methods section). As a comparison, we show a symmetric nanowire waveguide in the SEM image of <xref ref-type="fig" rid="f4">Fig. 4a</xref>. In case of an elongated cross-section the effective refractive indices are different for the polarizations along the short and the long axis of the nanowire. Here, we could imagine that the quantum dot emits photon pairs in the usual entangled quantum state <inline-formula id="d33e622"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e623" xlink:href="ncomms6298-m12.jpg"/></inline-formula>. As the emitted photons are guided along the nanowire, the two-photon state is modified by birefringence into <inline-formula id="d33e626"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e627" xlink:href="ncomms6298-m13.jpg"/></inline-formula>, as is illustrated in <xref ref-type="fig" rid="f3">Fig. 3c</xref>. Thus, <italic>HH</italic> and <italic>VV</italic> correlations rotate into predominantly <italic>RR</italic> and <italic>LL</italic> correlations, while <italic>RL</italic> and <italic>LR</italic> turn mostly into <italic>HV</italic> and <italic>VH</italic>, which explains the observations of <xref ref-type="fig" rid="f2">Fig. 2</xref>. For a nanowire waveguide of 6&#x02009;&#x003bc;m length a difference of effective refractive index of order 0.1 would be enough to explain the magnitude of the observed rotation. Apart from birefringence in the waveguide, the polarization state of the emitted photons could also have been influenced by &#x00393;₇ and &#x00393;₉ hole mixing in the wurtzite quantum dot.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In summary, we used a wurtzite nanowire quantum dot to generate single pairs of polarization-entangled photons with a fidelity as high as 0.859&#x000b1;0.006 and a concurrence up to 0.80&#x000b1;0.02. Furthermore, a high degree of entanglement is maintained (fidelity of 0.762&#x000b1;0.002) without any temporal post-selection. This first observation of entangled photon-pair generation from a nanowire quantum dot, which combines the desired properties of an ideal entangled photon source, opens new opportunities in quantum optics, integrated quantum photonic circuits<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref> and quantum information processing.</p><p>To realize an ideal entangled photon source in future work there are several properties of our source to consider. First, quantum dot-entangled photon sources have not yet reached the fidelity or concurrence values of parametric down-conversion sources<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b39">39</xref>. However, with recently available post-growth tuning methods to bring the fine-structure splitting of almost any quantum dot near zero<xref ref-type="bibr" rid="b25">25</xref> and two-photon resonant excitation<xref ref-type="bibr" rid="b3">3</xref>, the fidelity of these quantum dot sources are approaching that of parametric down-conversion sources. Second, the single-photon coherence of the emitted photon pairs is not yet Fourier-transform limited, which is needed for advanced quantum information-processing schemes. Such Fourier-transform-limited photons may be reached by combining two-photon resonant excitation techniques<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b40">40</xref>, cooling of the quantum dot sample to 300&#x02009;mK<xref ref-type="bibr" rid="b28">28</xref> and by accelerating the quantum dot emission via the Purcell effect<xref ref-type="bibr" rid="b20">20</xref>. Finally, the major advantage of tapered nanowire waveguides over other approaches is the light extraction efficiency, which promises entangled photon-pair extraction efficiencies exceeding 90% due to the broadband frequency of operation<xref ref-type="bibr" rid="b29">29</xref>. Such efficiencies would surpass the state-of-the-art entangled photon-pair efficiency of 12%<xref ref-type="bibr" rid="b20">20</xref>, without the stringent requirements needed to engineer both the exciton and biexciton into resonance with a cavity mode by using post-growth manipulation of pre-selected quantum dots.</p><p>Note: Similar work is reported by Huber <italic>et al</italic>.<xref ref-type="bibr" rid="b26">26</xref></p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Nanowire quantum dot growth</title><p>The InP nanowires containing single InAsP quantum dots were grown using chemical beam epitaxy with trimethylindium and pre-cracked PH₃ and AsH₃ sources. The nanowires were grown on a SiO₂-patterned (111)B InP substrate consisting of circular holes opened up in the oxide mask using electron-beam lithography and a hydrofluoric acid wet-etch. Au was deposited in these holes using a self-aligned lift-off process, which allows the nanowires to be positioned at known locations on the substrate<xref ref-type="bibr" rid="b41">41</xref>. The thickness of the deposited gold is chosen to give 20-nm to 40-nm diameter particles, depending on the size of the hole opening. The nanowires were grown at 420&#x02009;&#x000b0;C with a trimethylindium flux equivalent to that used for a planar InP growth rate of 0.1&#x02009;&#x003bc;m&#x02009;h^&#x02212;1 on (001) InP substrates at a temperature of 500&#x02009;&#x000b0;C. The growth is a two-step process: (i) growth of a nanowire core containing the quantum dot, nominally 200&#x02009;nm from the nanowire base, and (ii) cladding of the core to realize nanowire diameters for efficient light extraction (around 200&#x02009;nm). The quantum dot diameters are determined by the size of the nanowire core. In this study, we investigated quantum dot diameters ranging from ~25 to 30&#x02009;nm.</p><p>The nanowire core was grown for 26&#x02009;min at a PH₃ flow of 3&#x02009;s.c.c.m. The dot was incorporated by switching from a PH₃ to an AsH₃ overpressure for 3&#x02009;s after 15&#x02009;min of growth. This growth time results in a quantum dot height of ~6&#x02009;nm as determined in our previous studies<xref ref-type="bibr" rid="b12">12</xref>, using an energy-dispersive X-ray spectroscopy line scan along the nanowire, for a sample with nominally identical growth conditions. We note that our quantum dots are grown during 3&#x02009;s, resulting in taller quantum dots with longer emission wavelength, as compared with the work of Huber <italic>et al.</italic><xref ref-type="bibr" rid="b26">26</xref> who used a growth time of 2&#x02009;s. Our growth conditions result in very small fine-structure splittings as shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>.</p><p>The nanowire cladding was grown by increasing the PH₃ flow to 9&#x02009;s.c.c.m. The total growth time was 120&#x02009;min. To realize the smooth tapering towards the tip, the nanowire was made longer than the diffusion length of indium. Most nanowires, including the quantum dots, have a pure wurtzite crystal structure. The nanowire axis is the wurtzite <italic>c</italic> axis.</p></sec><sec disp-level="2"><title>Nanowire waveguide elongation</title><p>Radial growth is nominally constrained by the oxide opening, and the nanowire cross-section has a hexagonal symmetry. Optimal coupling of the quantum dot emission to the waveguide mode requires diameters exceeding that of the oxide opening. This is achieved by increasing the cladding growth time, which results in the nanowire overgrowing the oxide opening. No longer constrained by the opening, the hexagonal symmetry may be distorted (see <xref ref-type="fig" rid="f4">Fig. 4</xref>). This asymmetry results in a geometric birefringence and concomitant rotation of the polarization state emitted by the quantum dot.</p></sec><sec disp-level="2"><title>Optical measurements</title><p>The optical measurements were performed in a standard confocal microscopy setup where the quantum dot sample is cooled to a temperature of T=5&#x02009;K in a closed-cycle cryostat. The setup consists of two spectrometers both equipped with red-enhanced single-photon avalanche diodes having 75&#x02009;ps time resolution, dark count rates as low as 80 counts per second and quantum efficiency of 11.5% at 930&#x02009;nm<xref ref-type="bibr" rid="b42">42</xref>. A set of waveplates and polarizers placed in front of each spectrometer was used to perform polarization-dependent cross-correlation measurements. One spectrometer is set to the biexciton (XX) transition and the other to the exciton (X_B) transition. Each correlation measurement was done with 6,000&#x02009;s of integration to reach over 1,000 correlations in each side peak. For all photoluminescence and correlation measurements, we use a Ti:Sapphire laser emitting 3-ps-long pulses at 750&#x02009;nm with a repetition rate of 80&#x02009;MHz to excite the quantum dot.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>M.E.R., M.A.M.V. and V.Z. conceived and designed the experiments. K.D.J., M.E.R. and M.A.M.V. performed the experiments. D.D. and P.J.P. fabricated the sample. M.A.M.V., M.E.R. and K.D.J. analysed the data. A.Gu. and A.Gi. developed the detectors. M.E.R., M.A.M.V., K.D.J. and V.Z. wrote the manuscript with input from the other authors.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article</bold>: Versteegh, M. A. M. <italic>et al.</italic> Observation of strongly entangled photon pairs from a nanowire quantum dot. <italic>Nat. Commun.</italic> 5:5298 doi: 10.1038/ncomms6298 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-7 and Supplementary Note</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6298-s1.pdf"/></supplementary-material></sec>

Quantum signatures of a molecular nanomagnet in direct magnetocaloric measurements

<p>Geometric spin frustration in low-dimensional materials, such as the two-dimensional kagome or triangular antiferromagnetic nets, can significantly enhance the change of the magnetic entropy and adiabatic temperature following a change in the applied magnetic field, that is, the magnetocaloric effect. In principle, an equivalent outcome should also be observable in certain high-symmetry zero-dimensional, that is, molecular, structures with frustrated topologies. Here we report experimental realization of this in a heptametallic gadolinium molecule. Adiabatic demagnetization experiments reach ~200&#x02009;mK, the first sub-Kelvin cooling with any molecular nanomagnet, and reveal isentropes (the constant entropy paths followed in the temperature-field plane) with a rich structure. The latter is shown to be a direct manifestation of the trigonal antiferromagnetic net structure, allowing study of frustration-enhanced magnetocaloric effects in a finite system.</p>

<contrib contrib-type="author"><name><surname>Sharples</surname><given-names>Joseph W.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Collison</surname><given-names>David</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>McInnes</surname><given-names>Eric J. L.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Schnack</surname><given-names>J&#x000fc;rgen</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Palacios</surname><given-names>Elias</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Evangelisti</surname><given-names>Marco</given-names></name><xref ref-type="corresp" rid="c3">c</xref><xref ref-type="aff" rid="a3">3</xref></contrib><aff id="a1"><label>1</label><institution>School of Chemistry and Photon Science Institute, The University of Manchester</institution>, Manchester M13 9PL, <country>UK</country></aff><aff id="a2"><label>2</label><institution>Faculty of Physics, Bielefeld University</institution>, PO box 100131, D-33501 Bielefeld, <country>Germany</country></aff><aff id="a3"><label>3</label><institution>Departamento de F&#x000ed;sica de la Materia Condensada and Instituto de Ciencia de Materiales de Arag&#x000f3;n (ICMA), CSIC&#x02014;Universidad de Zaragoza</institution>, Pedro Cerbuna 12, 50009 Zaragoza, <country>Spain</country></aff>

Nature Communications

<p>Sub-Kelvin temperatures can be achieved via adiabatic demagnetization of paramagnetic salts<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. The underlying physics is the magnetocaloric effect (MCE) that can be evaluated by considering the adiabatic temperature change, which is when the system is driven on a constant entropy (<italic>S</italic>) curve (an isentrope):</p><p><disp-formula id="eq1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e171" xlink:href="ncomms6321-m1.jpg"/></disp-formula></p><p>where <italic>C</italic> is the heat capacity, <italic>T</italic> is the temperature and <italic>B</italic> is the applied magnetic field. For a paramagnet, the isentropes are straight lines in a <italic>T</italic>&#x02013;<italic>B</italic> plane that run through the origin. Interacting spin systems can show a much richer response to magnetic fields and thus very different isentropes. Importantly, the cooling rate can massively outperform those of paramagnets in certain regions of the <italic>T</italic>&#x02013;<italic>B</italic> plane<xref ref-type="bibr" rid="b3">3</xref>. The simplest illustration is an antiferromagnetically coupled dimer of <italic>s</italic>=1/2 spin (<xref ref-type="fig" rid="f1">Fig. 1</xref>) where extremes in the cooling rates (even changing sign) are found at the field-induced level crossing between singlet and triplet because the density of states (and hence the low-temperature entropy) peaks at this field.</p><p>Such a crossing belongs to the broader class of quantum phase transitions where the ground-state characteristics of a system change (for example, non-magnetic to magnetic, or from gapped to gapless) as a function of an external parameter such as magnetic field, pressure or doping<xref ref-type="bibr" rid="b4">4</xref>. For MCE, the drastic changes in entropy across a field-induced quantum critical point can give very efficient low-temperature magnetic cooling as recently demonstrated experimentally for a one-dimensional (1D) antiferromagnetic (AF) <italic>s</italic>=1/2 chain<xref ref-type="bibr" rid="b3">3</xref>. Geometric spin frustration can also give rise to regions of high density of states (and zero-temperature entropy), hence very high cooling rates should also be achievable, for example, when sweeping across the saturation field in such materials. The combination of these features in low-dimensional frustrated magnetic materials, for example, the famous 2D kagome or triangular AF lattices or the 1D saw-tooth AF chain<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>, makes them attractive targets for enhanced MCE and low-temperature refrigeration. In fact, such effects should be also observable in certain 0D systems, that is, molecular clusters of spins in frustrated geometries<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. These are a subset of the broader class of molecules known as molecular nanomagnets.</p><p>The molecular cluster [Gd₇(OH)₆(thmeH₂)₅(thmeH)(tpa)₆(MeCN)₂](NO₃)₂ (&#x02018;Gd₇&#x02019;; H₃thme=tris(hydroxymethyl)ethane; Htpa=triphenylacetic acid) consists of a planar centred hexagon of weakly AF-coupled Gd(III) ions (<xref ref-type="fig" rid="f2">Fig. 2</xref>; ref. <xref ref-type="bibr" rid="b14">14</xref>), each of which has an electronic spin <italic>s</italic>=7/2. Hence, this topology is a finite &#x02018;cutout&#x02019; of the 2D triangular AF lattice (<xref ref-type="fig" rid="f2">Fig. 2</xref>). Here we model all the magnetic observables of Gd₇, including sub-Kelvin susceptibility and heat capacity data. We then use this model to calculate the isentropes for Gd₇, revealing detailed structure in the <italic>T</italic>&#x02013;<italic>B</italic> landscape due to the frustration. Finally, we follow these isentropes experimentally by direct measurement of the temperature in applied magnetic field cycles under quasi-adiabatic conditions. The experimental data, reproduced by theoretical modelling, show the characteristics of frustration-enhanced MCE; moreover, we achieve cooling to ~200&#x02009;mK&#x02014;the first time sub-Kelvin cooling has been achieved with a molecular nanomagnet.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Magnetic properties</title><p>Low-temperature magnetic data of Gd₇ are summarized in <xref ref-type="fig" rid="f3">Fig. 3</xref>. The magnetization (<italic>M</italic>) saturates to the maximum possible 49/2 <italic>g&#x003bc;</italic>_B (where <italic>g</italic> is the electronic <italic>g</italic>-factor) per molecule at 2&#x02009;K, showing that the full magnetic entropy is accessible (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The <italic>&#x003c7;T</italic> product, where <italic>&#x003c7;</italic> is the molar magnetic susceptibility, has the value calculated for non-interacting Gd(III) ions at room temperature (56.2&#x02009;e.m.u.&#x02009;K&#x02009;mol^&#x02212;1) and decreases only slowly on cooling down to ~50&#x02009;K before decreasing rapidly on further cooling (<xref ref-type="fig" rid="f3">Fig. 3b</xref>), denoting a dominant AF interaction. That Gd₇ has a richer physics than a simple paramagnet is manifested in the very-low-temperature susceptibility, which goes through two shallow maxima, at 1&#x02013;2&#x02009;K and at 0.2&#x02013;0.3&#x02009;K (<xref ref-type="fig" rid="f3">Fig. 3b</xref>, inset). Above 4&#x02009;K, the molar heat capacity (<italic>C</italic>) in zero applied field is dominated by lattice phonon modes of the crystal, that is, non-magnetic contributions (<xref ref-type="fig" rid="f3">Fig. 3c</xref>). This is confirmed from <italic>C</italic>(<italic>T</italic>) data on the isostructural and diamagnetic yttrium analogue [Y₇(OH)₆(thmeH₂)₅(thmeH)(tpa)₆(MeCN)₂](NO₃)₂ (&#x02018;Y₇&#x02019;), which overlay those of Gd₇ at higher temperatures. The phonon heat capacity can be described by the Debye model, which simplifies to a <italic>C</italic>/<italic>R</italic>=<italic>aT</italic>³ dependence (<italic>R</italic> is the gas constant), where <italic>a</italic>=1.35 &#x000d7; 10^&#x02212;2&#x02009;K^&#x02212;3 for Gd₇ and Y₇, at the lowest temperatures. The magnetic contribution to the <italic>C</italic>(<italic>T</italic>) data for Gd₇ consists of a broad hump that shifts to higher temperature on increasing the applied magnetic field (<xref ref-type="fig" rid="f3">Fig. 3c</xref>).</p></sec><sec disp-level="2"><title>Magnetic modelling</title><p>We have modelled all these magnetic data assuming the simple Heisenberg spin Hamiltonian:</p><p><disp-formula id="eq2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e433" xlink:href="ncomms6321-m2.jpg"/></disp-formula></p><p>where <italic>J</italic>₁ is the exchange interactions between nearest neighbours on the hexagon (spins 1&#x02013;6), and <italic>J</italic>₂ is the interactions between each of these spins and the central Gd (spin 7). The huge matrix dimension of 8⁷ requires exploiting group theoretical methods<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref> (and the approximate C₆ molecular symmetry) for full matrix diagonalization. We find <italic>J</italic>₁=&#x02212;0.090(5)&#x02009;K, and <italic>J</italic>₂=&#x02212;0.080(5)&#x02009;K with <italic>g</italic>=2.02 reproduces all the experimental magnetic observables (<xref ref-type="fig" rid="f3">Fig. 3</xref>). Only at the very lowest temperatures, the weak-field susceptibility and zero-field heat capacity show slight deviations between calculated and experimental data. For instance, the calculated susceptibility reproduces the shallow two-peak structure, with the higher-temperature feature agreeing well but the lower temperature one calculated to be at ~0.05&#x02009;K rather than the experimental 0.2&#x02013;0.3&#x02009;K. Most likely, these discrepancies are due to weak magnetic dipolar interactions, which are not incorporated in the theoretical model. Dipolar interactions modify the structure of energy levels and can determine (on the mean-field level) an internal field; both become relevant in proximity of absolute zero and zero applied field.</p></sec><sec disp-level="2"><title>Experimental evaluation of the MCE</title><p>The MCE can be evaluated indirectly for a given applied field change from the experimental <italic>C</italic>(<italic>B</italic>,<italic>T</italic>) (for example, <xref ref-type="fig" rid="f3">Fig. 3d</xref>) and <italic>M</italic>(<italic>B,T</italic>) data via Maxwell&#x02019;s relations<xref ref-type="bibr" rid="b17">17</xref>: values for Gd₇ derived from these two observables are in very good agreement (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). Here we have also performed direct experimental measurements of the MCE for continuous field variations, that is, the temperature evolution via magnetization&#x02013;demagnetization cycles that we perform under controlled quasi-adiabatic conditions, using the set-up and protocols described in <xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref> and ref. <xref ref-type="bibr" rid="b18">18</xref>. <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref> displays a representative full magnetic field cycle, and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref> a representative demagnetization process from an initial temperature <italic>T</italic>₀=0.50&#x02009;K and field <italic>B</italic>₀=2&#x02009;T. We show both the raw temperature data and those for an ideal adiabatic process, that is, corrected for unavoidable thermal losses (non-adiabaticity) that have been evaluated independently (see <xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>). By this method, we experimentally follow isentropes in the <italic>T</italic>&#x02013;<italic>B</italic> plane for different <italic>B</italic>₀ and <italic>T</italic>₀ (up to 3&#x02009;T and 3&#x02009;K, respectively; <xref ref-type="fig" rid="f4">Fig. 4</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). The general trend is a decrease in <italic>T</italic> as <italic>B</italic> is decreased, as expected. There are two important results from these adiabatic demagnetization experiments. First, we achieve temperatures as low as <italic>~</italic>200&#x02009;mK. Despite many indirect MCE studies on molecular nanomagnets, this is the first direct experimental demonstration of sub-Kelvin cooling with such a species. Second, in contrast to the straight-line isentropes found for simple paramagnets, a rich structure is observed.</p><p>On demagnetization from <italic>B</italic>₀=3&#x02009;T, a minimum (at 2.2&#x02009;T) is found in the isentropes, that is, the sample cools rapidly (large positive slope) then heats (negative slope), strongly reminiscent of the behaviour observed recently for a 1D AF chain at a quantum critical point<xref ref-type="bibr" rid="b3">3</xref>. On decreasing the field further, the <italic>T</italic>(<italic>B</italic>) curves go through a second minimum (at ~0.7&#x02009;T). As far as we are aware, such multiple peak behaviour has not been observed previously. However, secondary minima have been predicted theoretically for ideal frustrated 2D lattices as a function of decreasing size<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b7">7</xref>, and also for very high-symmetry (cuboctahedral, icosidodecahedral) frustrated clusters<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>, that is, they arise as a function of finite-size effects.</p></sec><sec disp-level="2"><title>Comparison with calculated results</title><p>We have calculated theoretical isentropes from the entropy function <italic>S</italic>(<italic>T</italic>,<italic>B</italic>) based on the parameters from spin Hamiltonian (2) (see <xref ref-type="fig" rid="f5">Fig. 5c</xref>). We have done this for the experimental entropies that belong to the isentropes shown in <xref ref-type="fig" rid="f4">Fig. 4</xref> to allow a direct comparison, and for a lower entropy to emphasize the shape of the isentropes. The agreement with the experimental curves is remarkable, showing the double minimum in <italic>T</italic>(<italic>B</italic>) and consequent multiple cooling regimes. The agreement becomes poorer for the lowest temperatures and small fields because the aforementioned dipolar interactions become relevant. The latter, which are not included in our model, ultimately limit the base temperature reached by adiabatic demagnetization. Analysing the Zeeman diagram is difficult because of the massive (8⁷) number of levels; in <xref ref-type="fig" rid="f5">Fig. 5a</xref>, we plot the excitation energies (<italic>E</italic>*=<italic>E</italic>_i&#x02212;<italic>E</italic>₀, where <italic>E</italic>_i and <italic>E</italic>₀ are the energies of the ith and ground Zeeman states, respectively, at that field) to make the changes in density of states in certain field ranges more visible. The zero-temperature saturation field is ~2.9&#x02009;T (that is, above which the ground state is singly degenerate and the magnetic entropy is nil; <xref ref-type="fig" rid="f5">Fig. 5b</xref>). Below this saturation field, there is a high degeneracy of low-lying states (high entropy), hence rapid magnetic cooling is observed on demagnetizing towards 2.5&#x02009;T (positive slope isentrope; <xref ref-type="fig" rid="f5">Fig. 5c</xref>). Between about 2.2 and 1.4&#x02009;T, the density of states is much lower (<xref ref-type="fig" rid="f5">Fig. 5a</xref>), giving a plateau in the zero-temperature magnetization curve (<xref ref-type="fig" rid="f5">Fig. 5b</xref>), hence demagnetizing into this region decreases the entropy and leads to heating (negative slope isentrope; <xref ref-type="fig" rid="f5">Fig. 5c</xref>). Below 1.4&#x02009;T, the density of states increases again, and we are back in a region of cooling.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Several frustrated antiferromagnets, including 2D kagome and triangular lattices and certain 0D polytopes, have been predicted to show plateaus in their zero-temperature magnetization curves together with regions of lower densities of states<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. The uneven distributions of intervals between ground-state level crossings is a clear signature of frustration<xref ref-type="bibr" rid="b13">13</xref>, and is the reason for the peaks observed in the isentrope distribution. This frustration arises because <italic>J</italic>₁&#x02248;<italic>J</italic>₂, and test calculations show that the isentrope peaks are quickly destroyed by smaller values of <italic>J</italic>₂/<italic>J</italic>₁ (hence, weakening the frustration; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>).</p><p>Insight into the microscopic origin of the zero-Kelvin magnetization plateau in Gd₇ is gained from evaluating the ground-state nearest-neighbour spin&#x02013;spin correlation functions <inline-formula id="d33e693"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e694" xlink:href="ncomms6321-m3.jpg"/></inline-formula> as a function of the applied field (<xref ref-type="fig" rid="f6">Fig. 6</xref>), evaluated by numerical differentiation of the ground-state energy with respect to <italic>J</italic>₁ (<italic>S</italic>_{<italic>12</italic>}) or <italic>J</italic>₂ (<italic>S</italic>_{<italic>17</italic>}). Calculation from the ground-state eigenfunctions is prohibitive given the enormous Hilbert space. The <italic>S</italic>_{<italic>12</italic>} function, that is, for neighbouring spins on the hexagon, grows from a fully antiparallel alignment (maximum negative <italic>S</italic>_{<italic>12</italic>}) at <italic>B</italic>=0 to a saturated parallel alignment (maximum positive <italic>S</italic>_{<italic>12</italic>}) at <italic>B</italic>=1.4&#x02009;T. The <italic>S</italic>_{<italic>17</italic>} function, that is, for a spin on the hexagon correlated with the central spin, starts at a small negative value and becomes more negative with increasing <italic>B</italic>, reaching a fully antiparallel alignment at <italic>B</italic>=1.4&#x02009;T. <italic>S</italic>_{<italic>17</italic>} is then constant until <italic>B</italic>=2.2&#x02009;T after which it increases, reaching full parallel alignment at <italic>B</italic>=2.9&#x02009;T (and saturation of the magnetization at 49/2 <italic>g&#x003bc;</italic>_B per molecule). Hence, the magnetization plateau between 1.4 and 2.2&#x02009;T corresponds to a region of stability for the spin configuration with all the spins on the hexagon fully aligned parallel with each other but fully antiparallel to the central spin, consistent with the calculated plateau magnetization of 35/2 <italic>g&#x003bc;</italic>_B per molecule (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). In fact, the Gd₇ structural motif is one of the smallest fragments of the triangular AF net that would be predicted to show such effects. For example, the smallest possible frustrated fragment&#x02014;an equilateral triangle&#x02014;has no such &#x02018;meta-stable&#x02019; intermediate spin configuration, hence no magnetization plateau and a much simpler isentrope structure (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>).</p><p>Many molecular nanomagnets have now been proposed for low-temperature magnetic refrigeration (see, for example, refs <xref ref-type="bibr" rid="b17">17</xref>, <xref ref-type="bibr" rid="b19">19</xref>, <xref ref-type="bibr" rid="b20">20</xref>, <xref ref-type="bibr" rid="b21">21</xref>, <xref ref-type="bibr" rid="b22">22</xref>, <xref ref-type="bibr" rid="b23">23</xref>, <xref ref-type="bibr" rid="b24">24</xref>, <xref ref-type="bibr" rid="b25">25</xref>, <xref ref-type="bibr" rid="b26">26</xref>), even in principle to the single-molecule level<xref ref-type="bibr" rid="b27">27</xref>, due to the high magnetic degeneracies that can be built in by appropriate choice of metal ion and a favourable exchange coupling scheme. Almost all these studies have relied on indirect MCE measurements from magnetization or heat capacity data, which are analysed to predict some maximum magnetic entropy change for a maximum field change (typically 0&#x02013;5&#x02009;T on a conventional SQUID magnetometer) and certain initial temperature. Such indirect analyses can give impressive headline figures but ignore the details of the exchange coupling (other than, for example, being &#x02018;weak&#x02019;, hence giving large quasi-degeneracies in zero field). Hence, they are blind to the structure and true beauty of the isentropes that are a function of the exchange couplings. Here we have revealed the richness of the isentropes in Gd₇ via direct MCE studies, including the first experimental achievement of sub-Kelvin cooling with a molecular nanomagnet, with experimental and theoretical results in excellent agreement. Our results show that it is possible to design the cooling power of molecular materials by choosing an appropriate topology of magnetic couplings between the interacting spins, hence exploiting the great control of the latter given by molecular coordination chemistry.</p><p>The enhanced MCE we observe in certain regions of the <italic>T</italic>&#x02013;<italic>B</italic> plane for Gd₇ also confirms long-standing predictions about unusually large cooling rates in frustrated spin 0D polytopes as well as low-dimensional extended spin lattices<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. Indeed, the Gd₇ molecule is a cutout of the triangular AF lattice, with imposed geometric spin frustration giving exact or near degeneracies at certain applied magnetic fields, and serves as a finite-size realization of these predictions. Such finite systems are useful in their own right, as demonstrated here, but also enable exact numerical analysis, hence giving insight into the behaviour of infinitely extended systems. If bigger molecular fragments of the triangular AF net could be prepared (such molecules are known for some d-block ions, see refs <xref ref-type="bibr" rid="b28">28</xref>, <xref ref-type="bibr" rid="b29">29</xref>), this would allow fascinating insight into the transition from discrete to bulk behaviour in frustrated systems.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Materials</title><p>[Gd₇(OH)₆(thmeH₂)₅(thmeH)(tpa)₆(MeCN)₂](NO₃)₂ (&#x02018;Gd₇&#x02019;) was prepared as reported previously<xref ref-type="bibr" rid="b14">14</xref>. Its diamagnetic and isostructural analogue [Y₇(OH)₆(thmeH₂)₅(thmeH)(tpa)₆(MeCN)₂](NO₃)₂ (&#x02018;Y₇&#x02019;) was prepared by an identical method but with substitution of the appropriate metal precursor. Solvothermal reaction of Y(NO₃)₃&#x000b7;6H₂O (0.085&#x02009;g, 0.22&#x02009;mmol) with H₃thme (0.11&#x02009;mmol), Htpa (0.11&#x02009;mmol) and NEt₃ (0.165&#x02009;mmol) in MeCN (8&#x02009;ml) at 100&#x02009;&#x000b0;C for 12&#x02009;h, followed by slow cooling (0.05&#x02009;&#x000b0;C&#x02009;min^&#x02212;1) to room temperature, gave colourless crystals of the product in ~40% yield. The formulation is confirmed by elemental analysis, powder X-ray diffraction (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>) and a single-crystal unit cell determination, which show that Y₇ is isostructural with Gd₇. Elemental analysis (%) for Y₇C₁₅₄H₁₆₄N₄O₄₂ (found:calculated): C 53.26:54.96; H 4.45:4.91; N 1.74:1.66.</p></sec><sec disp-level="2"><title>Measurements</title><p>Magnetization measurements down to 2&#x02009;K and heat capacity measurements using the relaxation method down to 0.3&#x02009;K were carried out on powdered crystalline samples by means of commercial setups for the 0&#x02013;9&#x02009;T magnetic field range. Susceptibility measurements were extended down to 0.1&#x02009;K with a homemade susceptometer, installed in a ³He-⁴He dilution refrigerator. Direct MCE measurements were performed on a pressed pellet sample mounted on a sapphire plate attached to a Cernox resistance thermometer, attached by wires to a controlled thermal bath. Each MCE measurement started with the sample at zero applied magnetic field and at temperature <italic>T</italic>₀, and comprised: (a) gradual application of a magnetic field, up to a maximum <italic>B</italic>₀; (b) relaxation until the sample reached the thermal equilibrium with the bath; (c) gradual demagnetization down to <italic>B</italic>=0; and (d) relaxation at zero field until the sample reached thermal equilibrium at <italic>T</italic>₀. During the whole procedure, the temperature <italic>T</italic> and applied magnetic field <italic>B</italic> were recorded continuously. See <xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref> for full details.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>J.W.S. made and characterized the materials, under the supervision of D.C. and E.J.L.M. E.P. and M.E. designed and performed the quasi-adiabatic magnetocaloric and magnetic experiments. J.S. modelled the magnetic data. E.J.L.M., J.S. and M.E. wrote the manuscript with further contributions from all authors.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article</bold>: Sharples, J. W. <italic>et al.</italic> Quantum signatures of a molecular nanomagnet in direct magnetocaloric measurements. <italic>Nat. Commun.</italic> 5:5321 doi: 10.1038/ncomms6321 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-7, Supplementary Note 1 and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6321-s1.pdf"/></supplementary-material></sec>

The complex jujube genome provides insights into fruit tree biology

<p>The jujube (<italic>Ziziphus jujuba</italic> Mill.), a member of family Rhamnaceae, is a major dry fruit and a traditional herbal medicine for more than one billion people. Here we present a high-quality sequence for the complex jujube genome, the first genome sequence of Rhamnaceae, using an integrated strategy. The final assembly spans 437.65&#x02009;Mb (98.6% of the estimated) with 321.45&#x02009;Mb anchored to the 12 pseudo-chromosomes and contains 32,808 genes. The jujube genome has undergone frequent inter-chromosome fusions and segmental duplications, but no recent whole-genome duplication. Further analyses of the jujube-specific genes and transcriptome data from 15 tissues reveal the molecular mechanisms underlying some specific properties of the jujube. Its high vitamin C content can be attributed to a unique high level expression of genes involved in both biosynthesis and regeneration. Our study provides insights into jujube-specific biology and valuable genomic resources for the improvement of Rhamnaceae plants and other fruit trees.</p>

<contrib contrib-type="author"><name><surname>Liu</surname><given-names>Meng-Jun</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Jin</given-names></name><xref ref-type="aff" rid="a4">4</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Cai</surname><given-names>Qing-Le</given-names></name><xref ref-type="aff" rid="a5">5</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Guo-Cheng</given-names></name><xref ref-type="aff" rid="a5">5</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Jiu-Rui</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Zhi-Hui</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Ping</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Dai</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yan</surname><given-names>Guijun</given-names></name><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Wen-Jiang</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Xian-Song</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Yan</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Yu-Dong</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Zhi-Guo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Min-Juan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xiao</surname><given-names>Jing</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Ying-Ying</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Xiao-Feng</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Bin</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Ma</surname><given-names>Yong</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Jian</surname><given-names>Jian-Bo</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Wei</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Zan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Xue-Chao</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Wei</surname><given-names>Yan-Li</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Li-Li</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Chi</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Liao</surname><given-names>Sheng-Guang</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Rong-Jun</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Guang</surname><given-names>Xuan-Min</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Zhuo</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Yue-Yang</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Luo</surname><given-names>Long-Hai</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><aff id="a1"><label>1</label><institution>Research Center of Chinese Jujube, Agricultural University of Hebei</institution>, 071001 Baoding, <country>China</country></aff><aff id="a2"><label>2</label><institution>National Engineering Research Center for Agriculture in North Mountain Area (Ministry of Science and Technology of the People&#x02019;s Republic of China)</institution>, 071000 Baoding, <country>China</country></aff><aff id="a3"><label>3</label><institution>Jujube Working Group, International Society for Horticultural Science, Agricultural University of Hebei</institution>, 071001 Baoding, <country>China</country></aff><aff id="a4"><label>4</label><institution>College of Life Science, Agricultural University of Hebei</institution>, 071000 Baoding, <country>China</country></aff><aff id="a5"><label>5</label><institution>BGI-Shenzhen</institution>, 518083 Shenzhen, <country>China</country></aff><aff id="a6"><label>6</label><institution>College of Forestry, Agricultural University of Hebei</institution>, 071000 Baoding, <country>China</country></aff><aff id="a7"><label>7</label><institution>School of Plant Biology, Faculty of Science and The UWA Institute of Agriculture, The University of Western Australia</institution>, Perth, Western Australia 6009, <country>Australia</country></aff>

Nature Communications

<p>The jujube (<italic>Ziziphus jujuba</italic> Mill.) is the most economically important member of the Rhamnaceae, a large cosmopolitan family<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. It is one of the oldest cultivated fruit trees in the world, with evidence of domestication dating back to 7,000 years ago<xref ref-type="bibr" rid="b3">3</xref>. It is native to China and is now a major dry fruit crop with a cultivation area of 2 million&#x02009;ha, the main source of income for 20 million farmers as well as a traditional herbal medicine for more than one billion people in Asia<xref ref-type="bibr" rid="b4">4</xref>. It has been introduced into at least 47 countries from temperate to tropical zones throughout the five continents and is becoming increasingly popular worldwide<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>.</p><p>The jujube has a range of botanical and horticultural features<xref ref-type="bibr" rid="b6">6</xref> that gives it great potential in fruit tree molecular improvement, human health protection, and the economical development and ecological restoration of arid region. It is well-adapted to various biotic and abiotic stresses, especially drought and salinity (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>), and is considered an ideal cash crop for arid and semi-arid areas where common fruits and grain/oil crops do not grow well. Its fruit is an excellent source of vitamin C (higher than the well-known vitamin C-rich orange and kiwifruit) and sugar (25&#x02013;30%, twice as high as most common fruits and even higher than sugarcane and sugar beet)<xref ref-type="bibr" rid="b7">7</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). The jujube also has a very easy and quick flower bud differentiation (only ~7 days), a long flowering season lasting for 2 months, a very short period of ~6 months from planting or sowing to yielding fruits, and a very long lifecycle, even more than 1,000 productive years<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b6">6</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>).</p><p>Furthermore, jujube tree has evolved a distinct self-shoot-pruning system comprising four types of shoot namely primary shoot, secondary shoot, mother bearing shoot (MBS) and bearing shoot<xref ref-type="bibr" rid="b6">6</xref>, each of which has a very different function and developmental pattern. Primary shoot is the only normally extended shoot. Secondary shoot occurs from each node of the primary shoot and its tip dies back naturally. MBS is the branch producing bearing shoots, it is formed at each node of the secondary shoot and is extremely condensed elongating only ~1&#x02009;mm per year. Bearing shoot is the only fruiting shoot, it is deciduous and drops before winter normally, which is a very uncommon trait in tree plants. This self-shoot-pruning system makes it easy to control tree size, and the diversified shoot types offers a unique model for elucidating shoot development and function.</p><p>Sugar and vitamin C contents are the most common indicators of fruit quality, pruning is the most labour-consuming work of orchard management, earlier fruiting and more productive years are what farmers expect, and drought and salinity are the main abiotic stresses for fruit growing. Therefore, the aforementioned properties of the jujube are of great importance to the modern fruit production characterized by fast payback, easy management and labour-saving. In addition, the jujube is a close relative of Rosaceae (both belonging to the Rosales order in the widely accepted molecular taxonomy system of Angiosperm<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>), the most important fruit-producing family containing a large number of leading deciduous fruit species such as apple (<italic>Malus domestica</italic>), pear (<italic>Pyrus bretschneideri</italic>), peach (<italic>Prunus persica</italic>), strawberry (<italic>Fragaria vesca</italic>) and <italic>Rubus</italic>. Consequently, the jujube could be a rich source of genes for the molecular improvement of fruit trees, and a fundamental understanding of the genetics of the jujube is crucial.</p><p>So far, more than 70 plant genomes have been sequenced and assembled since the genome sequence of <italic>Arabidopsis thaliana</italic><xref ref-type="bibr" rid="b10">10</xref> was published in 2000. However, high level of heterozygosity and repeated sequences and low content of GC are still the main obstacles for genome sequencing and assembly using the next-generation-sequencing (NGS) technology. Owing to the short read length of the NGS technology, the assembling algorithm is always based on de Bruijn graph<xref ref-type="bibr" rid="b11">11</xref>, where heterozygous locus between haploid becomes a bubble resulting in the breakdown of the final assembly at the heterozygous locus. The repeated sequences make the assembly fragmental in the similar way. Bacterial artificial chromosome-to-bacterial artificial chromosome (BAC-to-BAC) strategy was reintroduced and a few genomes have been assembled at a reasonable level<xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. We revealed not only both high heterozygosity and high density of repeated sequence but also low GC content in the jujube genome, which indicated that new method should be applied to obtain a good quality sequence for this complex genome.</p><p>Knowledge of jujube genetics and genomics is very limited, and no genome-wide study (data on the genome size, heterozygosity and a completed molecular genetic linkage map) on any members of the family Rhamnaceae has so far been published, which has significantly hindered the molecular breeding, biological research and deep utilization of the jujube. In this research, we generate and analyze a high-quality genome sequence of one of the oldest and most widely cultivated Chinese jujube cultivars, &#x02018;Dongzao&#x02019; (2<italic>n</italic>=2<italic>x</italic>=24), using a novel strategy integrating whole-genome shotgun (WGS) sequencing, BAC-to-BAC and a PCR-free library. We also conduct comprehensive transcriptome analyses of 15 different tissues and evolutionary comparisons with related species to identify genetic characteristics that are likely to underpin some of the most valuable traits of jujube. Our study offers a rich resource of genetic information for the breeding of jujube and the molecular improvement of other Rhamnaceae plants and fruit species.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Sequencing and assembly of the complex jujube genome</title><p>Our analysis of the 17-mer frequency distribution based on short insert size (&#x0003c;1&#x02009;Kbp) clean data and simulation revealed a heterozygosity of 1.90% in the jujube genome (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>, <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). This is the highest degree of heterozygosity among the plants sequenced to date by NGS technology (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>), which is almost twice the second-highest level of heterozygosity in plants (1.02%, pear)<xref ref-type="bibr" rid="b14">14</xref>. In addition, the density of simple sequence repeats (SSRs) in the jujube genome reached 378.1 SSRs per Mb, which is 2.00 times and 2.84 times that found in its close relative species peach and apple, respectively (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 5</xref> and <xref ref-type="supplementary-material" rid="S1">6</xref>)<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>. The very high level of heterozygosity, very high density of SSRs and low GC content (33.41%) (<xref ref-type="table" rid="t1">Table 1</xref>) make the jujube genome a challenge for the WGS strategy using NGS technology.</p><p>A strategy combining WGS sequencing, BAC-to-BAC and WGS-PCR-free library was employed to reduce the impact of the complexity of the jujube genome. WGS was used to construct libraries of different insert sizes, ranging from 170&#x02009;bp to 40&#x02009;kb (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 7</xref>). A total of 21,504 BAC clones (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 8</xref>) were selected for sequencing at an average of 68 &#x000d7; coverage using the Illumina HiSeq 2000 system, representing a total of 5.86 &#x000d7; coverage of jujube genome size. These BAC sequences were taken as BAC end data to overcome the heterozygosity problem and to improve genome assembly. Paired-end-libraries of 170 to 800&#x02009;bp were constructed and sequenced at 318 &#x000d7; coverage to fill in the gaps. WGS mate-pair libraries of 2, 5, 10, 20 and 40&#x02009;kb were constructed and sequenced at 69 &#x000d7; coverage to build super scaffolds. However, parts of the genome were missing from the sequencing data owing to low GC content and the high density and even distribution of SSRs. Thus, WGS-PCR-free libraries were constructed and sequenced to reduce the bias in library construction caused by low GC content genome regions, then 30&#x02009;Mb sequences were added to the final assembly. Finally, we assembled all the above sequencing data into 28,930 contigs and 5,898 scaffolds with N50 sizes of 33.95&#x02009;kb and 301.04&#x02009;kb, respectively, spanning 437.65&#x02009;Mb of the genome sequence (<xref ref-type="table" rid="t1">Table 1</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 9</xref>). The assembly covered 98.6% of the jujube genome (444&#x02009;Mb) estimated by our 17-mer sequence analysis (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>, <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). In jujube genome, the percentage of low GC content sequences (with GC% range from 25&#x02013;30%) are much higher than that in grape vine and peach genomes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>).</p><p>To further evaluate the accuracy of the genome assembly, full-length sequences of four randomly selected BACs, later demonstrated to be located in pseudo-chromosomes 1, 2, 3 and 11 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>), were sequenced and assembled using Sanger sequencing technology. Each BAC was aligned to three scaffolds at most, with an average coverage ratio of 98.5% (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 10</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>). In addition, our assembled sequence covered &#x0003e;98.1% of the 1,942 published ESTs (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nucest/?term%20=jujube">http://www.ncbi.nlm.nih.gov/nucest/?term =jujube</ext-link>, downloaded on July 2013) and 97.8% of the transcriptome sequences (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 11</xref> and <xref ref-type="supplementary-material" rid="S1">12</xref>). Together, the above results indicate the high quality of our jujube genome sequence.</p></sec><sec disp-level="2"><title>Genome sequence anchoring and pseudo-chromosome construction</title><p>To anchor the assembled genome sequences to the jujube chromosomes, we mapped the scaffolds to a high density molecular genetic map that we constructed <italic>de novo</italic> using an inter-specific population (105 progenies) between <italic>Z. jujuba</italic> (female, <italic>2n</italic>=2<italic>x</italic>=24) and <italic>Z. acidojujuba</italic> (male, <italic>2n</italic>=2<italic>x</italic>=24). Each individual in the F1 population was genotyped by restriction site-associated DNA sequencing (RAD-Seq). Sequencing reads of parents and the population are aligned to the jujube genome sequences using SOAP2 (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 13</xref>). The final map spanned 974.01&#x02009;cM for the female parent and 935.40&#x02009;cM for the male parent across the 12 linkage groups, with a mean genetic distance between markers (single nucleotide polymorphisms (SNPs)) of 0.56&#x02009;cM and 0.43&#x02009;cM, respectively. The joint map across the 12 linkage groups was composed of 2,419 SNP markers spanning 1,020.22&#x02009;cM, with a mean marker distance of 0.42&#x02009;cM.</p><p>Combining the assembled genome sequences and the genetic linkage groups, we constructed pseudo-molecules for each of the 12 chromosomes and ordered them on the basis of genetic length (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). Using the mapped markers with known sequences that were uniquely aligned to the assembled scaffolds, a total of 1,120 scaffolds were anchored to the 12 linkage groups, comprising 73.56% (321.45&#x02009;Mb) of the jujube genome assembly (<xref ref-type="fig" rid="f1">Fig. 1</xref>). Of the anchored scaffolds, 784 could be oriented (267.98&#x02009;Mb, 83.37% of the total anchored sequences), suggesting high alignment accordance between the anchored genetic markers and the sequenced scaffolds.</p></sec><sec disp-level="2"><title>Genome annotation and characterization</title><p>We annotated the jujube genome by combining <italic>ab initio</italic> gene predictions, protein-based homology searches and experimental data (RNA-Seq). A total of 32,808 protein-coding genes with an average coding sequence length of 1,190&#x02009;bp and 4.5 exons per gene were predicted (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 14</xref> and <xref ref-type="supplementary-material" rid="S1">15</xref>). Overall, 78.26% of the gene models were predicted to contain two or more exons (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 16</xref>), and 89.80% of the predicted proteins were supported by the RNA-Seq data (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 17</xref>), showing the high accuracy of gene annotation. In addition, we identified a total of 410 ribosomal RNAs, 1,209 transfer RNAs, 286 small nuclear RNAs and 272 microRNAs in the jujube genome (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 18</xref>).</p><p>Tissue-specific genes and housekeeping genes were also screened in our 24&#x02009;Gb of RNA-Seq data from five tissues. We found that 613, 249, 382, 553 and 184 genes were specifically expressed in the root, shoot, leaf, flower or fruit, respectively. The 16,478 genes shared by all the five tissues (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>) accounted for 81.43% of the total recorded and 50.22% of the total predicted protein-coding genes in the jujube genome. Among these shared genes, 1,818 were constitutively expressed housekeeping genes (<italic>&#x003c4;</italic>&#x0003c;0.07, based on the tissue specificity index<xref ref-type="bibr" rid="b17">17</xref>), including 102 encoding ribosomal proteins and 21 encoding translation initiation factors (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 1</xref>).</p><p>We next characterized the genome by investigating genome duplication and GC content, gene density, repeat sequences, and SSR and SNP distribution along the pseudo-chromosomes (<xref ref-type="fig" rid="f2">Fig. 2</xref>). We identified 4.77 million SNPs in our jujube genome sequence. In total, 79.55% of them were anchored on the 12 pseudo-chromosomes, and the overall polymorphism density was 11 SNPs per kb (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 19</xref>). The SSRs were not only present at a high density (378.1 SSRs&#x02009;per&#x02009;Mb), they were also fairly evenly distributed (<xref ref-type="fig" rid="f2">Fig. 2a</xref>, vi). We observed large regions that alternate between high and low gene density (<xref ref-type="fig" rid="f2">Fig. 2a</xref>, iv), which was also noted in peach<xref ref-type="bibr" rid="b15">15</xref>. The density of repeated sequences also reflects gene density (<xref ref-type="fig" rid="f2">Fig. 2a</xref>, iv and v). The GC content is distributed unevenly in most pseudo-chromosomes (<xref ref-type="fig" rid="f2">Fig. 2a</xref>, iii).</p><p>We identified a total of 216.6&#x02009;Mb of repeated sequences (49.49% of the assembled genome) in the jujube genome (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 20</xref>). Among these sequences, 94.6% are transposable elements (TEs). The majority of TEs are retrotransposons (38.03% of the genome), whereas DNA transposons account for 8.08% of the genome. The most abundant (64%) retrotransposons are long-terminal repeat elements, of which 37% are Gypsy-type elements and 27% are of the Copia type (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 21</xref>). TE content in jujube genome (46.82%), compared with its close relatives, appears to be similar to that in mulberry (47%; ref. <xref ref-type="bibr" rid="b18">18</xref>), and higher than that in peach (37%; ref. <xref ref-type="bibr" rid="b15">15</xref>) and <italic>P. mume</italic> (45%; ref. <xref ref-type="bibr" rid="b19">19</xref>). About 99.41% of TEs had a divergence rate of &#x0003e;10% (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>), indicating that most jujube TEs are relatively ancient<xref ref-type="bibr" rid="b18">18</xref>.</p><p>In total, 23,996 genes (73% of the total number of annotated genes) were allocated on the 12 pseudo-chromosomes. Self-alignment of the jujube genome sequences based on the 23,996 gene models identified 943 paralogous gene groups (<xref ref-type="fig" rid="f2">Fig. 2a,i</xref>), indicating that the jujube genome may have undergone frequent inter-chromosome fusions and segmental duplication during its evolutionary history. Interestingly, the gene blocks located in the region from 9.20&#x02013;14.68&#x02009;Mb of pseudo-chromosome 1 showed obvious synteny with all other chromosomes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>), while the very low SNP density in this block (<xref ref-type="fig" rid="f2">Fig. 2a</xref>, vii) suggests that it is highly conserved. Further analysis revealed that this block contains many genes relating to sugar metabolism and stress tolerance (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). Consequently, it might be crucial for understanding the unusual characteristics of jujube.</p></sec><sec disp-level="2"><title>Genome comparison with closely related species</title><p>To estimate speciation times, we constructed a phylogenetic tree based on single-copy genes of jujube and the seven other sequenced species of Rosales, with <italic>A. thaliana</italic> (Brassicaceae) as the outgroup (<xref ref-type="fig" rid="f3">Fig. 3</xref>). The results suggest a speciation time of 79.9 million years ago (Mya) for jujube and the clades of mulberry (<italic>Morus alba</italic>, Moraceae) and <italic>Cannabis sativa</italic> (Cannabaceae), 87.2 Mya for jujube and Rosaceae (including <italic>P. bretschneideri</italic>, <italic>M. domestica</italic>, <italic>P. mume</italic>, <italic>P. persica</italic> and <italic>F. vesca</italic>), and 108.5 Mya for jujube and <italic>A. thaliana</italic>. The speciation time is consistent with the origin time inferred from the fossil records and the recent molecular estimation<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. Moreover, our data showed that jujube and the clade of <italic>M. alba</italic> and <italic>C. sativa</italic> diverged much earlier than the divergence of <italic>M. domestica</italic> and <italic>F. vesca</italic> in Rosaceae. Consequently, the jujube is the most ancient species among all of the sequenced species of Rosales (<xref ref-type="fig" rid="f3">Fig. 3</xref>). This result supports that Rhamnaceae has a long history and can be traced back to the Campanian<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. Thus, our analysis provides new insights into the phylogeny of Rosales plants on the basis of genome-scale data.</p><p>To further analyze the evolutionary divergence of jujube and other species, fourfold synonymous third-codon transversion (4DTv) rates were calculated (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). The 4DTv value peaked at only 0.50 in jujube, suggesting no recent whole-genome duplication occurred. The 4DTv value peaked at 0.06 and 0.50 for paralog pairs in pear, highlighting the recent whole-genome duplication and &#x003b1; and &#x003b2; duplication in this species. The orthologs between jujube and pear and between jujube and <italic>P. mume</italic> showed that 4DTv value peaked at 0.32 and 0.27, respectively, indicating that the divergence time between jujube and pear was earlier. All 4DTv values among paralogs in jujube, pear and <italic>P. mume</italic> peaked at ~0.50, indicating that the hexaploidy in these eudicots occurred at a similar time and before the split of Rhamnaceae and Rosaceae.</p><p>To study the evolutionary events leading to the modern genome structure of the jujube, we analyzed the syntenic relationships among jujube, pear and <italic>P. mume</italic> (all belong to order Rosales). About 4,954 (jujube versus pear) and 5,722 (jujube versus <italic>P. mume</italic>) orthologous genes in the jujube genome were selected. We then investigated the detailed orthologous chromosome-to-chromosome relationships between these species (<xref ref-type="fig" rid="f2">Fig. 2c</xref>). The complicated syntenic patterns, illustrated as mosaic chromosome-to-chromosome orthologous relationships, unveiled a high degree of chromosomal evolution and rearrangements among these three species. Even so, clear syntenies could be seen among chromosomes of the three species, such as pseudo-chromosome 12 of jujube, chromosome 4 of <italic>P. mume</italic> and chromosome 17 of pear. The analysis of the synteny blocks among jujube, strawberry (<italic>F. vesca</italic>), peach (<italic>P. persica</italic>) and grape (<italic>Vitis vinifera</italic>) indicated that the jujube genome shares high collinearity with strawberry and peach (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 22</xref>).</p><p>To better understand jujube-specific biology, we performed comparative analyses between the jujube genome and the other six sequenced species of Rosales (apple, pear, peach, <italic>P. mume</italic>, strawberry and mulberry). We then screened these genomes based on annotated genes in the public database. Of the 32,808 protein-coding genes (13,843 gene families) in the jujube genome, 1,043 gene families are specific to jujube, and the largest number of gene clusters (11,930 families) is shared with peach (<xref ref-type="fig" rid="f4">Fig. 4a</xref>), indicating a closer relationship between jujube and peach than between jujube and the other species analyzed.</p><p>Expanded gene families, unique genes and genes under positive selection have important roles in plant development and evolution. The jujube-specific gene sets compared with apple, pear, strawberry, peach, <italic>P. mume</italic> and mulberry were analyzed based on the KEGG orthology pathway database, to give an insight into jujube-specific biology and adaptation. A total of 2,791 unique genes, 254 genes under positive selection and 39 expanded gene families (2,650 genes) were found and assigned to KEGG pathways (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). A further functional characterization of the above genes revealed that most of them are involved in energy metabolism, vitamin C metabolism, sugar-related metabolism and secondary metabolism. This result is consistent with the special physiological characteristics of the jujube.</p></sec><sec disp-level="2"><title>Genes involved in extreme accumulation of vitamin C in fruit</title><p>To explore the molecular mechanism underlying the high content of vitamin C (or ascorbic acid (AsA)) in jujube fruit, we analyzed the genes encoding the key enzymes involved in all the four known AsA biosynthesis pathways and the recycling pathway during jujube fruit development. We found that those genes are involved in two of the four pathways and most of them are specific to L-galactose pathway (<xref ref-type="fig" rid="f5">Fig. 5a</xref> red part). Much fewer genes are associated with myo-inositol pathway, suggesting which maybe a compensatory pathway in jujube (<xref ref-type="fig" rid="f5">Fig. 5a</xref> blue part). Expression analyses showed that GDP-D-mannose 3,5-epimerase and GDP-L-galactose phosphorylase (two key enzymes in the <sc>L</sc>-galactose biosynthesis pathway) and monodehydroascorbate reductase (<italic>MDHAR</italic>, the key enzyme in the AsA recycling pathway) were expressed at continuously higher levels (<xref ref-type="fig" rid="f5">Fig. 5b</xref>).</p><p>Comparisons with the six other sequenced Rosales species at the genome level revealed that GDP-L-galactose phosphorylase is a positively selected gene and that <italic>MDHAR</italic> exhibits significant expansion in the jujube genome (<xref ref-type="fig" rid="f5">Fig. 5a,c</xref>). Based on the phylogenetic analysis of seven Rosales species (including jujube) and one AsA-rich species (sweet orange, <italic>Citrus sinensis</italic>) of the order Sapindales, we found that there are five major <italic>MDHAR</italic> gene subfamilies, of which subfamilies V and IV are specific to jujube and Rosaceae species, respectively (<xref ref-type="fig" rid="f5">Fig. 5c</xref>). There are eight copies in subfamily V, which are adjacently located on two scaffolds (six in No. 402 and two in No. 627, No. 402 positioned to pseudo-chromosome 1) in the jujube genome and likely to have been generated by a tandem duplication. Interestingly, there is only one <italic>MDHAR</italic> copy (subfamilies I, II and III) in the species without recent whole-genome duplication including <italic>F. vesca</italic> (strawberry), <italic>P. persica</italic> (peach), <italic>P. mume</italic>, <italic>M. alba</italic> (mulberry), <italic>Z. jujuba</italic> (jujube) and <italic>C. sinensis</italic> (sweet orange), but there are two or more copies in the species that have undergone recent whole-genome duplication, that is, <italic>M. domestica</italic> (apple) and <italic>P. bretschneideri</italic> (pear).</p><p>Collectively, our data indicate that the L-galactose pathway is the major route to AsA biosynthesis and that <italic>MDHAR</italic> contributes to AsA regeneration in jujube.</p></sec><sec disp-level="2"><title>Genes involved in high level accumulation of sugar in fruit</title><p>To investigate the molecular mechanism underlying the high content of sugar in jujube fruit, we analyzed the genomic and RNA-Seq data and the sugar contents of fruits at four crucial development stages (young, white mature, half-red and full red).</p><p>Our analysis showed that, fructose and glucose are the predominant sugars in young jujube fruits, whereas in mature fruits, sucrose and total sugar content are substantially increased, with sucrose becoming the dominant sugar (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 23</xref>). The annotated jujube genome contains 393 genes involved in starch and sucrose metabolism, 98 involved in galactose metabolism, 67 involved in fructose and mannose metabolism, and 195 involved in amino sugar and nucleotide sugar metabolism (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>). Compared with other sequenced species of the order Rosales, all these gene families are expanded in jujube to some extent (<xref ref-type="fig" rid="f4">Fig. 4b</xref>, IV and V), which is consistent with the high expression patterns of these genes in jujube fruit (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). The final sugar accumulation is determined by sugar being transported from the phloem into the fruit. We found that the expression of most genes related to the major facilitator superfamily sugar transporter was enhanced in parallel with jujube fruit ripening (<xref ref-type="fig" rid="f5">Fig. 5b</xref>).</p></sec><sec disp-level="2"><title>Genes involved in the distinct self-shoot-pruning system</title><p>We generated and analyzed RNA-Seq data on the four types of shoot to investigate the genetic basis underlying jujube shoot biology (<xref ref-type="fig" rid="f6">Fig. 6</xref>). Among the four types of shoots, the number of genes differentially expressed is the smallest between primary shoot and secondary shoot, which indicates the similar metabolism of the two predominant shoots in vegetative growth. The genes related to secondary metabolism, including those involved in arginine, lipid and polyamine metabolism, were expressed at much higher levels in these two types of shoot than in bearing shoot. These results are consistent with the environmental suitability of different types of shoot.</p><p>In MBS, the genes involved in abscisic acid (ABA) synthesis, such as the <italic>PYR/PYL</italic> genes, were much more highly expressed than in other shoot types, which might be a genetic basis of the extreme slow growth of MBS and the deciduous habit of bearing shoot attached on MBS. And the genes involved in porphyrin and chlorophyll metabolism were dramatically downregulated in the MBS, illustrating the decline in photosynthesis in this type of shoot.</p><p>In bearing shoot, the genes related to photosynthesis, such as those in the light-harvesting chlorophyll binding protein gene family and those involved in carbohydrate metabolism, were expressed at much higher levels than in all the other three shoot types, and the genes responsive to brassinosteroids and cytokinins were expressed at much lower levels in bearing shoots. The gene expression profiles meet the needs of its fruiting function and deciduous habit.</p><p>One of the more interesting things is that bearing shoots are normally deciduous, but could become lignified and persistent (not drop in the winter) in case they are extremely vigorous (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 9</xref>). The genes involved in plant hormone signal transduction were expressed in patterns, suggesting that they play key roles in this process. Small auxin-up RNA, cyclin D3 and two-component response regulator ARR-A family, which are responsive to auxin, brassinosteroids and cytokinins, respectively, were much more highly expressed in lignified bearing shoots (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 24</xref>). However, genes responsive to ethylene and ABA, such as ABA responsive element binding factor, serine/threonine-protein kinase SRK2 and ethylene-responsive transcription factor 1, were more strongly expressed in deciduous bearing shoots (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 24</xref>). In addition, genes responsive to jasmonic acid were expressed at lower levels in deciduous bearing shoots than in persistently lignified ones (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 24</xref>), indicating weaker stress resistance of the deciduous bearing shoot.</p></sec><sec disp-level="2"><title>Genes involved in the adaptation to abiotic/biotic stress</title><p>Gene Ontology annotation of the jujube-specific gene families showed that some of them may take part in defence and stress responses (<xref ref-type="fig" rid="f4">Fig. 4b</xref>, XI and <xref ref-type="supplementary-material" rid="S1">Supplementary Data 4</xref>). The number of the gene families functionally annotated as &#x02018;response to stress&#x02019; reaches up to 854. Arginine metabolism plays an important role in plants&#x02019; perception and adaptation to environmental disturbances, and many jujube-specific genes are also enriched in this pathway (<xref ref-type="fig" rid="f4">Fig. 4b</xref>, VI).</p><p>The genes responding to osmotic stress are expressed at very high levels at all stages of fruit development, which is consistent with jujube's salt tolerance and drought resistance (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 25</xref>). Chitinases are pathogenesis related proteins that are induced by biotic and abiotic stress in plants<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>. The high expression of chitinases in jujube shoots may contribute to its high stress tolerance (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 26</xref>).</p><p>There are 13 genes encoding homologues of autophagy-related protein 9 in the jujube genome, and only 1&#x02013;2 in the other 6 related species of Rosales (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 27</xref>). Autophagy has important roles in various cellular functions<xref ref-type="bibr" rid="b26">26</xref>. Specifically, in immunity, the stimulation of autophagy in infected cells helps the cells to degrade and eliminate intracellular pathogens. Therefore, autophagy may play important roles in jujube's defence responses.</p><p>Resistance (R) proteins function mainly in biotic stress responses, and they possess both a nucleotide-binding site (NBS) and a leucine rich repeat (LRR). Enrichment analyses of both IntrePro domains and gene ontology terms showed that the 849 <italic>R</italic> genes in jujube could be classified into 7 groups: CC-NBS-LRR, CC-NBS, LRR-RLK, NBS-LRR, NBS, TIR-NBS-LRR and TIR-NBS. The CC-NBS-LRR group (115 genes) was the most abundant among the 11 sequenced species from diversified taxa (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 28</xref>). A large number of <italic>R</italic> genes (140, 16%) are scattered throughout chromosome 9, indicating that this chromosome is an important candidate target for further research on disease resistance of jujube. Moreover, 17% of jujube genes with a nucleotide-binding adaptor shared by APAF-1, R proteins and CED-4 domain are unique genes or positively selected genes, which suggests that the function of disease resistance has been reinforced during jujube evolution (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 29</xref>).</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>High rate of heterozygosity remains a particular challenge for <italic>de novo</italic> assembly<xref ref-type="bibr" rid="b27">27</xref>. However, most perennial tree species are highly heterozygous<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>, and it is complicated and time-consuming to obtain their double haploid or pure line materials of very low heterozygosity. The jujube genome was characterized as high in heterozygosity, high in density of SSRs and low in GC content. High level of heterozygosity will fragment the assembly of diploid genome sequenced only using the NGS technology or WGS strategy, whereas BAC-to-BAC strategy could avoid this problem via sequencing and assembling of each haploid BAC sequence. Due to the high SSR density and low GC content of jujube genome, some genomic regions were missed in the WGS-PCR sequencing. To retrieve such sequences, WGS-PCR-free library was constructed and sequenced, and 30&#x02009;Mb sequences were added to the final assembly. The finished assembly covers 98.6% of the estimated jujube genome, &#x0003e;98% of the <italic>de novo</italic> transcriptome sequence and 1,942 published ESTs, and it shows an overall identity of 98.5% with four randomly chosen BAC sequences. Consequently, this integrated strategy provides a good solution for assembling the complex jujube genome based on the most widely used NGS technology nowadays.</p><p>Our genomic and RNA-Seq analyses of the jujube offers some insights into the molecular mechanisms underlying the extreme accumulation of vitamin C and sugar in fruit, the distinct self-shoot-pruning system and the outstanding tolerance to abiotic/biotic stress, which are what the main breeding objectives of fruit trees. Compared with orange and kiwifruit, two well-known vitamin C-rich fruits with expansion and high expression of genes involved in the biosynthesis and recycling of vitamin C (refs <xref ref-type="bibr" rid="b31">31</xref>, <xref ref-type="bibr" rid="b32">32</xref>), respectively, the gene involved in both the biosynthesis and the recycling of vitamin C are enhanced in jujube (<xref ref-type="fig" rid="f5">Fig. 5</xref>). This is the first report on such a kind of accumulation mechanism of vitamin C in fruit to our knowledge, which might elucidate why the vitamin C content of jujube is much higher than that in orange and kiwifruit (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). This study indicates that the jujube would be a useful source of genes of fruit nutrition, self-shoot-pruning and stress tolerance for introduction into other fruit crops. To better understand and use the specific traits of jujube, more studies should be carried out integrating further genomic and transcriptome analysis, physiological analysis and field experiment.</p><p>The newly obtained high-quality genome sequence and gene information linked to its valuable biological features, a relatively small genome of 444&#x02009;Mb similar to rice (466&#x02009;Mb) and diploid inheritance with 12 pairs of chromosomes as in rice, coupled with its suitable biological properties including very short generation time, relatively small tree size, easy and quick flower bud differentiation, long flowering season, ease of vegetative propagation and cultivation, and a well-established <italic>in vitro</italic> regeneration system<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>, will enable the use of the jujube as a potential reference genomic system for deciduous fruit trees.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>WGS sequencing and BAC sequencing</title><p>Genomic DNA was extracted from <italic>in vitro</italic> grown <italic>Z. jujuba</italic> Mill. &#x02018;Dongzao&#x02019; plantlets. Paired-end and mate-pair Illumina WGS libraries were constructed with multiple insert sizes (170&#x02009;bp to 40&#x02009;kb) according to the manufacturer&#x02019;s instructions (Illumina). For each short insert size (170, 250, 500 and 800&#x02009;bp) library construction about 5&#x02009;&#x003bc;g genomic DNA was used, and for large insert size (2, 5, 10, 20 and 40&#x02009;Kb) library construction 20~60&#x02009;&#x003bc;g DNA was used. Genomic DNA was fragmented, linked to adapters and extracted at specific size after agarose gel electrophoresis. For large insert size, first, fragments were biotin-labelled and circularized after size selection, sheared and enriched by magnetic beads (Invitrogen). Purified DNA was amplified by PCR, cloned to vector to yield 20 libraries for Illumina Hiseq 2000 sequencing.</p><p>We obtained 188.86&#x02009;Gb raw WGS sequencing data. In the raw data filtering process, several heuristic rules were applied: (1) we removed reads with &#x0003e;2% Ns or with poly-A structure; (2) we removed reads with &#x02265;40% low quality bases for short insert size libraries and &#x02265;60% for large insert size libraries; (3) we removed reads containing adapters; (4) we removed paired reads with mutual overlaps; (5) we removed PCR duplicates. After filtering, 109.88&#x02009;Gb high-quality data were retained, representing 249.72 &#x000d7; genome coverage.</p><p>To overcome the key issue of high heterozygosity, high repeat content, we also constructed BAC libraries with insert fragment size of 120&#x02009;kb length. A total of 21,504 BAC clones were randomly selected to extract plasmids. For each clone, unique index primer and adapter index were linked to fragment end, and a 500&#x02009;bp insert size library was constructed and used for Illumina sequencing to a coverage depth of ~5.8 &#x000d7; . A pooling of 2,688 libraries (equal to 28 &#x000d7; 96-well plates) was sequenced in one lane, totally 21,504 libraries were sequenced in eight lanes to generate 177.17&#x02009;Gb raw data for BAC sequencing (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 8</xref>).</p></sec><sec disp-level="2"><title><italic>De novo</italic> genome assembly</title><p>The short insert size library data were corrected by correct_error programme in the SOAPdenovo<xref ref-type="bibr" rid="b11">11</xref> software package, which changed error bases in low frequency K-mers to form high frequency K-mers. The jujube genome was <italic>de novo</italic> assembled using a hierarchical assembly strategy<xref ref-type="bibr" rid="b36">36</xref> along with WGS strategy. We assembled WGS sequencing data using SOAPdenovo-2.04 (ref. <xref ref-type="bibr" rid="b11">11</xref>) software with K-mer value set as 87. The procedure included three steps: (1) construct de Bruijn graph using the short insert size data; (2) construct scaffold by aligning reads onto the contigs and (3) fill gaps by local assembly through mapping reads to flanking sequences of gaps. For BAC sequencing data, we split each BAC library data by index sequences in SOAPdenovo-2.04 (ref. <xref ref-type="bibr" rid="b11">11</xref>) software to assemble each BAC clone. We combined the above WGS assembled sequences with the above assembled BAC sequences based on the overlap information by Rabbit<xref ref-type="bibr" rid="b37">37</xref>, and then used the paired-end relationships of the large insert size sequencing data to link the scaffolds/contigs into super-scaffold sequence using SSPACE-v1.1 (ref. <xref ref-type="bibr" rid="b38">38</xref>). We filled gaps by GapCloser v1.10 programme in the SOAPdenovo<xref ref-type="bibr" rid="b11">11</xref> software package. To overcome the problems associated with low GC content of the jujube, PCR-free libraries were also constructed and sequenced.</p></sec><sec disp-level="2"><title>RAD-seq for genetic map construction</title><p>The mapping population consisted of 105 inter-specific hybrids from a cross between <italic>Z. jujuba</italic> Mill. &#x02018;JMS2&#x02019; and <italic>Z. acidojujuba</italic> Cheng et Liu &#x02018;Xing 16&#x02019;. Each individual in the F1 population was genotyped with RAD-seq. The paired-end RAD reads was mapped by SOAP2 (ref. <xref ref-type="bibr" rid="b39">39</xref>). Based on the alignment result, the RAD-based SNP calling was done by SOAPsnp<xref ref-type="bibr" rid="b40">40</xref>. Two heterozygous SNP alleles or one heterozygous and one homozygous SNP allele between two parents were treated as potential SNP markers if the following criteria were satisfied: parents:sequencing depth &#x02265;10, base quality &#x02265;25, copy number &#x02264;1.5; progenies:sequencing depth &#x02265;6, base quality &#x02265;20, copy number &#x02264;1.5. The ratio of marker segregation was calculated by <italic>&#x003c7;</italic>²-test. Markers showing significantly distorted segregation (<italic>P</italic>-value &#x0003c; 0.01) were excluded from the map construction. The double pseudo-test cross strategy was applied<xref ref-type="bibr" rid="b41">41</xref>. Linkage analysis was performed for markers present at least 85% using JoinMap 4.0 software with CP population type codes<xref ref-type="bibr" rid="b42">42</xref>. An logarithm (base 10) of odds (LOD) score of 6.0 was initially set as the linkage threshold for linkage group identification. Twelve linkage groups that had the same number of jujube chromosomes were formed at a LOD threshold of 6.0 and ordered using the regression mapping algorithm. Linkage between markers, recombination rates and map distances were calculated using the Kosambi mapping function and the regression mapping algorithm with a recombination frequency threshold of 0.5. The mapping position of SNP markers were first aligned to the scaffolds and scaffolds with only one SNP marker could be anchored but not oriented owing to a lack of markers. Finally, we scanned all SNP markers to construct the linkage groups and to anchor scaffolds to chromosomes.</p></sec><sec disp-level="2"><title>Repeat annotation</title><p>Repetitive elements were identified by Repbase-based method<xref ref-type="bibr" rid="b43">43</xref> , <italic>de novo</italic> approach and TRF<xref ref-type="bibr" rid="b44">44</xref> software. We searched for repeats using RepeatMasker<xref ref-type="bibr" rid="b45">45</xref> and RepeatProteinMask<xref ref-type="bibr" rid="b45">45</xref> software against Repbase database, identifying TEs at DNA and protein level. Also, we employed Piler<xref ref-type="bibr" rid="b46">46</xref>, RepeatScout<xref ref-type="bibr" rid="b47">47</xref>, and LTR-FINDER<xref ref-type="bibr" rid="b48">48</xref> programmes to build the <italic>de novo</italic> repeat libraries and we ran RepeatMasker against the <italic>de novo</italic> library to find and classify the repeats.</p></sec><sec disp-level="2"><title>Gene prediction</title><p>We used homology-based and <italic>de novo</italic> methods, as well as RNA-seq data, to predict genes in the <italic>Z. jujuba</italic> genome. For homology-based gene prediction, protein sequences from six other plant species (<italic>C. sinensis</italic>, <italic>M. domestica</italic>, <italic>P. trichocarpa</italic>, <italic>G. max</italic>, <italic>P. persica</italic> and <italic>V. vinifera</italic>) were initially mapped onto the <italic>Z. jujuba</italic> genome using TBLASTN<xref ref-type="bibr" rid="b49">49</xref> (<italic>E</italic>-value&#x0003c;1e&#x02212;5), and the homologous genome sequences were aligned against the matching proteins using GeneWise<xref ref-type="bibr" rid="b50">50</xref> for accurate spliced alignments. Next, we used the <italic>de novo</italic> gene prediction methods Augustus<xref ref-type="bibr" rid="b51">51</xref> and GenScan<xref ref-type="bibr" rid="b52">52</xref>. We then integrated the homologues and those from <italic>de novo</italic> approaches using GLEAN<xref ref-type="bibr" rid="b53">53</xref> to produce a consensus gene set. In addition, we aligned all the RNA-seq reads to the reference genome using TopHat<xref ref-type="bibr" rid="b54">54</xref> and predicted the open reading frames (ORFs)s from the resulting data. Finally, we combined the GLEAN set with the gene models produced from RNA-seq to generate a high confidence gene set.</p><p>We did functional gene annotation by BlastP alignment to KEGG<xref ref-type="bibr" rid="b55">55</xref>, SwissProt<xref ref-type="bibr" rid="b56">56</xref> and TrEMBL<xref ref-type="bibr" rid="b56">56</xref> databases. Motifs and domains were determined by InterProScan<xref ref-type="bibr" rid="b57">57</xref> against protein databases such as ProDom, PRINTS, Pfam, SMART, PANTHER and PROSITE, and Gene Ontology<xref ref-type="bibr" rid="b58">58</xref> was obtained from the corresponding InterProScan entries.</p></sec><sec disp-level="2"><title>Gene family clusters</title><p>The protein-coding genes from seven Rosales species&#x02028;(<italic>F. vesca</italic>, <italic>P. persica</italic>, <italic>P. mume</italic>, <italic>M. notabilis</italic>, <italic>M. domestica</italic>, <italic>P. bretschneideri</italic> and <italic>C. sativa</italic>) and <italic>A. thaliana</italic> were downloaded from NCBI. The longest ORF was chosen to represent each gene, and ORF of genes encoding &#x0003c;50 amino acids were filtered out. The OrthoMCL<xref ref-type="bibr" rid="b59">59</xref> method was then used to cluster all the genes into paralogous and orthologous groups on the BLASTP alignment with pairwise comparison strategy (<italic>E</italic>-value &#x02264;1e&#x02212;5).</p></sec><sec disp-level="2"><title>Speciation time estimation</title><p>The phylogenetic tree of eight species including jujube was constructed. The divergence time between jujube and seven other sequenced species of Rosales (<italic>A. thaliana</italic> as the outgroup) was estimated using MrBayes<xref ref-type="bibr" rid="b60">60</xref> and the MCMCtree programme was implemented in the Phylogenetic Analysis by Maximum Likelihood (PAML)<xref ref-type="bibr" rid="b61">61</xref> software package. Calibration times was obtained from the TimeTree database (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.timetree.org/">http://www.timetree.org/</ext-link>).</p></sec><sec disp-level="2"><title>Transcriptome sequencing and analysis</title><p>Total RNA was extracted from 15 samples (root, primary shoot, secondary shoot, MBS, bearing shoot, leaf, flower, young fruit, white mature fruit, half-red fruit and full red fruit of &#x02018;Dongzao&#x02019;, deciduous and lignified bearing shoots of &#x02018;Dongzao&#x02019; and &#x02018;Lizao&#x02019; (four samples)). The deciduous and lignified bearing shoots were sampled on 26 July 2013, the stage at which we could confirm the shoots are deciduous or lignified according to the morphological characters. RNA-seq libraries were constructed to sequence the transcriptome of each sample. RNA-seq libraries were sequenced on an Illumina Genome Analyzer platform. The resulting reads were aligned to the <italic>Z. jujuba</italic> genome sequences using TopHat<xref ref-type="bibr" rid="b54">54</xref>. After alignment, the count of mapped reads from each sample was derived and normalized to reads per kilobase of exon per million reads mapped for each predicted transcript using the Cufflinks<xref ref-type="bibr" rid="b54">54</xref> package. In addition to conducting the analysis described above for the <italic>Z. jujuba</italic> transcriptome, we further assembled all reads from <italic>Z. jujuba</italic> using the Trinity<xref ref-type="bibr" rid="b62">62</xref> package to evaluate the gene region coverage ratio.</p></sec><sec disp-level="2"><title>Jujube-specific gene set analysis</title><p>We obtained three types of specific gene set for jujube (expansion-related genes, positively selected genes and unique genes) from seven Rosales species (<italic>Z. jujuba</italic>, <italic>F. vesca</italic>, <italic>P. persica</italic>, <italic>P. mume</italic>, <italic>M. notabilis</italic>, <italic>M. domestica</italic> and <italic>P. bretschneideri</italic>).</p><p>To obtain the jujube unique gene families, we clustered gene families of the seven sequenced Rosales plant genomes. The genes from these genomes were collected and aligned to each other using BLASTP. Pairwise protein sequence similarity was used as the distance for clustering genes.</p><p>To detect genes evolving under positive selection in jujube, we used gene clustering of the seven Rosales species to identify 1,420 single-copy orthologous genes and used the optimized branch-site model<xref ref-type="bibr" rid="b63">63</xref> to identify 254 genes that are positively selected in <italic>Z. jujuba</italic>.</p><p>To detect expansion gene categories (SwissProt, KEGG and InterPro), we annotated the gene functions of the seven sequenced Rosales species genes and analyzed over-represented categories. Each domain of the three categories (SwissProt, KEGG and InterPro) was retrieved from the seven Rosales species, and examples in which the abundance differed between jujube and all six other species were identified by applying Fisher&#x02019;s Exact test (significance was assumed if <italic>P</italic>&#x0003c;0.05).</p></sec><sec disp-level="2"><title>Fruit sugar analysis</title><p>The experiment was designed as a complete randomized block with three replicates, and four plants were used in each replicate. All samples (each 0.5&#x02009;kg fruits) were picked from <italic>Z. jujuba</italic> &#x02018;Dongzao&#x02019; at four developing stages including young (60 days after fruit set), white mature (90 days after fruit set), half-red (105 days after fruit set) and full red (120 days after fruit set) in National Jujube Germplasm Repository, Taigu, China.</p><p>After drying overnight at 60&#x02009;&#x000b0;C in the presence of silica gel, all samples were milled to powder. For each sample, 1&#x02009;g of powder was transferred to a 50&#x02009;ml volumetric flask and diluted to 40&#x02009;ml with water of ultrapure grade. The flask was sonicated for 1&#x02009;h at 80&#x02009;&#x000b0;C. After cooling to room temperature, the mixture was diluted to the volume. The extracts were filtered through filter paper and a fraction of the filtrate was filtered through a 0.45&#x02009;&#x003bc;m membrane filter. An HPLC system (Agilent 1200 HPLC Series, Waldbronn, Germany) equipped with a quaternary pump system, a refractive index detector (G1362A) was used for sucrose, glucose and fructose analysis<xref ref-type="bibr" rid="b64">64</xref>. Total sugar was determined by the anthrone method<xref ref-type="bibr" rid="b65">65</xref>. Three replicates were done for each sample and each analysis as well.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>M.-J.L. conceived the project. M.-J.L., J.Z. and Q.-L.C. coordinated the overall project. G.-C.L. and Y.C. directed sequencing data generation. Q.-L.C., Y.C., Y.-D.S. performed the assembly. M.-J.L., J.Z., Q.-L.C., Y.C., X.-F.L., Y.-L.W. and L.-L.Y. performed gene annotation, SNP analysis, transcriptome analysis and database management. M.-J.L., J.Z., G.-C.L., X.-F.L. and B.W. analyzed the genome evolution. Y.C., C.Z., G.-C.L., Y.M., X.-F.L., S.-G.L., R.-J.H. and X.-M.G. performed repetitive elements, genome anchor, gene synteny and evolutionary analyses. M.-J.L., J.-RW., J.Z., J.-B.J. and W.Y. provided the materials and constructed the genetic map. M.-J.L., J.Z., J.-RW., Z.-H.Z, P.L., L.D., Z.G.L., M.-J.Lin., J.X.,Y.-Y.C., Z.Y. and X.-C.S. performed sample preparation, fruit nutrition detection and data analysis. M.-J.L. and J.Z. wrote the manuscript with contributions from Q.-L.C., G.-C.L., Z.-H.Z, P.L. J.-R.W., G.Y., L.D., W.-J.W., X.-S.L. and Y.C. The manuscript was revised by M.-J.L., J.Z., G.Y., Z.W., Y.-Y.Z. and L.-H.L.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>Accession codes:</bold> The Jujube genome data has been deposited at DDBJ/EMBL/GenBank under the accession code JREP00000000. Sequence reads of transcriptome sequencing have been deposited in NCBI sequence read archive (SRA) under the accession code SRP046073.</p><p><bold>How to cite this article</bold>: Liu, M.-J. <italic>et al</italic>. The complex jujube genome provides insights into fruit tree biology. <italic>Nat. Commun.</italic> 5:5315 doi: 10.1038/ncomms6315 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-9, Supplementary Tables 1-29 and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6315-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Data 1</title><p>Housekeeping genes selected from 5 organs (&#x003c4;&#x0003c;0.07)</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6315-s2.xls"/></supplementary-material><supplementary-material id="d33e30" content-type="local-data"><caption><title>Supplementary Data 2</title><p>The location statistics of some genes in the super-block (9.20-14.68Mb) of LG1</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6315-s3.xls"/></supplementary-material><supplementary-material id="d33e36" content-type="local-data"><caption><title>Supplementary Data 3</title><p>Genes involved in sugar metabolism</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6315-s4.xls"/></supplementary-material><supplementary-material id="d33e42" content-type="local-data"><caption><title>Supplementary Data 4</title><p>Genes related to defense and stress responses in jujube</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6315-s5.xls"/></supplementary-material></sec>

Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole

<p>Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.</p>

<contrib contrib-type="author"><name><surname>Ohta</surname><given-names>Midori</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ashikawa</surname><given-names>Tomoko</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Nozaki</surname><given-names>Yuka</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kozuka-Hata</surname><given-names>Hiroko</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Goto</surname><given-names>Hidemasa</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Inagaki</surname><given-names>Masaki</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Oyama</surname><given-names>Masaaki</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kitagawa</surname><given-names>Daiju</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Centrosome Biology Laboratory, Center for Frontier Research, National Institute of Genetics</institution>, Mishima, Shizuoka 411-8540, <country>Japan</country></aff><aff id="a2"><label>2</label><institution>Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo</institution>, Minato-ku, Tokyo 108-8639, <country>Japan</country></aff><aff id="a3"><label>3</label><institution>Division of Biochemistry, Aichi Cancer Center Research Institute</institution>, Nagoya, Aichi 464-8681, <country>Japan</country></aff>

Nature Communications

<p>Centrosomes are the major microtubule organizing centre in most of animal cells and composed of a pair of centrioles surrounded by pericentriolar material. Centriole formation is indispensable for centrosome duplication and must be tightly coordinated with cell cycle progression to ensure robust formation of bipolar mitotic spindles and proper chromosome segregation. Centriole formation begins with the assembly of the cartwheel structure that mainly dictates the universal radial ninefold symmetry of centrioles, followed by attachment of peripheral centriolar microtubules and further centriole elongation<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref>. Despite the recent notable progress in our understanding of the molecular and structural principles of centriole assembly, the mechanisms ensuring formation of only one procentriole at the base of each parental centriole per cell division cycle remain incompletely understood.</p><p>An evolutionarily conserved core pathway for centriole assembly includes the following five major components: Cep192/DSpd-2/SPD-2, Plk4/Sak/ZYG-1, HsSAS-6/DSas-6/SAS-6, STIL/Ana2/SAS-5 and CPAP/DSas-4/SAS-4 (refs <xref ref-type="bibr" rid="b1">1</xref>, <xref ref-type="bibr" rid="b2">2</xref>, <xref ref-type="bibr" rid="b3">3</xref>). Among these components, Plk4 (refs <xref ref-type="bibr" rid="b4">4</xref>, <xref ref-type="bibr" rid="b5">5</xref>), HsSAS-6 (refs <xref ref-type="bibr" rid="b6">6</xref>, <xref ref-type="bibr" rid="b7">7</xref>) and STIL<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref> particularly may play an important role in controlling centriole number, since their overexpression induces concurrent formation of multiple procentrioles around a parental centriole<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>.</p><p>The conserved proteins of SAS-6 family are known to be a crucial element of a centriolar cartwheel structure<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. Whereas it seems that nine of SAS-6 rod-shaped homodimers self-assemble into the central part of the cartwheel<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>, there could be additional factors strictly regulating this process or other cartwheel components facilitating SAS-6 self-assembly at the onset of procentriole formation. Given that Plk4 acts upstream of HsSAS-6 and STIL<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>, and also that HsSAS-6 and STIL appear to be interdependent for their loading to the centrioles<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, Plk4 and STIL are plausible candidates for regulating HsSAS-6 oligomerization for cartwheel assembly. Although their relationship in other species appears to be mostly conserved<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref>, how their collaborative action regulates the onset of centriole formation remains elusive. Moreover, the critical substrates of Plk4, a key kinase for centriole duplication, and how the kinase reaction triggers the onset of procentriole formation remain to be discovered.</p><p>In this study, we identify STIL as a critical substrate of Plk4, and show that the phosphorylation event leads to formation of the STIL/HsSAS-6 complex and initiation of procentriole assembly. Furthermore, we demonstrate negative feedback in which centriolar recruitment of the STIL/HsSAS-6 complex in turn limits distribution of centriolar Plk4 through the ubiquitin&#x02013;proteasome pathway. This coordinated action of the three key factors triggers the onset of procentriole formation and, concurrently, restricts the occurrence of procentriole formation to one site per parental centriole.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Plk4 recruits STIL to the centrioles by direct binding</title><p>First, to investigate the physical interactions between the three key factors for centriole assembly, we conducted co-immunoprecipitation analysis with human 293T cells expressing FLAG-tagged full-length Plk4 or Plk4&#x00394;PEST lacking the first PEST destruction motif<xref ref-type="bibr" rid="b25">25</xref>, and tested whether the Plk4 proteins interact with endogenous STIL or HsSAS-6 proteins. This analysis revealed that Plk4-FLAG full-length interacts with endogenous STIL, but not with HsSAS-6 (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). Furthermore, we found that a higher amount of endogenous STIL could be co-immunoprecipitated with Plk4&#x00394;PEST-FLAG owing to the increase in the expression levels of Plk4&#x00394;PEST-FLAG compared with those of Plk4-FLAG full length (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). However, we could not detect such a robust interaction in the case of a kinase-dead mutant of Plk4&#x00394;PEST-FLAG, suggesting that STIL preferentially interacts with Plk4 wild-type (WT) rather than the kinase dead. We also found that the interaction requires the two tandem polo boxes, PB1 and PB2, but not the C-terminal PB3 of Plk4 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1a&#x02013;d</xref>)<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b26">26</xref>. Using human influenza hemagglutinin (HA) tagged deletion constructs of STIL, we narrowed down the STIL domain that is required for Plk4 binding to the short conserved coiled-coil domain<xref ref-type="bibr" rid="b23">23</xref> (<xref ref-type="fig" rid="f1">Fig. 1b&#x02013;d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1e,f</xref>). Moreover, yeast two-hybrid and <italic>in vitro</italic> binding assays established that Plk4 directly bound to the STIL fragments containing the CC domain presumably in a kinase-activity-dependent manner (<xref ref-type="fig" rid="f1">Fig. 1e</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1g,h</xref>).</p><p>We therefore reasoned that the physical interaction between the two proteins might be needed for centriolar recruitment of STIL. We depleted endogenous STIL using short interfering RNAs (siRNAs) against the 3&#x02032;-untranslated region (3&#x02032;UTR) and expressed STIL full-length or deletion constructs tagged with HA at comparable levels in human U2OS cells (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1a</xref>). We found that whereas centriole numbers were reduced in the majority of interphase cells upon depletion of endogenous STIL, expression of exogenous full-length STIL could functionally rescue this phenotype (~26% and ~57% of cells with &#x02265;4 centrioles, respectively; <xref ref-type="fig" rid="f1">Fig. 1f</xref>) and frequently induced formation of multiple procentrioles next to each pre-existing centriole. In contrast, expression of the N-terminal fragment of STIL rescued centriole formation in only ~13% of cells (<xref ref-type="fig" rid="f1">Fig. 1f</xref>) even though this fragment efficiently localized to the centrioles, indicating that the C-terminal region of STIL is crucial for centriole formation. Importantly, we determined that STIL proteins lacking the CC domain did not localize to the centrioles (~21% of cells with centriolar STIL&#x00394;CC, compared with ~76% of cells with centriolar STIL FL; <xref ref-type="fig" rid="f1">Fig. 1f</xref>) and failed to rescue centriole formation. Overall, these findings indicate that STIL is recruited to the centrioles by direct binding to Plk4 through its conserved CC domain, and that this interaction is necessary for centriole formation.</p></sec><sec disp-level="2"><title>STIL STAN motif is crucial for HsSAS-6 centriolar targeting</title><p>We then investigated how the C-terminal region of STIL contributes to centriole formation. Since centriolar loading of HsSAS-6 is dependent on STIL<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, we monitored whether HsSAS-6 proteins are present at centrioles when the C-terminal region or the conserved STAN motif<xref ref-type="bibr" rid="b23">23</xref> of STIL was depleted. We first confirmed that whereas centriolar loading of HsSAS-6 was significantly reduced upon depletion of endogenous STIL, expression of STIL full length restored the localization of HsSAS-6 around the mother centriole (~40% and ~85% of cells with centriolar HsSAS-6, respectively; <xref ref-type="fig" rid="f2">Fig. 2a</xref>). Intriguingly, we found that the STIL proteins lacking the STAN motif failed to recruit HsSAS-6 to the centrioles even though they robustly localized to the centrioles (~5% of cells with centriolar HsSAS-6; <xref ref-type="fig" rid="f2">Fig. 2a</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2a,b</xref>). These data indicate that the STAN motif is crucial for centriolar targeting of HsSAS-6.</p></sec><sec disp-level="2"><title>Plk4 kinase activity promotes STIL/HsSAS-6 interaction</title><p>It has been shown that the functional homologues of STIL: SAS-5 in <italic>Caenorhabditis elegans</italic><xref ref-type="bibr" rid="b7">7</xref> and Ana2 in <italic>Drosophila melanogaster</italic><xref ref-type="bibr" rid="b23">23</xref>, can directly bind to SAS-6 proteins. However, in recent studies, a direct interaction between STIL and HsSAS-6 could not be detected<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>. We therefore hypothesized that Plk4 might regulate the mode of their interaction in human cells. To test this idea, we conducted co-immunoprecipitation experiments using 293T cells expressing Plk4&#x00394;PEST-FLAG and Myc-HsSAS-6 proteins. Interestingly, we found that endogenous STIL was detected in the immunoprecipitated fraction of HsSAS-6 in cells expressing Plk4&#x00394;PEST-FLAG and Myc-HsSAS-6, whereas this was not the case in cells expressing kinase-dead Plk4&#x00394;PEST-FLAG and Myc-HsSAS-6 (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). This result prompted us to address whether phosphorylation of STIL by Plk4 facilitates a direct interaction between STIL and HsSAS-6.</p><p>To examine this, we used a combined biochemical approach using <italic>in vitro</italic> kinase assays with recombinant STIL N3C and Plk4&#x00394;PEST-FLAG, followed by <italic>in vitro</italic> pull-down assays with recombinant maltose-binding protein (MBP) tagged HsSAS-6 (<xref ref-type="fig" rid="f2">Fig. 2c</xref>). We found that Plk4&#x00394;PEST efficiently phosphorylates STIL N3C <italic>in vitro</italic> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3c</xref>). Remarkably, we demonstrated that the phosphorylated STIL N3C directly bound to MBP-HsSAS-6 <italic>in vitro</italic> (<xref ref-type="fig" rid="f2">Fig. 2d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2c,d</xref>). By contrast, this interaction was abolished when the STAN motif was removed from STIL N3C (<xref ref-type="fig" rid="f2">Fig. 2e</xref>). This is in line with the observation that the STAN motif is crucial for centriolar loading of HsSAS-6 (<xref ref-type="fig" rid="f2">Fig. 2a</xref>). To further narrow down the region of STIL for directly binding to HsSAS-6, we generated several deletion mutants within the C-terminal fragment of STIL (<xref ref-type="fig" rid="f2">Fig. 2f,g</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2e</xref>). As expected, amino acid (a.a.) 1,061&#x02013;1,083 and a.a. 1,106&#x02013;1,147 regions in the STAN appeared to be required for the interaction between the phosphorylated STIL and HsSAS-6 (<xref ref-type="fig" rid="f2">Fig. 2f</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 2e and 3a</xref>). Furthermore, we demonstrated that the STIL STAN motif phosphorylated by Plk4 is sufficient for binding to HsSAS-6 (<xref ref-type="fig" rid="f2">Fig. 2g</xref>). Overall, these findings support the notion that phosphorylation of STIL by Plk4 facilitates the direct interaction between the conserved STAN motif of STIL and HsSAS-6, leading to centriolar loading of HsSAS-6.</p></sec><sec disp-level="2"><title>STIL is phosphorylated by Plk4 <italic>in vitro</italic> and <italic>in vivo</italic></title><p>We next sought to analyse the phosphorylation of STIL by Plk4 to further investigate its biological relevance for centriole formation. We first conducted <italic>in vitro</italic> kinase assays with Plk4&#x00394;PEST-FLAG and the indicated four STIL fragments, and found that the fragments STIL N3 and C were efficiently phosphorylated by Plk4&#x00394;PEST-FLAG <italic>in vitro</italic> (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). Intriguingly, we found a significant shift in the mobility of the phosphorylated STIL C fragment due to hyper-phosphorylation by Plk4&#x00394;PEST (<xref ref-type="fig" rid="f3">Fig. 3a,b</xref>). Using mass spectrometry (MS) analysis and phospho-specific antibodies, we identified seven and five phosphorylated serine/threonine residues within the N3 and C fragments, respectively (<xref ref-type="fig" rid="f3">Fig. 3a,c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3a&#x02013;c</xref>). We next investigated whether STIL is also phosphorylated by Plk4 <italic>in vivo</italic>. We observed a shift in the mobility of endogenous STIL when expressing Plk4&#x00394;PEST-FLAG in U2OS cells (<xref ref-type="fig" rid="f3">Fig. 3d</xref>). The shift was abolished upon treatment of the cell lysate with &#x003bb;-phosphatase, indicating that endogenous STIL proteins were phosphorylated by Plk4 <italic>in vivo</italic> (<xref ref-type="fig" rid="f3">Fig. 3d</xref>). Similarly, we found a significant band shift of STIL N3C fragment in U2OS cells expressing Plk4&#x00394;PEST-FLAG, which is suggestive of the occurrence of multiple phosphorylations on the STIL fragment (<xref ref-type="fig" rid="f3">Fig. 3e</xref>). Importantly, in addition, the shift of STIL N3C was drastically attenuated by mutating all the identified phosphorylated residues to alanine, indicating that these sites can be phosphorylated <italic>in vivo</italic> (<xref ref-type="fig" rid="f3">Fig. 3e</xref>).</p><p>We then set out to examine the biological relevance of these phosphorylation sites for STIL function. We first found that the mutation of all seven S/T residues in the STIL N3 region or all five S/T residues in the STIL C region to alanine did not affect the interaction between Plk4 and STIL (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a&#x02013;d,f</xref>). Moreover, since the N3 region of STIL is sufficient for binding to Plk4 (<xref ref-type="fig" rid="f1">Fig. 1e</xref>), we further analysed deletion mutants within the N3 and confirmed that the conserved coiled-coil domain is essential for the Plk4/STIL interaction (<xref ref-type="fig" rid="f1">Fig. 1b&#x02013;d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a&#x02013;c,f</xref>). However, when mutating the sole S/T residue, T727, within the CC to alanine, we still detected the Plk4/STIL interaction (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a,g</xref>). These data suggest that while Plk4 kinase activity itself seems to be critical for the interaction, the phosphorylation of STIL by Plk4 is possibly dispensable for the interaction. We speculate that Plk4 autophosphorylation<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref> can modulate the state of Plk4 self-assembly, leading to the direct binding to the CC domain of STIL.</p></sec><sec disp-level="2"><title>Critical sites in the STAN for HsSAS-6 centriolar targeting</title><p>Given that the phosphorylated STAN motif of STIL itself is sufficient for interacting with HsSAS-6 <italic>in vitro</italic> (<xref ref-type="fig" rid="f2">Fig. 2f,g</xref>), we reasoned that S1061 and S1116 that are within the STAN motif and phosphorylated by Plk4 <italic>in vitro</italic> could be critical residues to mediate the STIL/HsSAS-6 interaction (<xref ref-type="fig" rid="f4">Fig. 4a</xref>). To address this idea, we first conducted an alanine mutational scan for the S/T sites within the STAN motif, which led to the identification of three critical sites (S1061, S1116 and T1119) for the STIL/HsSAS-6 interaction <italic>in vitro</italic> (<xref ref-type="fig" rid="f4">Fig. 4b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5a</xref>). As we could not find any evidence of phosphorylation at T1119 by MS, we assume that T1119 could be important for the structural arrangement of the STAN motif rather than being a phosphorylation site. Interestingly, S1061 and S1116 are the specific residues that are highly conserved from human STIL to <italic>Drosophila</italic> Ana2 (<xref ref-type="fig" rid="f4">Fig. 4a</xref>), implying their biological significance throughout evolution. Second, we investigated whether alanine substitution mutants of the two phosphorylation sites of STIL have the ability to rescue the defect in the centriolar loading of HsSAS-6 and centriole formation when endogenous STIL proteins are depleted. Strikingly, we found that although mutating both residues to alanine did not affect centriolar targeting of STIL (<xref ref-type="fig" rid="f4">Fig. 4c,d</xref>), expression of the S1061A, S1116A or 2A double mutant of STIL failed to rescue centriolar loading of HsSAS-6 and centriole formation in the cells depleted of endogenous STIL (centriolar HsSAS-6; ~4% for STIL 2A and ~78% for STIL WT: &#x02265;4 centrin foci; ~2% for STIL 2A and ~50% for STIL WT; <xref ref-type="fig" rid="f4">Fig. 4c,d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5b&#x02013;f</xref>). On the other hand, expression of the STIL alanine mutants at three other MS-identified phosphorylation sites (S1181, T1238 or T1250) or the STIL deletion mutant lacking all the seven MS-identified phosphorylation sites in the N3 region mostly rescued the phenotype provoked by depletion of endogenous STIL (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 4e and 5d&#x02013;f</xref>). To further characterize the two critical phosphorylation sites in STIL, S1061 and S1116, we generated phosphomimetic mutants at these residues and investigated their function for centriole formation. Importantly, we found that expression of a phosphomimetic STIL mutant at S1061 under an attenuated human cytomegalovirus (CMV) promoter induced centriole overduplication more efficiently than that of STIL WT in the same condition (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6a,b</xref>). Consistently, a phosphomimetic mutant at S1061 of the STIL STAN could interact with MBP-HsSAS-6 <italic>in vitro</italic> even without being phosphorylated by Plk4 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6c</xref>). On the other hand, introducing of a phosphomimetic mutation at S1116 impaired Plk4-mediated STIL/HsSAS-6 interaction <italic>in vitro</italic>, and centriolar targeting of HsSAS-6 in human cells (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6d</xref> and data not shown). Given that the MS analysis revealed that this residue was phosphorylated by Plk4 <italic>in vitro</italic> and also that it is found to be phosphorylated in mammalian cells (PhosphoSitePlus database), we favour the possibility that substitution of S1116 to D/E could not mimic the phosphorylation state, but rather reduced the function of STIL. However, alternatively, it is also possible that S1116 is just critical for structural integrity of the STAN motif and/or its function independently of being phosphorylated. Taken together, we propose that S1061 and S1116 of STIL can be the most important phosphorylation sites for the STIL/HsSAS-6 interaction and resulting centriolar targeting of HsSAS-6 in procentriole formation.</p></sec><sec disp-level="2"><title>Bimodal centriolar distribution of Plk4 during cell cycle</title><p>We next hypothesized that the interaction between Plk4 and STIL might have an influence on the centriolar recruitment and/or maintenance of Plk4. To explore this possibility, we examined the distribution of endogenous Plk4 (ref. <xref ref-type="bibr" rid="b29">29</xref>) in U2OS cells released from a nocodazole arrest and fixed at successive time points. Intriguingly, we found a bimodal distribution of Plk4 at centrioles depending on the cell cycle progression (<xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c</xref>, <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1b and 7a,b</xref>). As previously reported<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>, we observed that most of the cells exhibited one intense focus of Plk4 on each parental centriole during prophase/metaphase (~90%, ~0.3&#x02009;&#x003bc;m in diameter; <xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7a,b</xref>), and also that the signal intensity of the foci substantially declined around telophase. However, in the next cell cycle, we detected endogenous Plk4 localized in a ring-like manner around the parental centrioles (~0.62&#x02009;&#x003bc;m in diameter; <xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7a</xref>) and partially overlapped with a Cep152 ring, as a marker for parental centrioles (<xref ref-type="fig" rid="f5">Fig. 5a</xref>)<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>. Quantitative analyses indicated that the majority of cells in G1 phase harboured a ring-like pattern of endogenous Plk4 (~65% at 8&#x02013;10&#x02009;h after the release; <xref ref-type="fig" rid="f5">Fig. 5a,c</xref>). Remarkably, we further found that the ring-like pattern of centriolar Plk4 changed back into a dot on the parental centriole wall in G1/S phase (~52% as a dot at 13&#x02009;h after the release; <xref ref-type="fig" rid="f5">Fig. 5a,c</xref>). Co-staining of endogenous Plk4 and STIL revealed that when STIL localized to centrioles in G1/S phase, centriolar distribution of Plk4 was always restricted to a spot and largely overlapped with centriolar STIL foci (<xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c</xref>). We could hardly detect the existence of a ring-like pattern of Plk4 with STIL foci at centrioles, suggesting that they are mutually exclusive. In addition, we noted that STIL and HsSAS-6 appeared to be loaded to the centrioles almost concomitantly and precisely co-localized with each other (data not shown)<xref ref-type="bibr" rid="b31">31</xref>. We therefore assumed that the existence of STIL and HsSAS-6 might allow the conversion of the centriolar Plk4 ring into a dot.</p><p>To test this, we examined the centriolar distribution of Plk4 when STIL or HsSAS-6 was depleted from U2OS cells. Interestingly, we found that most of the interphase cells depleted of STIL or HsSAS-6 harboured the ring-like arrangement of centriolar Plk4 while the cell cycle progression was not affected (Plk4 ring; ~18% for siCnt, ~91% for siHsSAS-6 and ~71% for siSTIL; <xref ref-type="fig" rid="f5">Fig. 5d,e</xref>, and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1a and 7c,d</xref>). In addition, the expression of STIL &#x00394;CC in STIL siRNA-treated cells did not suppress the increase in the number of cells with a ring-like pattern of centriolar Plk4 (~59% for siSTIL+empty vector and ~60% for siSTIL+&#x00394;CC; <xref ref-type="fig" rid="f5">Fig. 5e</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7e</xref>), indicating that the existence of STIL and HsSAS-6 at centrioles is required for the conversion of the centriolar Plk4 ring into a dot.</p></sec><sec disp-level="2"><title>The interaction of Plk4 with STIL protects centriolar Plk4</title><p>Given that the expression levels of Plk4 are known to be regulated by trans-autophosphorylation that induces proteolytic degradation by the E3 ubiquitin ligase Skp-Cullin-F-box containing complex, SCF^Slimb^{/&#x003b2;-TrCP} and the ubiquitin&#x02013;proteasome-dependent pathway in <italic>D. melanogaster</italic> and mammalian cells<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>, we hypothesized that the conversion of centriolar Plk4 followed by centriolar STIL/HsSAS-6 loading might involve protein degradation through the ubiquitin&#x02013;proteasome pathway mediated by the SCF complex or other E3 ligases. To address this, we synchronized U2OS cells in G1/S phase with aphidicolin and transiently treated with MG132, a proteasome inhibitor. In the cells treated without MG132, endogenous Plk4 localized mostly as a dot and resided with STIL foci at centrioles (Plk4 ring; ~8%; <xref ref-type="fig" rid="f6">Fig. 6a,b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7f</xref>). In stark contrast, when treated with MG132, majority of the cells harboured a complete ring of Plk4 with STIL foci at the centrioles, which we could hardly detect in normal cycling cells (Plk4 ring; ~93%; <xref ref-type="fig" rid="f6">Fig. 6a,b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7c,f</xref>). In this situation, Plk4 proteins were primarily enriched at the position overlapping with STIL foci as compared with the rest of the ring. This implies that centriolar STIL could interact with and protect Plk4 proteins from protein degradation, whereas the residual centriolar Plk4 proteins are normally degraded.</p><p>To address this model, we next investigated whether overexpression of STIL full-length or mutant proteins stabilizes centriolar Plk4. We found that whereas low expression of HA-STIL full length in the cells depleted of endogenous STIL restored the population of cells containing Plk4 dots at the centrioles, overexpression of HA-STIL full-length, &#x00394;STAN or 5A mutant proteins stabilized centriolar Plk4 as a ring overlapping with the STIL proteins (<xref ref-type="fig" rid="f6">Fig. 6c</xref>). Considering that the centriolar loading of HsSAS-6 and procentriole formation were inhibited in the cells expressing HA-STIL &#x00394;STAN or 5A mutant, this result suggests that centriolar presence of STIL might be sufficient for stabilizing centriolar Plk4 in this situation. Intriguingly, when expressing the PACT-STIL &#x00394;CC mutant that fails to interact with Plk4, but still localizes to centrioles, we found that expression of the STIL mutant proteins maintained centriolar Plk4 as a ring overlapping with the STIL mutant proteins and recruited HsSAS-6 to centrioles (<xref ref-type="fig" rid="f6">Fig. 6c</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7g</xref>). This result suggests that PACT-STIL &#x00394;CC can bypass the requirement of STIL function for centriolar loading of HsSAS-6, and also that close-range presence of STIL and Plk4 at centrioles, or their transient interaction could be critical for protecting centriolar Plk4 from protein degradation and for centriolar recruitment of HsSAS-6. However, we cannot exclude the possibility that residual binding activity of overexpressed STIL &#x00394;CC to Plk4 somehow managed to facilitate those events.</p><p>Using biochemical analysis in human 293T cells, we next sought to investigate whether STIL protects Plk4 from protein degradation. Importantly, we demonstrated that expression of STIL efficiently inhibited ubiquitination of Plk4&#x00394;PEST-FLAG WT (<xref ref-type="fig" rid="f6">Fig. 6d</xref>). Given that the first PEST domain of Plk4 contains the site recognized by the SCF complex<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b34">34</xref>, the degradation of Plk4&#x00394;PEST proteins might be regulated by another E3 ubiquitin ligase. Furthermore, we found that expression of STIL full-length stabilized Plk4&#x00394;PEST-FLAG WT, whereas that was not the case when co-expressing STIL&#x00394;N3, which lacks the binding region to Plk4 (<xref ref-type="fig" rid="f6">Fig. 6e</xref>). We also noted that STIL seemed to stabilize activated Plk4&#x00394;PEST-FLAG WT proteins (p-Plk4&#x00394;P) (<xref ref-type="fig" rid="f6">Fig. 6e</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7h</xref>). This is in agreement with the observation that STIL preferentially interacts with Plk4 WT rather than the kinase dead (<xref ref-type="fig" rid="f1">Fig. 1</xref>). Taken together, these data suggest that the interaction between STIL and Plk4 protects Plk4 from protein degradation mediated by the ubiquitin&#x02013;proteasome pathway.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In conclusion, our findings uncover a molecular basis for the onset of centriole formation by demonstrating that direct association of STIL with Plk4 and STIL phosphorylation by Plk4 lead to centriolar loading of HsSAS-6 for cartwheel assembly (<xref ref-type="fig" rid="f7">Fig. 7</xref>). Furthermore, our study suggests a negative feedback model in which centriolar STIL/HsSAS-6 recruitment limits centriolar distribution of Plk4 to one site per parental centriole (<xref ref-type="fig" rid="f7">Fig. 7</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7i</xref>). This coordinated action promotes formation of a single procentriole and, concurrently, could inhibit formation of another procentriole, thus presumably contributing to maintenance of one procentriole next to each parental centriole.</p><p>How could phosphorylated STIL facilitate cartwheel assembly by direct binding to HsSAS-6? It could be structurally critical for the spatial arrangement and connection between HsSAS-6 homodimers. Indeed, it has been shown in <italic>D. melanogaster</italic> that co-overexpression of DSas-6 and Ana2 induces formation of highly ordered tubules with the structures reminiscent of the centriolar cartwheel hub<xref ref-type="bibr" rid="b20">20</xref>. However, to drive centriole overduplication through efficient centriolar recruitment of extra DSas-6 and Ana2, additional co-expression with Plk4/Sak is needed<xref ref-type="bibr" rid="b20">20</xref>. These observations are compatible with our findings, implying that the interplay between the three key factors that we demonstrated may underlie the centriole assembly pathway across species. On the other hand, in <italic>C. elegans</italic>, given that the interaction between SAS-5 and SAS-6 seems to be detectable presumably even in the absence of ZYG-1 (refs <xref ref-type="bibr" rid="b7">7</xref>, <xref ref-type="bibr" rid="b36">36</xref>), and also that ZYG-1 can directly bind to<xref ref-type="bibr" rid="b37">37</xref> and phosphorylate SAS-6 (ref. <xref ref-type="bibr" rid="b38">38</xref>) for centriole assembly, the regulatory mechanism for cartwheel assembly may be somehow different in this organism along with its structural divergence of the centriole structure.</p><p>It has been recently shown that the expression levels of Plk4 are regulated by trans-autophosphorylation that mediates proteolytic degradation by the E3 ubiquitin ligase SCF^{Slimb/&#x003b2;-TrCP} and the ubiquitin&#x02013;proteasome-dependent pathway in <italic>D. melanogaster</italic><xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref> and mammalian cells<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. The reduction of centriolar Plk4 followed by centriolar recruitment of the STIL/HsSAS-6 complex seems to involve protein degradation through the ubiquitin&#x02013;proteasome pathway (<xref ref-type="fig" rid="f5">Fig. 5d</xref>). It will be therefore important in the future study to further investigate the detailed mechanisms how the recruitment of STIL/HsSAS-6 allows SCF^{Slimb/&#x003b2;-TrCP} and/or other E3 ubiquitin ligases to target for degradation of centriolar Plk4 proteins that do not form a complex with STIL. Considering that physical association and co-localization of Plk4 and STIL at one site on the parental centriole wall is dependent on the kinase activity of Plk4, it is possible that a complex formation between active Plk4 and STIL prevents Plk4 from undergoing protein degradation. Alternatively, any other protein that STIL brings to centrioles may protect Plk4 from the protein degradation. Furthermore, given that the presence of the SAS-5/SAS-6 complex at centrioles is needed for the diminution of centriolar ZYG-1 during interphase<xref ref-type="bibr" rid="b18">18</xref>, it is tempting to speculate that the feedback mechanism that we demonstrated in this study is a conserved system for tight control of centriole copy number throughout evolution.</p><p>Based on the findings in this study, further study will be needed to establish the structural model how the sequential physical interactions between Plk4/STIL/HsSAS-6 proteins lead to the assembly of a core structure for initiating cartwheel assembly.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Cell culture and cell lines</title><p>Human U2OS and HEK293T cells were obtained from the European Collection of Cell Culture (ECACC). U2OS cells stably expressing GFP-centrin1 were gifted from Bornens<xref ref-type="bibr" rid="b41">41</xref>. These cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37&#x02009;&#x000b0;C in 5% CO₂ incubator.</p></sec><sec disp-level="2"><title>Cell cycle synchronization and flow cytometry analysis</title><p>For cell synchronization at prometaphase, cells were treated with 100&#x02009;ng&#x02009;ml^&#x02212;1 nocodazole for 14&#x02009;h, washed three times with PBS and released in fresh medium. For cell cycle arrest in G1/S phase, cells were treated with 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 aphidicolin for 24&#x02009;h.</p><p>For flow cytometry analyses, cells cultured on dishes were trypsinized, washed twice with PBS and fixed in 70% cold ethanol at &#x02212;20&#x02009;&#x000b0;C at each time point. The fixed cells were washed with PBS twice and incubated with Muse Cell Cycle reagents at room temperature (RT) for 30&#x02009;min. The DNA contents of the cells were then measured using Muse Cell Analyzer (Merck Millipore). Flow cytometry analysis was repeated at least two times.</p></sec><sec disp-level="2"><title>Molecular biology and RNA interference</title><p>The following siRNAs were used: Stealth siRNA (Life Technologies) against 3&#x02032;UTR of HsSAS-6 (5&#x02032;-GAGCUGUUAAAGACUGGAUACUUUA-3&#x02032;) and negative control Low GC Duplex no. 2 (12935110); custom siRNA (Sigma Genosys) against 3&#x02032;UTR of Plk4 (5&#x02032;-CTCCTTTCAGACATATAAG-3&#x02032;); custom siRNA (JBios) against 3&#x02032;UTR of STIL (5&#x02032;-GTTTAAGGGAAAAGTTATT-3&#x02032;).</p><p>pcDNA3 constructs encoding Plk4 full-length FLAG, a kinase-dead Plk4 [K41M]-FLAG, Plk4Plk4&#x00394;PEST-FLAG and Plk4[K41M]&#x00394;PEST-FLAG were gifts from Dr Hiroyuki Mano. The mammalian expression constructs for HA-STIL full-length, deletion mutants, alanine substitution mutants and phosphomimetic mutants were created by insertion of subcloned fragments into SpeI-digested modified pCMV5-HA vector or using PrimeSTAR mutagenesis basal kit (TaKaRa). pcDNA3-Plk4&#x00394;PEST&#x02013;[&#x00394;PB1]/[&#x00394;PB2]/[&#x00394;PB3]&#x02013;FLAG expression constructs were created using PrimeSTAR mutagenesis basal kit (TaKaRa). Since expression levels of Plk4&#x00394;P&#x00394;PB3-FLAG were high compared with those of the other Plk4 mutants used in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1d</xref>, the Plk4&#x00394;P&#x00394;PB3-FLAG plasmid was transfected into cells with half the amount of the other Plk4 plasmids.</p><p>Transfection of siRNA or DNA constructs into U2OS and HEK293T cells was performed using Lipofectamine RNAiMAX (Life Technologies) or Lipofectamine 2000 (Life Technologies) according to the manufacturer&#x02019;s instructions. Unless otherwise noted, transfected cells were analysed 48&#x02013;72&#x02009;h after transfection with siRNA and 24&#x02009;h after transfection with DNA constructs.</p></sec><sec disp-level="2"><title>Antibodies</title><p>The following primary antibodies were used in this study: rabbit polyclonal antibodies against STIL (Abcam, ab89314, indirect immnunofluorescence (IF) 1:500, western blotting (WB) 1:1,000), Cep152 (Bethyl Laboratories, A302-480A, IF 1:1,000), HA-tag (Abcam, ab9110, IF 1:1,000, WB 1:1,000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1,000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, WB 1:1,000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), FLAG-tag (Sigma, F1804, IF 1:1,000, WB 1:1,000), HA-tag (Covance, MMS-101P, WB 1:500) and &#x003b1;-tubulin (Sigma, DM1A, WB 1:2,000). P-S1061 rabbit antibodies were raised against C+NGVDL[pS]MEAN, where [pS] is a phosphorylated serine residue (Eurofins Operon). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular Probes, 1:500), Alexa Fluor 568 goat anti-rabbit IgG (H+L) (Molecular Probes, 1:500) for IF; goat polyclonal antibodies horseradish peroxidase against mouse IgG (Promega, W402B, 1:5,000), rabbit IgG (Promega, W401B, 1:5,000) for WB.</p></sec><sec disp-level="2"><title>Indirect immunofluorescence and immunoblotting</title><p>For indirect immunofluorescence microscopy, the cells cultured on coverslips were fixed using &#x02212;20&#x02009;&#x000b0;C methanol for 10&#x02009;min. The cells were then permeabilized with PBS/0.05% Triton X-100 (PBSX) for 5&#x02009;min, washed with PBS three times and incubated for blocking in 1% BSA in PBSX for 30&#x02009;min at RT. The cells were then incubated with primary antibodies for 3&#x02009;h at RT, washed with PBSX three times and incubated with secondary antibodies for 1&#x02009;h at RT. The cells were thereafter washed with PBSX twice, stained with 0.2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 Hoechst 33258 (Dojindo) in PBS for 5&#x02009;min at RT, washed again with PBSX and mounted onto glass slides. Counting the number of immunofluorescence signals was performed by using an Axioplan2 fluorescence microscope (Carl Zeiss) with a &#x000d7; 100/1.4 numerical aperture plan-APOCHROMAT objective. Data acquisition for the images and quantification of the signal intensity were performed using DeltaVision Personal DV-SoftWoRx system (Applied Precision) equipped with a CoolSNAP CH350 CCD camera. The images were acquired as serial sections along the <italic>z</italic> axis and stacked using the &#x02018;quick projection&#x02019; algorithm in SoftWoRx. The signal intensities of centriolar Plk4, STIL and HsSAS-6 were quantified using the Data Inspector tool in SoftWorx. The captured images were processed with Adobe Photoshop CS5.1 (version 12.1). We assessed cells from several fields for each experiment, and we were normally blinded to the sample ID during experiments and outcome assessment. Once a field was determined, we counted all cells that matched with the criteria within the field. In the experiments using the cells expressing HA-tagged full-length or mutants of STIL, we counted cells adequately expressing the STIL proteins at comparable levels and excluded cells expressing the STIL proteins at low levels or cells excessively expressing the STIL proteins.</p><p>For preparation of human cell lysates for immunoblotting, cells were collected, washed in PBS and lysed by vortexing at 4&#x02009;&#x000b0;C in lysis buffer (20&#x02009;mM Tris/HCl pH 7.5, 50&#x02009;mM NaCl, 1% Triton X-100, 5&#x02009;mM EGTA, 1&#x02009;mM dithiothreitol (DTT), 2&#x02009;mM MgCl₂ and 1/1,000 protease inhibitor cocktail (Nakalai Tesque)). Lysates were cleared by centrifugation for 10&#x02009;min at 13,000&#x02009;r.p.m. at 4&#x02009;&#x000b0;C and the supernatant was collected. SDS&#x02013;polyacrylamide gel electrophoresis (SDS&#x02013;PAGE) was performed using 7&#x02013;12% polyacrylamide gels, followed by transfer on Immobilon-P membrane (Millipore Corporation). The membrane was probed with the primary antibodies, followed by incubation with their respective horseradish peroxidase-conjugated secondary antibodies (Promega). Washes were performed in PBS containing 0.02% Tween. The signal was detected as Chemi Doc XRS+ (Bio-Rad). Signal intensity of immunoreactive bands was measured using Adobe Photoshop. Full scan images of the western blots and gels used in the main figures are shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8</xref>. Unless otherwise specified, the experiments of western blotting were repeated at least three times. In <xref ref-type="fig" rid="f3">Figs 3c,e</xref> and <xref ref-type="fig" rid="f4">4b</xref>, and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 2c&#x02013;e, 4b,c, 5a and 6a</xref> were repeated at least two times.</p></sec><sec disp-level="2"><title>Immunoprecipitation</title><p>For preparing whole-cell lysates of HEK293T cells, cells were washed by PBS and lysed in ice-cold lysis buffer. The lysates were vortexed for 40&#x02009;min at 4&#x02009;&#x000b0;C, and insoluble material was removed after centrifugation for 10&#x02009;min. For immunoprecipitation of FLAG-tagged Plk4 proteins, whole-cell lysates were incubated with FLAG antibody-conjugated M2 agarose (Sigma) for 2&#x02009;h at 4&#x02009;&#x000b0;C. Since the expression levels of FLAG-Plk4 proteins in the cells were very low, we monitored them by using the Flag immunoprecipitation instead of using the input materials. For HsSAS-6 immunoprecipitation, whole-cell lysates were incubated with protein G sepharose for 1&#x02009;h at 4&#x02009;&#x000b0;C for preclear, and then incubated for 2&#x02009;h at 4&#x02009;&#x000b0;C with protein G agarose that had been incubated with anti-HsSAS-6 antibodies. In both cases, the beads were washed at least four times with lysis buffer and resuspended in SDS sample buffer before loading onto a SDS&#x02013;PAGE gel.</p></sec><sec disp-level="2"><title><italic>In vitro</italic> kinase assay and MBP pull-down assay</title><p>For <italic>in vitro</italic> kinase assay, HEK293T cells were transfected with Plk4&#x00394;PEST-FLAG WT or kinase-dead using Lipofectamine 2000 (Invitrogen). After 24&#x02009;h, cells were harvested, treated with lysis buffer (20&#x02009;mM Tris/HCl, pH 7.5, 150&#x02009;mM NaCl, 0.5% Triton X-100, 1&#x02009;mM DTT, 2&#x02009;mM MgCl₂ and 1/1,000 protease inhibitor cocktail (Nacalai Tesque)) and the lysates were immunoprecipitated with beads conjugated to FLAG antibodies. The beads were washed four times with lysis buffer supplemented with additional 500&#x02009;mM NaCl and twice with kinase buffer (20&#x02009;mM Tris HCl (pH 7.5), 150&#x02009;mM NaCl and 1&#x02009;mM DTT). The beads were then incubated with bacterially expressed recombinant proteins of STIL fragments thereof in 30&#x02009;&#x003bc;l kinase buffer containing 10&#x02009;mM MgCl₂ and 30&#x02009;&#x003bc;M ATP without or with 5&#x02009;&#x003bc;&#x02009;Ci [&#x003b3;-³²P] ATP. Kinase reactions were performed at 30&#x02009;&#x000b0;C for 15&#x02013;90&#x02009;min and terminated by adding SDS sample buffer. Proteins were separated by SDS&#x02013;PAGE, stained with SimplyBlue Safe (Invitrogen) and phosphorylation was visualized by autoradiography (Typhoon FLA 9000, GE Healthcare). After the kinase reaction, the resulting materials were subsequently processed for <italic>in vitro</italic> binding assay with MBP-HsSAS-6 proteins. <italic>In vitro</italic> kinase assays for <xref ref-type="fig" rid="f3">Fig. 3a</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3b</xref> were repeated three times.</p><p>For <italic>in vitro</italic> MBP pull-down assays in <xref ref-type="fig" rid="f2">Fig. 2d,e</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2c</xref>, after the kinase reaction, the supernatant and eluted fraction with FLAG peptides (Sigma) both of which contained phosphorylated STIL N3C proteins were collected. For other <italic>in vitro</italic> MBP pull-down assays, only the supernatant was collected. The resulting fractions were then incubated with MBP-HsSAS-6 full length purified from baculovirus/insect cell expression system and thereafter pulled down using amylose resin (New England Biolabs). Input and the protein complexes pulled down with the resins were analysed by western blotting using STIL, HA, FLAG or HsSAS-6 antibodies.</p><p>DNAs encoding fragments of human STIL were cloned in pGEX system vectors (GE Healthcare) encoding for glutathione S-transferase (GST) tags. The recombinant protein expression of the fragments was performed in <italic>E. coli</italic> strain BL21 gold (DE3) in LB medium. Protein expression was induced at 22&#x02009;&#x000b0;C by addition of 0.3&#x02009;mM isopropyl-&#x003b2;-<sc>D</sc>-thiogalactoside and allowed to proceed for 18&#x02009;h. Cell pellets were lysed by lysozyme treatment and sonication, resuspended in lysis buffer containing 50&#x02009;mM Tris HCl (pH 7.5), 150&#x02009;mM NaCl, 2&#x02009;mM MgCl₂, 5&#x02009;mM EDTA, 1&#x02009;mM DTT, 1:500 protease inhibitor cocktail (Nacalai Tesque) and 0.5% Triton X-100. The lysates were incubated with Glutathion sepharose beads (GE Healthcare). The beads were then washed 10 times with lysis buffer supplemented with additional 500&#x02009;mM NaCl. For preparing STIL fragments, proteins were eluted from the beads by removal of the GST tags by PreScission Protease (GE Healthcare) in a cleave buffer containing 20&#x02009;mM Tris HCl (pH 7.5), 150&#x02009;mM NaCl and1&#x02009;mM DTT. For <italic>in vitro</italic> MBP pull-down assays in <xref ref-type="fig" rid="f2">Figs 2g</xref> and <xref ref-type="fig" rid="f4">4b</xref>, and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 2e and 6c,d</xref>, GST-fused STIL fragments were eluted by 10&#x02009;mM glutathione in the cleave buffer.</p><p>Since it was not feasible to obtain soluble fraction of GST-fused HsSAS-6 full-length proteins from bacteria, we generated MBP-fused HsSAS-6 full-length proteins using Baculovirus Expression System with Gateway Technology (Invitrogen). In brief, DNA encoding HsSAS-6 full length was cloned into a modified pENTR-1A-5Myc-MBP vector. The expression clone was obtained from the entry clone and a pDEST vector through gateway cloning strategy. The recombinant bacmid was then obtained from DH10Bac <italic>E. coli</italic> cells transformed with the expression vector, and subsequently transfected to Sf9 insect cells with Cellfectin II reagent (Invitrogen). The titre of recombinant baculovirus was amplified by repeated infection to Sf9 cells. MBP-5Myc-HsSAS-6 full-length proteins were purified from 1&#x02009;l (~2 &#x000d7; 10⁶&#x02009;ml^&#x02013;1) culture of the Sf9 cells infected with sufficiently amplified baculovirus for 3 days. The procedure for protein purification was similarly done with amylose resin as described above for GST-fusion proteins.</p></sec><sec disp-level="2"><title>Mass spectrometry</title><p>For MS analysis, to identify Plk4-phosphorylated residues of STIL, the STIL proteins phosphorylated by Plk4&#x00394;PEST-FLAG <italic>in vitro</italic> were digested into shorter peptides in solution by trypsin. The peptides were subsequently desalted and analysed by a nanoLC-linear ion trap-orbitrap mass spectrometer. MS analysis was repeated two times.</p></sec><sec disp-level="2"><title>Yeast two-hybrid analysis</title><p>Yeast strain L40 (a gift from Masato Kanemaki) was grown in complete medium (yeast extract peptone dextrose; (YPD)) and transformed with a modified version of the vectors pSM671 (bait) and pSM378 (prey; gifts from Satoru Mimura) that contained full length or fragments of Plk4 or STIL. Positive colonies were cultured on yeast plate without leucine and tryptophan (SD&#x02013;L/&#x02013;W) in the presence of histidine overnight. On the next day, cells were streaked on SD&#x02013;L/&#x02013;W without histidine plates supplemented with 50&#x02009;mM 3-amino-triazol. Two independent colonies were streaked per sample. Plates were placed at 30&#x02009;&#x000b0;C for 3 days. Yeast two-hybrid analysis was repeated at least three times.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>M.Oh. and D.K. designed the study; M.Oh., T.A., Y.N. and D.K. performed experiments; H.K.-H. and M.Oy. performed mass spectrometry analysis; H.G. and M.I. provided reagents for the baculovirus/sf9 expression system; M.Oh. and D.K. designed experiments and analysed data; and M.Oh. and D.K. wrote the manuscript, which was commented on by all authors.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Ohta, M. <italic>et al.</italic> Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole. <italic>Nat. Commun.</italic> 5:5267 doi: 10.1038/ncomms6267 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-8</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6267-s1.pdf"/></supplementary-material></sec>

Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins

<p>Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, <italic>SORT1/CELSR2/PSRC1</italic> and <italic>SLCO1B1</italic>, not previously identified in GWAS. Moreover, we confirm the previously described associations with <italic>APOE</italic> and <italic>LPA.</italic> Our findings advance the understanding of the pharmacogenetic architecture of statin response.</p>

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A.</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Tardif</surname><given-names>Jean-Claude</given-names></name><xref ref-type="aff" rid="a11">11</xref></contrib><contrib contrib-type="author"><name><surname>Colhoun</surname><given-names>Helen M.</given-names></name><xref ref-type="aff" rid="a4">4</xref><xref ref-type="aff" rid="a72">72</xref></contrib><contrib contrib-type="author"><name><surname>Hitman</surname><given-names>Graham</given-names></name><xref ref-type="aff" rid="a73">73</xref></contrib><contrib contrib-type="author"><name><surname>Krauss</surname><given-names>Ronald M.</given-names></name><xref ref-type="aff" rid="a74">74</xref></contrib><contrib contrib-type="author"><name><surname>Wouter Jukema</surname><given-names>J</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a3">3</xref><xref ref-type="aff" rid="a75">75</xref><xref ref-type="aff" rid="a76">76</xref><xref ref-type="author-notes" rid="n3">&#x02021;</xref></contrib><contrib contrib-type="author"><name><surname>Caulfield</surname><given-names>Mark J.</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a6">6</xref><xref ref-type="aff" rid="a8">8</xref><xref ref-type="author-notes" rid="n3">&#x02021;</xref></contrib><contrib contrib-type="author"><collab>Membership of Wellcome Trust Case Control Consortium

Nature Communications

<p>The 3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are widely prescribed and are highly effective in the management and prevention of cardiovascular disease. Statin therapy results in a lowering of low-density lipoprotein cholesterol (LDL-C) levels by up to 55%<xref ref-type="bibr" rid="b1">1</xref> and a 20&#x02013;30% reduction of cardiovascular events<xref ref-type="bibr" rid="b2">2</xref>. Despite the clinical efficacy of statins in a wide range of patients<xref ref-type="bibr" rid="b2">2</xref>, interindividual variability exists with regard to LDL-C-lowering response as well as efficacy in reducing major cardiovascular events<xref ref-type="bibr" rid="b3">3</xref>. The suggestion that some of this variability may be due, in part, to common pharmacogenetic variation is supported by previous studies that have identified genetic variants associated with differential LDL-C response to statin therapy<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>.</p><p>A small number of genome-wide association studies (GWAS) have previously identified loci associated with statin response on a genome-wide level. A GWAS in the JUPITER trial identified three genetic loci, <italic>ABCG2</italic> (rs2199936), <italic>LPA</italic> (rs10455872) and <italic>APOE</italic> (rs7412), that were associated with percentage LDL-C reduction following rosuvastatin therapy<xref ref-type="bibr" rid="b7">7</xref>. In the CARDS and ASCOT studies, single nucleotide polymorphisms (SNPs) at <italic>LPA</italic> (rs10455872) and <italic>APOE</italic> (rs445925 and rs4420638) were associated with LDL-C response to atorvastatin treatment<xref ref-type="bibr" rid="b8">8</xref>. A combined GWAS in three statin trials identified a SNP within <italic>CLMN</italic> (rs8014194) that is associated with the magnitude of statin-induced reduction in plasma cholesterol<xref ref-type="bibr" rid="b9">9</xref>. However, two other GWAS identified no genetic determinants of LDL-C response to statin therapy at a genome-wide significant level<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b10">10</xref>.</p><p>On the basis of these studies, as well as previous candidate gene studies<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b6">6</xref>, the only genetic variants that have been consistently identified to be associated with variation in LDL-C response to statin therapy, irrespective of statin formulation, are located at or nearby <italic>APOE</italic> and <italic>LPA</italic>. To determine whether additional loci may influence LDL-C response to statins, we formed the Genomic Investigation of Statin Therapy (GIST) consortium and conducted a pharmacogenetic meta-analysis using GWAS data sets from randomized controlled trials (RCTs) and observational studies. We identify two loci not previously identified in GWAS, <italic>SORT1/CELSR2/</italic>PSRC1 and <italic>SLCO1B1.</italic> In addition, we confirm the associations within the <italic>APOE</italic> and <italic>LPA</italic> genes. These findings will extend the knowledge of the pharmacogenetic architecture of statin response.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>First-stage meta-analysis</title><p>The GIST consortium includes 6 RCTs (<italic>n</italic>=8,421 statin recipients) and 10 observational studies (<italic>n</italic>=10,175 statin recipients) that participated in the first stage (see Methods; <xref ref-type="supplementary-material" rid="S1">Supplementary Tables 1 and 2</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Notes 1 and 2</xref>). To search for genetic variants associated with differential LDL-C response to statin therapy, each study independently performed a GWAS among statin users, using the difference between the natural log-transformed LDL-C levels on- and off-treatment as the response variable (see Methods).</p><p>The first-stage meta-analysis identified three loci, including 13 SNPs, that attained genome-wide significance (<italic>P</italic>&#x0003c;5 &#x000d7; 10^&#x02212;8) for association with LDL-C response to statin treatment (<xref ref-type="fig" rid="f1">Fig. 1</xref>; <xref ref-type="table" rid="t1">Table 1</xref>). The most significant association was for a SNP on chromosome 19, at <italic>APOE</italic> (rs445925, minor allele frequency (MAF)=0.098, <italic>&#x003b2;</italic>=&#x02212;0.043, s.e.=0.005, <italic>P</italic>=1.58 &#x000d7; 10^&#x02212;18; <xref ref-type="fig" rid="f2">Fig. 2a</xref>), indicating that carriers of the rs445925 SNP respond to statins with an additional 4.3% increase per allele in LDL-C lowering effect compared with non-carriers. The second strongest association was with a SNP at <italic>LPA</italic> on chromosome 6 (rs10455872, MAF=0.069, <italic>&#x003b2;</italic>=0.041, s.e.=0.006, <italic>P</italic>=1.95 &#x000d7; 10^&#x02212;11; <xref ref-type="fig" rid="f2">Fig. 2b</xref>), indicating a 5.9% smaller LDL-C lowering per minor allele for carriers of the SNP compared with non-carriers. Associations at both loci have previously been described<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>. A third genome-wide significant association was found with a SNP at <italic>RICTOR</italic> on chromosome 5 (rs13166647, MAF=0.230, <italic>&#x003b2;</italic>=&#x02212;0.253, s.e.=0.046, <italic>P</italic>=4.50 &#x000d7; 10^&#x02212;8), although genotypes for this SNP were only available in two studies within the first stage (<italic>n</italic>=2,144).</p></sec><sec disp-level="2"><title>Second-stage meta-analysis</title><p>We selected 246 SNPs with <italic>P</italic> &#x0003c;5 &#x000d7; 10^&#x02212;4 from 158 loci for further investigation in three additional studies comprising up to 22,318 statin-treated subjects (see Methods; <xref ref-type="supplementary-material" rid="S1">Supplementary Tables 1 and 5</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Note 3</xref>). This second stage confirmed the genome-wide significant associations between variations within the <italic>APOE</italic> and <italic>LPA</italic> loci and LDL-C response, as observed in the first stage (<xref ref-type="table" rid="t1">Table 1</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>). In addition, SNPs at two new loci with <italic>P</italic> values between 6.70 &#x000d7; 10^&#x02212;7 and 2.26 &#x000d7; 10^&#x02212;6 in the first phase were shown to be significantly associated with statin-induced LDL-C lowering after statin treatment in the total combined meta-analysis at a genome-wide level: <italic>SORT1/CELSR2/PSRC1</italic> (rs646776, <italic>&#x003b2;</italic>=&#x02212;0.013, s.e.=0.002, <italic>P</italic>=1.05 &#x000d7; 10^&#x02212;9 and rs12740374, <italic>&#x003b2;</italic>=&#x02212;0.013, s.e.=0.002, <italic>P</italic>=1.05 &#x000d7; 10^&#x02212;9; <xref ref-type="fig" rid="f2">Fig 2c</xref>) and <italic>SLCO1B1</italic> (rs2900478, <italic>&#x003b2;</italic>=0.016, s.e.=0.003, <italic>P</italic>=1.22 &#x000d7; 10^&#x02212;9; <xref ref-type="fig" rid="f2">Fig 2d</xref>), indicating an additional 1.5% increase per allele in LDL-C lowering effect for carriers of the <italic>SORT1/CELSR2/PSRC1</italic> SNP and a 1.6% smaller LDL-C lowering per minor allele for carriers of the <italic>SLCO1B1</italic> SNP.</p><p>The six next-ranked SNPs with <italic>P</italic> values just below 5 &#x000d7; 10^&#x02212;8 in the combined meta-analysis, including the two SNPs at <italic>RICTOR</italic> (rs13166647 and rs13172966), were selected for additional genotyping in the Scandinavian ASCOT participants (see Methods). None of these six SNPs reached genome-wide significance after this additional genotyping (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 6</xref>). Therefore, our overall genome-wide significant findings were the SNPs at <italic>APOE</italic>, <italic>LPA</italic>, <italic>SORT1/CELSR2/PSRC1</italic> and <italic>SLCO1B1</italic>.</p></sec><sec disp-level="2"><title>Subfraction analyses</title><p>To extend our results for the novel GWAS finding <italic>SORT1/CELSR2/PSRC1</italic>, we performed additional association analyses, using measurements of cholesterol levels in four LDL subfractions (large, medium, small and very small) from two of the trials in GIST, CAP and PRINCE (<xref ref-type="table" rid="t2">Table 2</xref>; see Methods). The minor allele of <italic>SORT1</italic> rs646776 was associated with greater statin-induced reductions in levels of all LDL subfractions, and there was a nonsignificant trend for larger effect sizes and greater statistical significance for lowering of small and very small LDL (<xref ref-type="table" rid="t2">Table 2</xref>). In contrast, the <italic>APOE</italic> SNP associated with greater LDL-C response to statins (rs445925) showed a small and nonsignificant association with change in very small LDL (<xref ref-type="table" rid="t2">Table 2</xref>). For the minor allele of rs2900478 (<italic>SLCO1B1</italic>), the borderline significant association with smaller magnitude of LDL-C reduction showed a trend for preferential association with larger versus smaller LDL subfractions. The lack of association of rs10455872 (<italic>LPA</italic>) with changes in LDL subfractions is consistent with evidence discussed below that this locus affects levels of lipoprotein(a) (Lp(a)) and not LDL particles. Using generalized estimating equations, we tested the association of log change in each of the LDL subfractions with interactions of the four SNPs. For very small LDL, the association with the rs646776 minor allele was significantly different from that of the other minor alleles (<italic>P</italic>=0.03 after adjustment for multiple testing).</p></sec><sec disp-level="2"><title>Effects of off-treatment LDL-C</title><p>To demonstrate that our findings for LDL-C response to statin treatment are unlikely to be explained through associations with baseline LDL-C levels, we performed a number of additional analyses (see Methods). First, <xref ref-type="supplementary-material" rid="S1">Supplementary Table 7</xref> shows regression coefficients for baseline-adjusted and measurement noise-corrected estimates of the direct effect of genotype on on-treatment LDL-C at the strongest SNPs in the GIST meta-analysis (<italic>P</italic>&#x0003c;1 &#x000d7; 10^&#x02212;8), which were available in the CARDS data set. Correcting our effect size estimate further and modelling measurement noise at baseline reduced the apparent effect only slightly for all the markers, suggesting that there is little effect of measurement noise. Next, within the JUPITER trial, additional analyses were performed to determine whether there was an interaction between LDL-C change and statin or placebo allocation. <xref ref-type="supplementary-material" rid="S1">Supplementary Table 8</xref> shows significant <italic>P</italic> values for interaction (all &#x0003c;5 &#x000d7; 10^&#x02212;2) for SNPs at the four genome-wide significant loci in the GIST meta-analysis, also suggesting that genetic effects on baseline LDL-C as manifested in the placebo group contribute at most only in part to genetic effects on LDL-C response in the statin group.</p></sec><sec disp-level="2"><title>Genome-Wide Conditional Analysis</title><p>To investigate whether there were multiple SNPs within any gene and multiple loci associated with differential LDL-C lowering to statin therapy, we performed a conditional analysis across the genome using the summary statistics of the combined meta-analysis. The results of the Genome-Wide Conditional Analysis (GWCA; see Methods; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 9</xref>) showed 14 SNPs independently associated with statin response and these explained ~5% of the variation in LDL-C response to statin treatment. Of the 14 independent SNPs, 6 were genome-wide significant in the combined GWAS meta-analysis (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>).</p></sec><sec disp-level="2"><title>Previous findings</title><p>In <xref ref-type="supplementary-material" rid="S1">Supplementary Table 10</xref>, we performed a look-up in our GWAS meta-analysis for SNPs previously described in the literature (NHGRI Catalogue<xref ref-type="bibr" rid="b11">11</xref> of Published GWAS and Candidate gene studies) to be associated with statin response, besides the loci associated at a genome-wide level in the current study. None of these SNPs was associated with statin response in our GWAS after correcting for multiple testing.</p></sec><sec disp-level="2"><title>Functional analyses</title><p>Functional characterization of the 246 SNPs selected for the second stage was performed using a range of bioinformatics tools (see Methods). A total of 420 expression quantitative trait loci (eQTL) associations were identified across a wide range of tissues (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 1</xref>), which comprised 67 independent gene eQTL associations. Eleven genes, including <italic>APOE</italic>, <italic>SORT1</italic>, <italic>CELSR2</italic> and <italic>PSRC1</italic>, showed eQTLs in liver, which considering its primary role in mediating statin-induced LDL reduction may be particularly relevant to statin response. Putative gene eQTLs were combined with genes annotated to variants in linkage disequilibrium (LD) with LDL-C response-associated variants, resulting in a list of 185 candidate gene loci, defined by 2,681 SNPs (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 2 and 3</xref>). To identify statin responsive genes among the candidate loci, gene expression data measured in response to statin treatment in a range of cell lines was retrieved from the Connectivity Map resource<xref ref-type="bibr" rid="b12">12</xref> (see Methods). Five genes (<italic>APOE, BRCA1, GRPEL1, ADRB2</italic> and <italic>ETV1</italic>) showed convincing evidence of statin responsiveness on the basis of greater than twofold differential expression in response to statin treatment. Eight genes showed suggestive evidence (1.5- to 2-fold change; <italic>TOMM40</italic>, <italic>SREBP1</italic>, <italic>PSRC1</italic>, <italic>BCL3</italic>, <italic>BCAM</italic>, <italic>ANK3</italic>, <italic>SIVA1</italic> and <italic>RANBP9</italic>; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>).</p><p>Finally, involvement in statin response was investigated at a pathway level using GeneGo Metacore (Thomson Reuters<xref ref-type="bibr" rid="b13">13</xref>). Briefly, 87 literature-reported genes linked to statin response were combined with the 185 candidate gene loci reported here (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>). A conservative network of direct interactions was constructed between query genes (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 4</xref>). The network included 24 genes located in the LDL-C-associated loci (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). Collectively, our functional and pathway analysis confirms a strong biological and functional role in statin response for several strongly associated gene loci, including <italic>APOE/TOMM40/PVRL2</italic> and <italic>SORT1/CELSR2/PSRC2.</italic></p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>We have performed a meta-analysis of GWAS including more than 40,000 subjects, investigating genetic variants associated with variation in LDL-C lowering on statin treatment independent from associations with baseline LDL-C. We identified four loci at genome-wide significance, including the previously identified <italic>APOE</italic> and <italic>LPA,</italic> and the novel GWAS loci <italic>SORT1/CELSR2/PSRC1</italic> and <italic>SLCO1B1</italic>.</p><p>Nine SNPs in the <italic>APOE</italic> gene region reached genome-wide significance for LDL-C response. The minor allele of the lead SNP rs445925, which is a proxy for the apoE &#x003b5;2 protein variant defining SNP rs7412 (ref. 14)<xref ref-type="bibr" rid="b14">14</xref>, was associated with a larger LDL-C-lowering response to statins compared with carriers of the major allele. The magnitude and direction of the effect size was similar to previously reported findings for the rs445925 variant in the GWAS study performed in CARDS and ASCOT<xref ref-type="bibr" rid="b8">8</xref> and of the SNP rs7412 in JUPITER<xref ref-type="bibr" rid="b7">7</xref>. Since the apoE &#x003b5;2 protein results in increased hepatic cholesterol synthesis, it may also predispose to stronger inhibition of cholesterol synthesis by statin treatment<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b10">10</xref>.</p><p>Three independent SNPs at <italic>LPA</italic> were significantly associated with LDL-C response to statins. The minor G allele of the lead SNP rs10455872 was associated with smaller LDL-C reduction than the major allele. This result was similar to the previous GWAS findings for this SNP in the JUPITER trial and the combined ASCOT and CARDS study<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>. The rs10455872 SNP was strongly associated with the KIV-2 copy number variant in Lp(a), which encodes variability in apo(a) size and is responsible for ~30% of variance in Lp(a) levels<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b15">15</xref>. Furthermore, rs10455872 was shown to be strongly associated with plasma Lp(a) levels<xref ref-type="bibr" rid="b16">16</xref>. Standard assays of LDL-C, as well as the Friedewald formula, include cholesterol that resides in Lp(a)<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b8">8</xref>. Carriers of this <italic>LPA</italic> variant are characterized by higher Lp(a) levels and a larger proportion of their measured LDL-C resides in Lp(a) particles<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b10">10</xref>. Since statin therapy does not reduce the number of Lp(a) particles<xref ref-type="bibr" rid="b17">17</xref>, their presence attenuates the measured LDL-C response to statins.</p><p>Two SNPs at <italic>SORT1/CELSR2/PSRC1</italic> (rs646776 and rs12740374) on chromosome 1p were associated with an enhanced statin LDL-C response. A similar association was previously observed in a large candidate gene study in HPS<xref ref-type="bibr" rid="b6">6</xref>; however, we demonstrate this finding now first at a genome-wide significance level. The minor allele of rs12740374 has been shown to generate a binding site for the transcription factor C/EBPa<xref ref-type="bibr" rid="b18">18</xref>. Transcription results in upregulation of hepatic expression of three genes at this locus, <italic>SORT1</italic>, <italic>CELSR2</italic> and <italic>PSRC1</italic> (ref. <xref ref-type="bibr" rid="b18">18</xref>), which we also showed in our eQTL analysis (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 1</xref>). Of these, <italic>SORT1</italic> is most notable, in that it encodes the multifunctional intracellular trafficking protein sortilin, which has been shown to bind tightly to apoB<xref ref-type="bibr" rid="b19">19</xref>. Sortilin-induced lowering of plasma LDL-C results from two mechanisms: reduced secretion of apoB-containing precursors, and, perhaps of greater importance, increased hepatic LDL uptake via binding to sortilin at the cell surface, with subsequent internalization and lysosomal degradation<xref ref-type="bibr" rid="b19">19</xref>. Notably, the minor allele of rs646776 is preferentially associated with lower levels of small and very small LDL (<xref ref-type="table" rid="t2">Table 2</xref>), suggesting that sortilin is of particular importance for regulating levels of these particles<xref ref-type="bibr" rid="b18">18</xref>. Smaller LDL subfractions have been shown to be relatively enriched in particles with reduced LDL receptor binding affinity and cellular uptake<xref ref-type="bibr" rid="b20">20</xref>, a property that may contribute to their associations with increased risk for cardiovascular disease<xref ref-type="bibr" rid="b21">21</xref>. This property may also underlie the diminished efficacy of statins for reduction of these particles (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>)<xref ref-type="bibr" rid="b22">22</xref>, since statins act to reduce LDL-C levels to a large extent by increasing LDL receptor expression as a result of upregulation of the transcription factor SREBP2, whereas <italic>SORT1</italic> is not regulated by this mechanism. Hence, the greater statin-mediated reduction of LDL-C among carriers of the rs646776 minor allele could be attributed to relative depletion of LDL particles dependent on sortilin for clearance and hence a residually greater proportion of those LDL particles whose uptake is more dependent on the LDL receptor than on sortilin.</p><p>Notably, the strong association of rs646776 with statin-induced reductions in small and very small LDL particles contrasts to the weaker associations of changes in these particles with rs445925, likely the result of differing mechanisms underlying the effects of these SNPs on statin response. As noted above, rs445925 is a proxy for the SNP defining the apoE &#x003b5;2 protein variant that is thought to predispose to heightened statin response as a result of greater statin inhibition of cholesterol synthesis and hence upregulation of SREBP and LDL receptor activity.</p><p>The <italic>SLCO1B1</italic> rs2900478 minor allele was associated with a smaller LDL-C reduction in response to statin treatment. <italic>SLCO1B1</italic> encodes the organic anion-transporting polypeptide OATP1B1 and facilitates the hepatic uptake of statins<xref ref-type="bibr" rid="b23">23</xref>. SNP rs2900478 is in strong LD (<italic>r</italic>²=0.89) with rs4149056, which represents the Val174Ala substitution resulting in complete loss of function. In the HPS trial, which used simvastatin, this candidate gene SNP was associated with a 1% lower LDL-C reduction per allele<xref ref-type="bibr" rid="b6">6</xref>. Single-dose studies have shown that the observed area under the curve of plasma level of active simvastatin after a dose of 40&#x02009;mg was 221% higher in rs4149056 CC homozygotes compared with rs4149056 TT homozygotes, as compared with atorvastatin 20&#x02009;mg (144% higher for CC versus TT) and rosuvastatin 40&#x02009;mg (117% higher for CC versus TT)<xref ref-type="bibr" rid="b24">24</xref>. This finding results from the slower hepatic uptake of statins caused by the genetic variant, which would also be expected to result in a reduction in the cholesterol-lowering effect<xref ref-type="bibr" rid="b25">25</xref>. In a GWAS of the genetic risk factors for simvastatin-induced myopathy, <italic>SLCO1B1</italic> showed the strongest association<xref ref-type="bibr" rid="b25">25</xref>. Homozygous carriers of the <italic>SLCO1B1</italic> variant had a 16.9 times higher risk for myopathy compared with non-carriers. This might have led to a decrease in study medication adherence, and consequently a decreased effect on LDL-C in carriers of this SNP. In addition, previous analysis in the GoDARTS study showed that the effect of the <italic>SLCO1B1</italic> gene on statin efficacy was abolished after removal of individuals who showed signs of intolerance<xref ref-type="bibr" rid="b26">26</xref>.</p><p>GWCA identified three independent loci in the <italic>APOE</italic> gene region and two loci in the <italic>LPA</italic> gene region (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 9</xref>). GWCA also showed several other loci with <italic>P</italic> &#x0003c;5 &#x000d7; 10^&#x02212;8 that were not GWAS significant on single-SNP analysis (<italic>HGD</italic>, <italic>RNF175</italic>, <italic>ISCA1L-HTR1A</italic>, <italic>GLIS3-SLC1A1</italic>, <italic>LOC100128657</italic>, <italic>NKX2-3-SLC25A28</italic> and <italic>PELI2</italic>). These findings will require replication in independent, larger data sets. The significant SNPs in the GWCA analysis explained ~5% of the variation in LDL-C response to statin treatment. Whether this 5% is clinically relevant should be investigated by other studies. For example, it would be of interest to investigate whether this differential LDL-C lowering is also associated with differential event reduction by statin treatment.</p><p>In the current study, we combined the results of 6 randomized clinical trials and 10 observational studies in the first stage. This approach resulted also in combining several types of statins, since different statins were studied in the trials and within the observational studies (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). This, and the variation in statin dosage during follow-up for an individual, is a limitation of the current study, since, for example, the impact of the <italic>SLCO1B1</italic> variant on statin pharmacogenetics is known to be highly dependent on statin type and dose<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b27">27</xref>. To overcome this limitation, the individual study analyses were adjusted for statin dose. Dividing the actual statin dose given by the statin-specific dose equivalent (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>) gives the statin-adjusted equivalent based on the daily dosages required to achieve a mean 30% LDL-C reduction. Using this table, we made the different statin dosages and types comparable within the studies. To correct for between-study variance, we used a fixed effect meta-analysis with inverse variance weighting. Since we observed that the <italic>SLCO1B1</italic> gene was genome-wide significantly associated with LDL lowering, this highlights the thoroughness of our analytical approach, in which the analyses were correctly adjusted for the type and dose of statins used (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). Moreover, a comparison of the estimates of the SNPs between the RCTs (where there are no intra-individual differences in dosages) with the estimates of the SNPs in the observational studies showed large homogeneity between the estimates in the various study designs (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>), indicating that our adjustment for dosage seems to be sufficient within this study.</p><p>Another possible limitation of the current study is the influence of the identified genetic variants on baseline LDL-C levels. In pharmacogenetic studies investigating the LDL-C-lowering response to statins, it is important to eliminate the effect of association between the genetic variant and baseline LDL-C levels, since those findings may confound the response to treatment associations. Previous large GWAS studies have shown strong associations between baseline LDL-C levels and genetic variants in <italic>SORT1/CELSR2/PSRC1</italic>, <italic>APOE</italic> and <italic>LPA</italic><xref ref-type="bibr" rid="b28">28</xref>. To eliminate those possible confounding effects, our response to treatment analyses were adjusted for baseline LDL-C levels. In addition, additional analysis in CARDS and JUPITER suggests no or little influence of genetic associations with baseline LDL-C on the genetic effects on LDL-C-lowering response.</p><p>In conclusion, this study is the largest meta-analysis of GWAS for LDL-C response to statin therapy conducted to date. Our results demonstrate that apart from the previously identified <italic>APOE</italic> and <italic>LPA</italic> loci, two new loci, <italic>SORT1/CELSR2/PSRC1</italic> and <italic>SLCO1B1</italic>, also have a modest but genome-wide significant effect on LDL-C response. The minor alleles of the <italic>APOE</italic> rs445925 and <italic>SORT1</italic>/<italic>CELSR2/PSRC1</italic> rs646776 SNPs were associated with a larger statin response, whereas the minor alleles of the <italic>LPA</italic> rs10455872 and <italic>SLCO1B1</italic> rs2900478 SNPs were associated with a smaller statin response. Our findings advance the understanding of the pharmacogenetic architecture of statin response.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Study populations</title><p>The meta-analysis was conducted in the GIST consortium, which includes data from 8 randomized controlled statin trials (RCTs) and 11 prospective, population-based studies. The initial analysis (first stage) was performed in 8,421 statin-treated subjects from 6 RCTs (ASCOT, CARDS, CAP, PRINCE, PROSPER and TNT) and 10,175 statin-treated subjects from 10 observational studies (AGES, ARIC, BioVU, CHS, FHS, GoDARTS I, GoDARTS II, Health ABC, HVH and MESA). Further investigation (second stage) was performed in 21,975 statin-treated subjects from two randomized trials (HPS and JUPITER) and one observational study (Rotterdam Study). Six SNPs were additionally genotyped in the Scandinavian participants of the ASCOT study. The details of the first- and second-stage studies can be found in the <xref ref-type="supplementary-material" rid="S1">Supplementary Tables 1 and 2</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Notes 1 and 2</xref>.</p></sec><sec disp-level="2"><title>Subjects</title><p>Response to statin treatment was studied in statin-treated subjects only and not in those treated with placebo. Subjects included in the observational studies&#x02019; analysis should be treated with statins and have LDL-C measurements before and after start of statin treatment. Subjects of reported or suspected non-European ancestry were excluded. All participants gave written informed consent and the study was approved by all institutional ethics committees.</p></sec><sec disp-level="2"><title>Outcome measurements</title><p>The response to statin treatment was defined as the difference between the natural log-transformed on- and off-treatment LDL-C levels. The beta of the corresponding regression thus reflects the fraction of differential LDL lowering in carriers versus non-carriers of the SNP. For observational studies, the on-treatment LDL-C levels were taken into account for all kinds of prescribed statins, at any dosage, for any indication and for at least 4 weeks before measurement. Characteristics of on- and off-treatment LDL-C levels and statins used in each study are shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>. For each individual, at least one off-treatment LDL-C measurement and at least one on-treatment LDL-C measurement were required. When multiple on- or off-treatment measurements were available, the mean of the cholesterol measurements was used. Subjects with missing on- or off-treatment measurements were excluded, with the exception of the GoDARTS cohorts for which missing off-treatment LDL-C levels were estimated using imputation methods (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 2</xref>). In the HPS, proportional LDL-C response was defined by the changes in natural log lipid levels from the screening visit before starting statin therapy to the randomization visit<xref ref-type="bibr" rid="b6">6</xref>.</p></sec><sec disp-level="2"><title>Genotyping and imputation</title><p>Genotyping, quality control, data cleaning and imputation were performed independently in each study using different genetic platforms and software as outlined in <xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>. In all studies, genotyping was performed using Illumina, Affymetrix or Perlegen genotyping arrays, and MACH, Impute or BIMBAM software was used for imputation.</p></sec><sec disp-level="2"><title>GWAS analysis</title><p>Each study independently performed the GWAS on the difference between natural log-transformed on- and off-treatment LDL-C levels. To control for possible associations with off-treatment LDL-C levels, analyses were adjusted for the natural log-transformed off-treatment LDL-C level. An additive genetic model was assumed and tested using a linear regression model. For imputed SNPs, regression analysis was performed onto expected allele dosage. Analyses were additionally adjusted for age-, sex- and study-specific covariates (for example, ancestry principal components or country). Analyses in the observational studies were, if available, additionally adjusted for the statin dose by the natural logarithm of the dose equivalent as defined in <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>. This table shows the dose equivalent per statin type; dividing the statin dosage of an individual by the dose equivalent shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref> will give the adjusted statin dosage.</p></sec><sec disp-level="2"><title>Quality control and meta-analysis</title><p>Centrally, within each study, SNPs with MAF &#x0003c;1% or imputation quality &#x0003c;0.3 were excluded from the analysis. QQ-plots were assessed for each study to identify between-study differences (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). The software package METAL was used for performing the meta-analysis ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.sph.umich.edu/csg/abecasis/Metal/index.html">http://www.sph.umich.edu/csg/abecasis/Metal/index.html</ext-link>). A fixed effects, inverse variance weighted approach was used. Using an inverse variance weighted meta-analysis will give smaller weights to studies with large s.e.. To correct for possible population stratification, genomic control was performed by adjusting the within-study findings and the meta-analysis results for the genomic inflation factor.</p></sec><sec disp-level="2"><title>Second stage</title><p>SNPs with <italic>P</italic> values &#x0003c;5 &#x000d7; 10^&#x02212;4 in the first-stage meta-analysis were selected for further investigation in a second stage. A maximum of two SNPs per locus were selected, based on statistical significance, except for the <italic>APOE</italic> locus, for which all genome-wide significant associated SNPs were selected for validation. A total of 246 SNPs, within 158 independent loci, were selected for the second stage, which was performed in the JUPITER trial, HPS study and the Rotterdam Study, which all had GWAS data and response to statin treatment available. For 2 of the 246 SNPs, a proxy was used in the JUPITER trial, and 31 SNPs were not available, nor was a proxy SNP. HPS provided data on 151 directly genotyped SNPs from GWAS and IPLEX experiments, including 48 of the requested SNPs and 103 proxy SNPs (<italic>r</italic>²&#x0003e;0.8). Analysis in HPS was not adjusted for ln baseline LDL-C levels. In addition, the number of subjects with data varied from SNP-to-SNP and ranges from ~4,000 for variants with GWAS data to ~18,000 for some candidate genes. Results of the first and second stage were combined using fixed effects, inverse variance weighted meta-analysis and analysed by METAL. As a third stage, six SNPs with <italic>P</italic> values 5 &#x000d7; 10^&#x02212;8&#x0003c;<italic>P</italic>&#x0003c;5 &#x000d7; 10^&#x02212;7 in the combined meta-analysis were selected for additional genotyping in the Scandinavian participants of the ASCOT study. Kaspar assays were designed for four of the SNPs using the KBioscience Primerpicker software, and oligos were provided by Intergrated DNA technologies ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://eu.idtdna.com/site">http://eu.idtdna.com/site</ext-link>). Full Kaspar methodology is available from LGC SNP genotyping ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.lgcgenomics.com/genotyping/kasp-genotyping-reagents/">http://www.lgcgenomics.com/genotyping/kasp-genotyping-reagents/</ext-link>). Two SNPs (rs981844 and rs13166647) were genotyped using Taqman assays supplied by Life Technologies ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.lifetechnologies.com/uk/en/home.html">http://www.lifetechnologies.com/uk/en/home.html</ext-link>) using the standard Taqman protocol. Results of the additional genotyping were combined with results from the first and second stages using a fixed effects, inverse variance weighted meta-analysis and analysed by METAL.</p></sec><sec disp-level="2"><title>Determination of changes in LDL subfractions</title><p>LDL subclasses were analysed as described previously<xref ref-type="bibr" rid="b29">29</xref> using non-denaturing gradient gel electrophoresis of fasting plasma samples taken at baseline and after 6 weeks of simvastatin 40&#x02009;mg per day (CAP study, <italic>n</italic>=579) or 12 weeks of pravastatin 40&#x02009;mg per day (PRINCE study, <italic>n</italic>=1,284). Aliquots of 3.0&#x02009;ml of whole plasma were mixed 1:1 with a sampling buffer of 20% sucrose and 0.25% bromophenol blue. Electrophoresis of samples and size calibration standards was performed using 2&#x02013;14% polyacrylamide gradients at 150&#x02009;V for 3&#x02009;h following a 15-min pre-run at 75&#x02009;V. Gels were stained with 0.07% Sudan black for 1&#x02009;h and stored in a 0.81% acetic acid, 4% methanol solution until they were scanned by computer-assisted densitometry for determination of areas of LDL IVb (22.0&#x02013;23.2&#x02009;nm), LDL IVa (23.3&#x02013;24.1&#x02009;nm), LDL IIIb (24.2&#x02013;24.6&#x02009;nm), LDL IIIa (24.7&#x02013;25.5&#x02009;nm), LDL IIb (25.6&#x02013;26.4&#x02009;nm), LDL IIa (26.5&#x02013;27.1&#x02009;nm) and LDL I (27.2&#x02013;28.5&#x02009;nm). The cholesterol concentrations of the subfractions (mg&#x02009;dl^&#x02212;1 plasma) were determined by multiplying percent of the total stained LDL area for each subfraction by the LDL-C for that sample. For genetic association analyses, subfractions were grouped into large LDL (LDL I+IIa), medium LDL (LDL IIb), small LDL (LDL IIIa) and very small LDL (LDL IIIb+IVa+IVb) as described previously<xref ref-type="bibr" rid="b18">18</xref>. A generalized estimating equation method was used to test the association of log change with the interaction of the four SNPs by LDL subfraction.</p></sec><sec disp-level="2"><title>Effect of off-treatment LDL-C</title><p>Effects of genetic variation on treatment response as measured by on-treatment LDL-C could be mediated through effects on the off-treatment LDL-C. To evaluate whether genetic on-treatment LDL-C likely reflects residual effect on off-treatment LDL-C, it is necessary to adjust for the off-treatment LDL-C levels and to correct the maximum likelihood estimate of the adjusted effect of genotype on on-treatment value for the noise in off-treatment values (the noise is both random measurement error and intra-individual variation in usual LDL-C). This analysis was only carried out in CARDS in which multiple baseline measurements were available. From the rules of path analysis, we calculated the direct effect <italic>&#x003b3;</italic> of genotype on an on-treatment trait value as <italic>&#x003b2;</italic>&#x02212;<italic>&#x003b1;&#x003b4;</italic> (1&#x02212;<italic>&#x003c1;</italic>)/<italic>&#x003c1;</italic>, where <italic>&#x003b2;</italic> is the coefficient of regression for on-treatment trait value on genotype adjusted for measured off-treatment value, <italic>&#x003b1;</italic> is the coefficient of regression of baseline LDL on genotype, <italic>&#x003c1;</italic> is the intraclass correlation between replicate measurements of off-treatment values and <italic>&#x003b4;</italic> is the coefficient of regression for on-treatment value on observed off-treatment value<xref ref-type="bibr" rid="b8">8</xref>. For these calculations, we used <italic>&#x003c1;</italic>=0.8 as a plausible value for the intraclass correlation based on the within-person correlation in LDL-C values taken over two off-treatment visits in CARDS. The interaction of candidate SNPs with statin versus placebo allocation was assessed in the JUPITER trial, since this study was not involved in the first-stage meta-analysis. Regression models were applied to the combined population of statin- and placebo-treated subjects by including extra terms encoding placebo allocation and the product of placebo allocation with SNP minor allele dose<xref ref-type="bibr" rid="b7">7</xref>.</p></sec><sec disp-level="2"><title>GWCA using Genome-Complex Trait Analysis</title><p>There may be multiple causal variants in a gene and the total variation that could be explained at a locus may be underestimated if only the most significant SNP in the region is selected. To identify independent SNPs, we ideally can perform a conditional analysis, starting with the top associated SNP, across the whole genome followed by a stepwise procedure of selecting additional SNPs, one by one, according to their conditional <italic>P</italic> values. Such a strategy would allow the discovery of more than two associated SNPs at a locus. To identify independent SNPs across the genome-wide data, we used an approximate conditional and joint analysis approach implemented in Genome-Complex Trait Analysis (GCTA) software ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.complextraitgenomics.com/software/gcta/">http://www.complextraitgenomics.com/software/gcta/</ext-link>). We used summary-level statistics from the first- and second-stage-combined meta-analysis and LD corrections between SNPs estimated from CARDS GWAS data. SNPs on different chromosomes or more than 10&#x02009;Mb distant are assumed to be in linkage equilibrium. The model selection process in GCTA starts with the most significant SNP in the single-SNP meta-analysis across the whole genome with <italic>P</italic> value &#x0003c;5 &#x000d7; 10^&#x02212;7. In the next step, it calculates the <italic>P</italic> values of all the remaining SNPs conditional on the top SNP that have already been selected in the model. To avoid problems due to colinearity, if the squared multiple correlations between a SNP to be tested and the selected SNP(s) is larger than a cut-off value, such as 0.9, the conditional <italic>P</italic> value for that SNP will be set to 1. Select the SNPs with minimum conditional <italic>P</italic> value that is lower than the cut-off <italic>P</italic> value. Fit all the selected SNPs jointly in a model and drop the SNPs with the <italic>P</italic> value that is greater than the cut-off <italic>P</italic> value. This process is repeated until no SNPs can be added or removed from the model.</p></sec><sec disp-level="2"><title>Pathway analysis and construction of a statin response network</title><p>Genes showing evidence of association (based on direct association or LD (HapMap CEU <italic>r</italic>²&#x0003e;0.8)) were reviewed for evidence of involvement in statin response at a pathway level using GeneGo Metacore (Thomson Reuters (portal.genego.com)). A statin response network was constructed in two stages. First, all genes with a literature-reported involvement in statin response (based on Medical Subject Headings (MeSH)) were identified using GeneGo MetaCore (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>). Second, these genes were combined with all genes in associated loci (including genes in LD) and a network was constructed based on direct interactions only. By including direct interactions only, we created a conservative network of direct gene interactions that have been consistently linked to statin response in the literature.</p></sec><sec disp-level="2"><title>eQTL analysis</title><p>LDL-C-associated index SNPs (246 SNPs) were used to identify 1,443 LD proxy SNPs displaying complete LD (<italic>r</italic>²=1) across four HapMap builds in European ancestry samples (CEU) using the SNAP tool ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.broadinstitute.org/mpg/snap/">http://www.broadinstitute.org/mpg/snap/</ext-link>). The primary index SNPs and LD proxies were searched against a collected database of expression SNP (eSNP) results, including the following tissues: fresh lymphocytes<xref ref-type="bibr" rid="b30">30</xref>, fresh leukocytes<xref ref-type="bibr" rid="b31">31</xref>, leukocyte samples in individuals with Celiac disease<xref ref-type="bibr" rid="b32">32</xref>, whole-blood samples<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>, lymphoblastoid cell lines (LCL) derived from asthmatic children<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref>, HapMap LCL from three populations<xref ref-type="bibr" rid="b39">39</xref>, a separate study on HapMap CEU LCL<xref ref-type="bibr" rid="b40">40</xref>, additional LCL population samples<xref ref-type="bibr" rid="b41">41</xref><xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref> (Mangravite <italic>et al.</italic>, unpublished), CD19+ B cells<xref ref-type="bibr" rid="b44">44</xref>, primary phytohaemagglutinin-stimulated T cells<xref ref-type="bibr" rid="b41">41</xref>, CD4+ T cells<xref ref-type="bibr" rid="b45">45</xref>, peripheral blood monocytes<xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b46">46</xref><xref ref-type="bibr" rid="b47">47</xref>, CD11+ dendritic cells before and after <italic>Mycobacterium</italic> tuberculosis infection<xref ref-type="bibr" rid="b48">48</xref>, omental and subcutaneous adipose<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b49">49</xref>, stomach<xref ref-type="bibr" rid="b49">49</xref>, endometrial carcinomas<xref ref-type="bibr" rid="b50">50</xref>, ER+ and ER&#x02212; breast cancer tumour cells<xref ref-type="bibr" rid="b51">51</xref>, brain cortex<xref ref-type="bibr" rid="b46">46</xref><xref ref-type="bibr" rid="b52">52</xref><xref ref-type="bibr" rid="b53">53</xref>, prefrontal cortex<xref ref-type="bibr" rid="b54">54</xref><xref ref-type="bibr" rid="b55">55</xref>, frontal cortex<xref ref-type="bibr" rid="b56">56</xref>, temporal cortex<xref ref-type="bibr" rid="b53">53</xref><xref ref-type="bibr" rid="b56">56</xref>, pons<xref ref-type="bibr" rid="b56">56</xref>, cerebellum<xref ref-type="bibr" rid="b53">53</xref><xref ref-type="bibr" rid="b56">56</xref>, three additional large studies of brain regions including prefrontal cortex, visual cortex and cerebellum, respectively<xref ref-type="bibr" rid="b57">57</xref>, liver<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b58">58</xref><xref ref-type="bibr" rid="b59">59</xref>, osteoblasts<xref ref-type="bibr" rid="b60">60</xref>, ileum<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b61">61</xref>, lung<xref ref-type="bibr" rid="b62">62</xref>, skin<xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b63">63</xref> and primary fibroblasts<xref ref-type="bibr" rid="b41">41</xref>. Micro-RNA QTLs were also queried for LCL<xref ref-type="bibr" rid="b64">64</xref> and gluteal and abdominal adipose<xref ref-type="bibr" rid="b65">65</xref>. The collected eSNP results met the criteria for association with gene expression levels as defined in the original papers. In each case where a LDL-C-associated SNP or proxy was associated with a transcript, we further examined the strongest eSNP for that transcript within that data set (best eSNP), and the LD between the best eSNP and GIST-selected eSNPs to estimate the concordance of the LDL-C and expression signals.</p></sec><sec disp-level="2"><title>Statin response connectivity map analysis</title><p>The Connectivity Map (Cmap) data set is available at the Broad Institute ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.broadinstitute.org/cmap">www.broadinstitute.org/cmap</ext-link>) and contains more than 7,000 expression profiles representing 1,309 compounds used on five different cultured human cancer cell lines (MCF7, ssMCF7, HL60, PC3 and SKMEL5). We selected (prostate tumour-derived) PC3 cells as they showed the most responsiveness to statins at a genome-wide level. Four statins were included in our analysis, including pravastatin, atorvastatin, simvastatin and rosuvastatin. PC3 Instance reference files for each statin treatment were extracted (as defined by Lamb <italic>et al.</italic><xref ref-type="bibr" rid="b12">12</xref>), that is, a treatment associated to its control pair. Transcripts were considered to show evidence of differential expression with a fold change &#x0003e;2. A fold change &#x0003e;1.5 was considered to be suggestive of differential expression only.</p></sec><sec disp-level="2"><title>Exploration of functional impact among directly and indirectly associated variants</title><p>Genes and variants across all LDL-C-associated loci were investigated for evidence of functional perturbation using a range of bioinformatics tools and databases. Variants showing LD (CEU <italic>r</italic>²&#x0003e;0.8) with associated variants were explored for impact on coding gene function using Annovar<xref ref-type="bibr" rid="b66">66</xref> and regulatory function using a combination of HaploReg<xref ref-type="bibr" rid="b67">67</xref> and Regulomedb<xref ref-type="bibr" rid="b68">68</xref>, which both draw on comprehensive data from the Encyclopedia of DNA Elements (ENCODE)<xref ref-type="bibr" rid="b69">69</xref> and the NIH Roadmap Epigenomics consortium<xref ref-type="bibr" rid="b70">70</xref>. Building on the functional annotation, we also identified variants that were shown to mediate eQTLs. Genes in associated loci were also used to query the NIH connectivity map for evidence of differential expression in PC3 cell lines treated with pravastatin, simvastatin and rosuvastatin. By combining a wide range of functional data and pathway support, we were able to build up a view of genes with the highest level of support in statin response.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>I.P., S.T., H.A.D., M.R.B., X.L., H.R.W., D.I.C., K.Z., B.J.A., B.M.P., G.H., R.M.K., J.W.J. and M.J.C. constituted the writing and analysis group. I.P., S.T., H.A.D. and K.Z. performed quality control on the individual study summary results. I.P. and S.T. performed meta-analysis. I.P., H.A.D., M.R.B., X.L., H.R.W., D.I.C. and R.M.K. performed additional analyses. All analysis and writing group authors extensively discussed the analysis, results, interpretation and presentation of results. All authors contributed to the research and reviewed the manuscript.</p><p>Study concept and design of contributing studies by (PROSPER) J.W.J., D.J.S., B.M.B., I.F., N.S. and R.G.J.W.; (ASCOT) M.J.C., P.S., N.P., A.S., D.C.S. and E.O.; (CARDS) H.A.D., H.M.C., P.M.M., J.B., P.N.D., A.D. and G.H.; (PARC) X.L., Y.-D.I.C., J.I.R. and R.M.K.; (TNT) J.J.P.K.; (AGES) L.J.L., T.B.H. and V.G.; (ARIC) C.L.A., E.A.W., T.S., E.B. and C.M.B.; (BioVU) Q.F., W.-Q.W., R.A.W. and J.C.D.; (CHS, HVH) N.S., K.R., T.L., J.I.R., B.M.P. and S.R.H.; (FHS) L.A.C. and V.R.; (GoDARTS) C.N.A.P. and H.M.C.; (HABC) Y.L.; (MESA) X.G., S.R.H., W.P. and J.I.R.; (Rotterdam Study) C.E.d.K., B.H.S., A.G.U., A.H. and F.R.; and (JUPITER) D.I.C., B.J.B., F.N. and P.M.R.</p><p>Phenotype data acquisition of contributing studies by (PROSPER) J.W.J., D.J.S., B.M.B., I.F., A.J.M.d.C., N.S. and R.G.J.W.; (ASCOT) M.J.C., P.B.M., P.S., N.P., A.S., D.C.S., E.O. and S.S.H.; (CARDS) H.A.D., H.M.C., P.M.M., J.B., P.N.D., A.D. and G.H.; (PARC) X.L., Y.-D.I.C., J.I.R. and R.M.K.; (TNT) J.J.P.K.; (AGES) G.E.; (ARIC) C.M.B.; (BioVU) W.W.; (CHS, HVH) K.L.W., J.C.B., A.M.A., N.L.S., B.M.P. and S.R.H.; (FHS) L.A.C., C.J.O., V.R.; (GoDARTS) C.N.A.P., L.A.D., K.Z., A.D., A.M. and H.M.C.; (HABC) D.M.H. and S.B.K.; (MESA) W.P. and J.I.R.; (Rotterdam Study) C.E.K., B.H.S., A.H. and O.H.F.; (JUPITER) D.I.C., F.G., J.G.M. and P.M.R.</p><p>Genotype data acquisition of contributing studies by (PROSPER) S.T., J.W.J., A.J.M.C. and P.E.S.; (ASCOT) M.J.C., P.B.M., P.S., A.S. and S.S.H.; (CARDS) H.A.D., H.M.C., P.M.M., P.N.D., A.D. and G.H.; (PARC) Y.-D.I.C., J.I.R., D.A.N. and J.D.S.; (TNT) B.J.A., M.P.D., S.M.B., G.K.H. and J.-C.T.; (AGES) A.V.S.; (ARIC) E.B.; (BioVU) Q.F., J.C.D., C.T.L. and F.S.; (CHS, HVH) G.L., J.C.B., K.D.T., J.I.R., K.R., T.L. and S.R.H.; (FHS) C.J.O.; (GoDARTS) C.N.A.P., K.Z., A.D., F.C., H.M.C., M.I.M., L.G., E.A. and WTCCC2; (HABC) Y.L.; (MESA) K.D.T. and J.I.R.; (Rotterdam Study) A.G.U. and F.R.; and (JUPITER) D.I.C., F.G., B.J.B., F.N. and P.M.R.</p><p>Primary analysis from contributing studies by (PROSPER) I.P., S.T., A.J.M.C. and P.E.S.; (ASCOT) M.J.C., M.R.B. and H.R.W.; (CARDS) H.A.D., H.M.C. and P.M.M.; (PARC) X.L., Y.-D.I.C. and J.I.R.; (TNT) B.J.A., M.P.D., S.M.B., G.K.H. and J.-C.T.; (AGES) A.V.S.; (ARIC) C.L.A., E.A.W. and T.S.; (BioVU) Q.F., W.W., C.T.L. and F.S.; (CHS, HVH) K.L.W. and G.L.; (FHS) L.A.C., P.G. and J.S.N.; (GoDARTS) C.N.A.P., L.A.D., K.Z. and H.M.C.; (HABC) D.S.E., J.M.S. and J.D.; (MESA) K.D.T., X.G., X.L. and J.I.R.; (Rotterdam Study) C.E.K. and B.H.S.; and (JUPITER) D.I.C., A.Y.C., F.G., J.G.M. and P.M.R.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Postmus, I. <italic>et al.</italic> Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins. <italic>Nat. Commun.</italic> 5:5068 doi: 10.1038/ncomms6068 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References</title><p>Supplementary Figures 1-4, Supplementary Tables 1-10, Supplementary Notes 1-3 and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6068-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Data 1</title><p>Expression Quantitative trait loci (eQTLs) linked to LDL-C associated SNPS</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6068-s2.xlsx"/></supplementary-material><supplementary-material id="d33e30" content-type="local-data"><caption><title>Supplementary Data 2</title><p>Functional characterization of all variants showing LD with with LDL-C associated SNPs</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6068-s3.xlsx"/></supplementary-material><supplementary-material id="d33e36" content-type="local-data"><caption><title>Supplementary Data 3</title><p>Candidate loci defined by association and eQTLs identified in this study</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6068-s4.xlsx"/></supplementary-material><supplementary-material id="d33e42" content-type="local-data"><caption><title>Supplementary Data 4</title><p>GeneGo custom network of known statin interacting genes</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6068-s5.xlsx"/></supplementary-material></sec>

A developmental cell-type switch in cortical interneurons leads to a selective defect in cortical oscillations

<p>The cellular diversity of interneurons in the neocortex is thought to reflect subtype-specific roles of cortical inhibition. Here we ask whether perturbations to two subtypes&#x02014;parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons&#x02014;can be compensated for with respect to their contributions to cortical development. We use a genetic cell fate switch to delete both PV+ and SST+ interneurons selectively in cortical layers 2&#x02013;4 without numerically changing the total interneuron population. This manipulation is compensated for at the level of synaptic currents and receptive fields (RFs) in the somatosensory cortex. By contrast, we identify a deficit in inhibitory synchronization <italic>in vitro</italic> and a large reduction in cortical gamma oscillations <italic>in vivo</italic>. This reveals that, while the roles of inhibition in establishing cortical inhibitory/excitatory balance and RFs can be subserved by multiple interneuron subtypes, gamma oscillations depend on cellular properties that cannot be compensated for&#x02014;likely, the fast signalling properties of PV+ interneurons.</p>

<contrib contrib-type="author"><name><surname>Takada</surname><given-names>Naoki</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Pi</surname><given-names>Hyun Jae</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Sousa</surname><given-names>Vitor H.</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n2">&#x02020;</xref></contrib><contrib contrib-type="author"><name><surname>Waters</surname><given-names>Jack</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Fishell</surname><given-names>Gord</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kepecs</surname><given-names>Adam</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Osten</surname><given-names>Pavel</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Cold Spring Harbor Laboratory</institution>, Cold Spring Harbor, New York 11724, <country>USA</country></aff><aff id="a2"><label>2</label><institution>Smilow Neuroscience Program, the Department of Cell Biology, New York University</institution>, New York, New York 10016, <country>USA</country></aff><aff id="a3"><label>3</label><institution>Department of Physiology, Feinberg School of Medicine, Northwestern University</institution>, Chicago, Illinois 60611, <country>USA</country></aff>

Nature Communications

<p>GABAergic interneurons have traditionally been classified into distinct subtypes based on their cellular properties, including firing properties, morphological features and expression of proteins linked to synaptic functions, such as the calcium-binding proteins parvalbumin (PV) and calretinin (CR) or co-neurotransmitters somatostatin (SST) and vasoactive intestinal peptide (VIP)<xref ref-type="bibr" rid="b1">1</xref>. The extraordinary diversity of interneuron subtypes in the mammalian neocortex suggests that inhibition plays particularly important and complex roles in controlling the development and adult functions of cortical excitatory circuits.</p><p>Genetic lineage-tracing studies have revealed detailed information about the embryonic origins of the main cortical interneuron subtypes, as well as their migration and final distribution across cortical layers. The most abundant cortical subtypes are the PV+ and SST+ interneurons, which arise from the medial ganglionic eminence (MGE) and account for nearly 70% of cortical inhibitory cells<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>. The PV+ interneurons are characterized as fast spiking and include basket cells populating cortical layers 2&#x02013;6 and chandelier cells found in layers 2 and 5/6, whereas the SST+ interneurons comprise the Martinotti cells and distribute to layers 2/3 and 5. The remaining ~30% of cortical interneurons arise from the caudal ganglionic eminence and can be broadly classified as bipolar VIP-positive (VIP+), the CR-positive (CR+) interneurons and the reelin-expressing neurogliaform cells, all populating mainly superficial cortical layers 2/3 (ref. <xref ref-type="bibr" rid="b4">4</xref>). Remarkably, a single transcription factor, called <italic>Nkx2-1</italic>, acts as a &#x02018;master switch&#x02019; in promoting the cell fate of the MGE-derived interneurons: Nkx2.1 is expressed only in the MGE and its genetic deletion leads to a cell fate switch of the PV+ and SST+ subtypes into caudal ganglionic eminence-like VIP+, CR+ and reelin+ subtypes as defined by the expression of the respective marker proteins, firing properties and axonal arborizations<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>.</p><p>In contrast to the detailed knowledge about the embryonic origins, we know much less about subtype-specific roles of cortical interneurons during postnatal development, when excitatory and inhibitory circuits are established and shaped by sensory experience. On the basis of the studies of a knockout mouse model lacking the glutamic acid decarboxylase 65 (GAD65) protein, one of the two GABA-synthesizing enzymes in the brain, the onset of normal receptive field (RF) development is proposed to depend on the maturation of GABAergic circuits that is needed to reach a necessary threshold level of inhibition and establish normal excitation/inhibition (E/I) balance<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>. The developmental increase in inhibition has been proposed to be mediated by the PV+ interneurons, because their maturation in the cortex approximately parallels RF development<xref ref-type="bibr" rid="b7">7</xref>. However, it is not known whether specific cellular properties of PV+ interneurons are required for normal cortical development, including establishing the E/I balance and sensory RFs, or whether the overall increase in inhibition (independent of subtype-specific roles) is the critical developmental factor.</p><p>In the adult cortex, subtype-specific roles of inhibition have recently begun to be elucidated by optogenetic manipulations. The PV+ interneurons are proposed to drive gamma (30&#x02013;80&#x02009;Hz) oscillations, thought to be a substrate for neuronal coordination underlying cortical processing. The fast signalling properties of PV+ interneurons, such as the ability to fire short-duration action potentials, rapid and highly synchronized GABA release and short inhibitory postsynaptic current (IPSC) decay time constant, have been proposed as the underlying cellular mechanisms that enable this cell type to organize the fast network oscillations<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. This can occur either via the activation of reciprocally connected inhibitory circuits, termed interneuron gamma, or via an excitatory-inhibitory loop, termed the pyramidal-interneuron gamma<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. In support of the PING model in the cortex, optogenetic activation of either the PV+ interneurons or layer 2/3 pyramidal neurons was shown to be sufficient to initiate cortical gamma activity<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>. Other optogenetic studies showed that the activity of PV+ interneurons can control the size as well as the gain of sensory RFs<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>, while the activity of SST+ interneurons can regulate the RF size in the visual cortex<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>. These studies suggest that the adult requirements for modes of cortical inhibition are allocated in a subtype-specific manner. They do not, however, address the importance of interneuron subtype diversity during development, including the question whether the PV+ and SST+ subtypes are necessary for the proposed roles or whether some inhibitory circuit functions may be compensated for by other interneurons.</p><p>Here we study the developmental cell type-specific roles of cortical inhibition by genetically deleting the PV+ and SST+ interneurons in the upper layers of the neocortex. This manipulation enabled us to test whether the innervation by the two interneuron subtypes is necessary for normal development of pyramidal neuron cellular (intrinsic) and synaptic properties, cortical E/I balance, sensory RFs and network synchronization.</p><sec disp-level="1" sec-type="results"><title>Results</title><p>We used a genetic approach based on a conditional deletion of the MGE-specific Nkx2.1 in a loss-of-function (<italic>Nkx2-1</italic>^{<italic>LOF</italic>}) mouse model<xref ref-type="bibr" rid="b6">6</xref>, which allowed us to test the roles of the PV+ and SST+ interneurons during development and in the young adult. We timed the Nkx2-1 deletion at embryonic day (E) 12.5 (<italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>}), which restricts the cell fate switch to the cortical layers 1&#x02013;4 and causes the substitution of the PV+/SST+ interneurons by the VIP+, CR+ and neurogliaform subtypes without affecting the total interneuron cell count<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>. Given that the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice have normal lifespans and no seizures<xref ref-type="bibr" rid="b6">6</xref>, we hypothesized that the loss of PV+ and SST+ interneurons may be compensated for at the level of gross cortical development and, therefore, that these mice may allow us to directly test whether these interneuron subtypes are necessary for the development of cortical E/I balance and sensory RFs, as well as the generation of gamma cortical oscillations.</p><sec disp-level="2"><title>Characterization of the Nkx2-1 deletion <italic>in vitro</italic></title><p>In the first set of experiments, we confirmed by immunohistochemistry that the E12.5 deletion of Nkx2-1 reliably induces the cell fate switch as intended<xref ref-type="bibr" rid="b6">6</xref>, demonstrating ~93% reduction in PV+ and ~85% reduction in SST+ without any changes in the total interneuron count as revealed by anti-GABA immunostaining in the superficial layers of the somatosensory cortex (<xref ref-type="fig" rid="f1">Fig. 1</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>).</p><p>We then considered the consequences of this manipulation on the development of excitatory layer 2/3 pyramidal neurons. First, we tested whether the development of biophysical and cellular properties of pyramidal neurons is affected by the altered interneuron composition. If so, such changes would need to be taken into account when interpreting more complex cortical functions in later experiments. Whole-cell recordings in acute brain slices from control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice revealed no differences in intrinsic membrane properties, including input resistance, rectification index and membrane time constant (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 2</xref> and <xref ref-type="supplementary-material" rid="S1">3</xref>). Several other cellular properties, including action potential properties, resting membrane potential and excitability, were altered during the second postnatal week (P11&#x02013;13), but returned mostly to normal by the end of the third week (P20&#x02013;22) (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 2</xref> and <xref ref-type="supplementary-material" rid="S1">3</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1</xref> and <xref ref-type="supplementary-material" rid="S1">2</xref>). These data show that the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} manipulation induces transient changes in the development of cellular properties of cortical pyramidal neurons, which are largely compensated for by the end of the third postnatal week marking the end of the critical period of sensory development in the somatosensory barrel cortex<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref>.</p><p>Next, we determined whether normal balance of synaptic excitation and inhibition is preserved in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} somatosensory cortex, using whole-cell recordings in brain slices from 3- to 4-week-old animals. First, recordings of spontaneous miniature excitatory postsynaptic currents (EPSCs) and IPSCs (mEPSCs and mIPSCs, respectively) in layer 2/3 pyramidal neurons revealed no changes in mEPSC frequencies and comparably increased mEPSC and mIPSC amplitudes, by ~24 and ~32%, respectively, resulting in no change in the mEPSC/mIPSC amplitude ratio in <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} compared with control slices (<xref ref-type="fig" rid="f2">Fig. 2a&#x02013;g</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>). Second, we also tested evoked EPSCs and IPSCs by incubating the brain slices in a bath solution with higher potassium and lower magnesium concentration<xref ref-type="bibr" rid="b28">28</xref>. As shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>, this analysis also did not detect any differences between the two conditions (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>). These data thus indicate that the cortical E/I balance is normal in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice at 4 weeks of age. The transient changes in cellular properties and the comparable increase in mEPSC and mIPSC amplitudes suggest that some forms of homeostatic synaptic plasticity<xref ref-type="bibr" rid="b29">29</xref> likely contribute to the normalization of synaptic currents and E/I balance in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} cortex.</p></sec><sec disp-level="2"><title>Nkx2-1 deletion does not affect RF properties</title><p>Having demonstrated largely normal properties of excitatory pyramidal neurons and normal E/I balance in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} cortex, we turned our attention to the study of sensory RF development, by mapping sensory responses in the somatosensory barrel cortex representing inputs from the rodent facial vibrissae (whiskers). The critical period in the barrel cortex occurs during the second to third postnatal week, when the sensory responses of layer 2/3 neurons increase in strength and their RFs are sensitive to sensory deprivation<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref>. Mature RFs of layer 2/3 pyramidal neurons in the barrel cortex have the strongest response to the deflection of the principal whisker and weaker responses to the deflections of the adjacent surround whiskers<xref ref-type="bibr" rid="b30">30</xref>. Whole-cell recordings <italic>in vivo</italic> in anaesthetized mice (~4 weeks old) revealed normal layer 2/3 RF properties in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} barrel cortex (<xref ref-type="fig" rid="f2">Fig. 2h&#x02013;k</xref>). The amplitude of whisker-evoked subthreshold postsynaptic potentials was (mV): principal whisker: 16.4&#x000b1;3.1 and 16.3&#x000b1;2.4 (<italic>P</italic>=0.996; Student&#x02019;s <italic>t</italic>-test); and surround whisker: 8.2&#x000b1;1.7 and 8.7&#x000b1;1.9 (<italic>P</italic>=0.843; mean&#x000b1;s.e.m.), in control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} recordings, respectively. In addition, no differences were observed in the postsynaptic potential kinetics (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>) or in the number of sensory-evoked action potentials (spikes per trial): 0.064&#x000b1;0.032 and 0.067&#x000b1;0.040 (<italic>P</italic>=0.954), control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} barrel cortex, respectively. On the basis of these data, we conclude that the cell fate switch of the PV+ and SST+ interneurons is compensated for at the level of somatosensory RF development in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} barrel cortex.</p></sec><sec disp-level="2"><title>Nkx2-1 deletion prevents fast cortical oscillations</title><p>Next, we turned our attention to the study of cortical circuit dynamics, first using an optogenetic model of cortical oscillations driven by activation of layer 2/3 pyramidal neurons<xref ref-type="bibr" rid="b20">20</xref>. Channelrhodopsin-2 (ChR2) was targeted to layer 2/3 pyramidal neurons by stereotaxic injections of an adeno-associated virus expressing yellow fluorescent protein (YFP)-tagged ChR2 (ChR2-YFP) under the control of the alpha-CaMKII promoter<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b31">31</xref>. Driving ChR2-expressing pyramidal neurons with a ramp of blue light (2&#x02009;s of increasing intensity) was sufficient to induce a robust synchronization in control brain slices, as manifested by an increase in IPSC power in the 20&#x02013;30&#x02009;Hz frequency band in whole-cell recordings from layer 2/3 pyramidal neurons (<xref ref-type="fig" rid="f3">Fig. 3</xref>). In contrast, the same manipulation evoked variable activity with a broadly reduced peak power in layer 2/3 neurons in <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} brain slices (<xref ref-type="fig" rid="f3">Fig. 3</xref>), indicating an impaired capacity of local inhibitory circuits to synchronize at higher frequencies. This suggests that the inhibitory circuits in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} cortex may not be able to sustain fast oscillations <italic>in vivo</italic> in behaving mice.</p><p>In the final sets of experiments, we addressed the question whether fast cortical oscillations persist in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice during behaviour. Local field potentials (&#x00394;LFP in bipolar configuration between supragranular and infragranular layers) were recorded in the barrel cortex in control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice during a novelty-induced exploration paradigm. During exploration, behavioural measures were not different between control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} animals (mean&#x000b1;s.e.m.): occupancy of a novel object (novel object visit/total time): 0.07&#x000b1;0.018 and 0.07&#x000b1;0.042 (<italic>P</italic>=0.917); speed of movement (m&#x02009;s^&#x02212;1): 0.046&#x000b1;0.0055 and 0.037&#x000b1;0.0014 (<italic>P</italic>=0.146); and distance covered (m): 8.26&#x000b1;0.741 and 6.92&#x000b1;0.151 (<italic>P</italic>=0.081), respectively. However, we observed a dramatic difference between control and <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice in terms of oscillations (<xref ref-type="fig" rid="f4">Fig. 4c,d</xref>). To focus on the rhythmic components of the LFP (instead of total spectral power that includes the non-rhythmic 1/f background), we used the better oscillation detection (BOSC) spectral analysis method. This technique detects oscillatory episodes based on power and duration threshold of the wavelet-filtered signal<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>. The probability of oscillatory episodes was highest around the gamma frequency range in control mice, whereas in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice the gamma frequency component was nearly absent and the peak frequency was shifted to the lower beta frequency range (<xref ref-type="fig" rid="f4">Fig. 4</xref>). These results thus demonstrate that the PV+/SST+ cell-type switch indeed interferes with the capacity of cortical circuits to generate gamma oscillations in the somatosensory barrel cortex during behaviour, as predicted based on our optogenetic experiments in brain slices.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Our study answered three questions regarding the role of GABAergic inhibition during development and in the young adult cortex. First, we showed that genetic deletion of two major interneuron cell types, the PV+ and SST+ interneurons, can be largely compensated for at the level of cortical E/I balance development when the total interneuron cell count remains unchanged. This is in contrast to the PV+/SST cell fate switch induced by the Nkx2.1 deletion at E10.5, just 2 days earlier than the timing used here, which induces a significant reduction in the number of cortical interneurons and leads to pronounced spontaneous seizures<xref ref-type="bibr" rid="b6">6</xref>. We have also observed transient changes in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} pyramidal neuron cellular properties during the second postnatal week, which were largely compensated for by the end of the third week. The second-to-third week period marks the critical period of RF development in the somatosensory barrel cortex<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref>. This suggests that some forms of homeostatic plasticity, known to play important roles during postnatal development<xref ref-type="bibr" rid="b29">29</xref>, are activated and contribute to the establishment of E/I balance.</p><p>Second, the finding of normal sensory-evoked responses in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} barrel cortex suggests that the PV+/SST+ cell types can be also compensated for during somatosensory RF development. The role of inhibition in RF development was examined first in the visual cortex in the GAD2 knockout mice, in which the lack of the GAD65 isoform was shown to lead to a reduction in fast inhibitory transmission, enhanced sensory-evoked responses and a deficit in monocular deprivation-induced plasticity<xref ref-type="bibr" rid="b9">9</xref>. PV+ interneurons are likely to play a critical role in mediating normal RF development and plasticity, because their maturation occurs approximately in parallel to critical periods of RF development in sensory cortices and their responses are modulated by monocular deprivation<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>. Thus, our data do not exclude a role of PV+ interneurons in this process, but support a model in which the overall level of inhibition mediated by PV+ and possibly other interneuron subtypes and E/I balance<xref ref-type="bibr" rid="b7">7</xref>, rather than specific cellular properties of the PV+ interneurons, is necessary for normal RF development. In addition, although our data show that the PV+ and SST+ interneurons are not necessary for normal RF development in the barrel cortex, this finding should not be interpreted to mean that these cell types do not regulate RFs in the normal brain. For example, optogenetic studies showed that PV+ interneurons can regulate both the size and the gain of sensory RFs<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>, while the activity of SST+ interneurons was proposed to regulate the RF size by the suppressive surround mechanism in the adult visual cortex<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>.</p><p>Third, in contrast to the overall normal synaptic currents and sensory-evoked responses, the PV+/SST+ cell fate switch induced large changes at the level of cortical network activity, including a loss of cortical gamma frequency and an augmentation of lower-frequency oscillations in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mice. While several studies have demonstrated that cortical gamma activity can be externally manipulated by multiple means, including optogenetic activation of pyramidal neurons and PV+ interneurons, glutamate AMPA/kainate and metabotropic receptor-mediated excitation and cholinergic neuromodulation<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b36">36</xref><xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref><xref ref-type="bibr" rid="b42">42</xref>, our study demonstrates that the MGE-derived interneuron subtypes are necessary for the generation of cortical gamma rhythms.</p><p>Our study does not directly address the question of which interneuronal cellular properties are necessary for the generation of gamma oscillations. On the basis of modelling studies, the lack of fast signalling properties of the PV+ interneurons, including synaptic kinetics, rapid action potentials and high intrinsic resonance frequency, is likely to play a critical role in the observed oscillatory phenotype<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b43">43</xref>. In addition, the SST+ interneurons, which innervate distal dendrites of cortical pyramidal neurons, have also been shown to synchronize even though at lower (&#x0003c;30&#x02009;Hz) frequencies<xref ref-type="bibr" rid="b44">44</xref>. Therefore, the genetic deletion of SST+ interneurons may also contribute to the oscillatory phenotype observed in the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} cortex.</p><p>In summary, our study describes distinct developmental roles of cortical inhibition&#x02014;cell type-independent regulation of E/I balance and sensory RFs and cell type-dependent regulation of fast cortical oscillations. To our knowledge, this is the first demonstration of a selective deficit in neuronal network synchronization, in the absence of other synaptic network changes. Since the E12.5 <italic>Nkx2.1-1</italic> deletion does not affect interneuron composition in the hippocampus or elsewhere in the brain<xref ref-type="bibr" rid="b6">6</xref>, our study opens the door to future investigations using the <italic>Nkx2-1</italic>^{<italic>E12.5LOF</italic>} mouse model to probe the roles of cortical gamma oscillations in cognitive behaviours.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><p>All data are presented as mean&#x000b1;s.e.m. Statistical significance was tested using the Student <italic>t</italic>-test or Mann&#x02013;Whitney <italic>U</italic>-test, as stated in the Figure legend texts (significance level &#x0003c;0.05). Animal procedures were approved by the Cold Spring Harbor Laboratory Animal Care and Use Committee. All animals were housed under constant temperature and light conditions (12&#x02009;h cycle lights ON: 0600, lights OFF: 1800) and given food and water <italic>ad libitum</italic>.</p><sec disp-level="2"><title>Generation of Nkx2-1^E12.5LOF mice</title><p>Triple-heterozygote male mice (<italic>Nkx2-1</italic>^{<italic>+/&#x02212;</italic>} (ref. <xref ref-type="bibr" rid="b45">45</xref>); <italic>Olig2</italic>^{<italic>CreER/+</italic>} (ref. <xref ref-type="bibr" rid="b46">46</xref>); <italic>Z/EG</italic>^{<italic>+/&#x02212;</italic>} (ref. <xref ref-type="bibr" rid="b47">47</xref>)) were intercrossed with Nkx2-1 conditional homozygote females (<italic>Nkx2-1</italic>^{<italic>C/C</italic>}) (ref. <xref ref-type="bibr" rid="b48">48</xref>) to generate experimental control (Nkx2-1^C/+; Olig2^CreER/+; Z/EG^+/&#x02212; or Z/EG^{&#x02212;/&#x02212;}) and mutant (Nkx2-1^C/&#x02212;; Olig2^CreER/+; Z/EG^+/&#x02212; or Z/EG^{&#x02212;/&#x02212;}) mice. Tamoxifen (4&#x02009;mg, Sigma) was administered to pregnant mice at E12.5 to induce the Cre recombinase, leading to a cell fate switch of superficial cortical interneurons derived from the MGE<xref ref-type="bibr" rid="b6">6</xref>.</p></sec><sec disp-level="2"><title>Immunocytochemistry</title><p>The mice were killed by transcardial perfusion with 4% paraformaldehyde and the brains were dissected and sectioned coronally at 50&#x02009;&#x003bc;m. Sections comprising the somatosensory cortex were incubated in a blocking solution containing 5% donkey serum and 0.2% Triton X-100 in phosphate buffer for 1&#x02009;h, followed by the incubation with the blocking solution containing mouse anti-parvalbumin (Sigma, 1:1,000) or rat anti-somatostatin (Millipore, 1:200) antibodies or fluorescein-labelled Wisteria floribunda lectin (Vector Laboratories, 1:500), in combinations with rabbit anti-GABA (Sigma, 1:500) or guinea pig anti-type 2 vesicular glutamate transporter (VGluT2, Millipore, 1:1,000) antibodies overnight at 4&#x02009;&#x000b0;C. Primary antibodies were fluorescently labelled by incubation with appropriate secondary antibodies conjugated with Alexa Fluor-405, -488 and -594 (1:200, Invitrogen). Fluorescence images were taken using LSM710 confocal laser-scanning microscope (Zeiss). VGluT2 staining was used to visualize barrel structures of the somatosensory barrel cortex, based on the dense labelling of axon terminals from thalamocortical projections<xref ref-type="bibr" rid="b49">49</xref>. The specific interneuron cell types were counted manually using FIJI image processing software.</p></sec><sec disp-level="2"><title><italic>In vitro</italic> whole-cell patch-clamp recordings</title><p>Acute brain slices including the barrel cortex were made by cutting the brain at 45&#x000b0; angle between the horizontal and sagittal plane<xref ref-type="bibr" rid="b50">50</xref> in an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1 CaCl₂, 4 MgCl₂, 25 NaHCO₃, 1.25 NaH₂PO₄, 25 glucose, 1 kynurenic acid, equilibrated with a mixture of 95% O₂ and 5% CO₂. The thickness of slices is 300&#x02009;&#x003bc;m and the age of animals is postnatal day 20&#x02013;25 (P20&#x02013;26), unless otherwise noted. The holding chamber was maintained at 29&#x000b1;1&#x02009;&#x000b0;C for about 30&#x02009;min and then kept at room temperature.</p><p>Membrane currents and potentials were recorded by using whole-cell patch-clamp techniques (MultiClamp 700B patch-clamp amplifier, Molecular Devices) at 29&#x02009;&#x000b0;C. The ACSF contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 25 NaHCO₃, 1.25 NaH₂PO₄, 25 glucose (pH 7.4), unless otherwise noted. The slices were observed under an upright microscope (BX50WI, Olympus) equipped with a &#x000d7; 40 water immersion objective and IR-DIC optics via a CCD (charge-coupled device) camera (ORCA, Hamamatsu). Glass micropipettes fabricated from borosilicate glass capillaries were filled with the intracellular solution (tip resistance: 4&#x02013;7&#x02009;M&#x003a9;) containing (in mM): 135 K-gluconate, 4 KCl, 10 HEPES, 4 MgATP, 0.3 Na₃GTP, 10 phosphocreatine-2Na (pH 7.35) for current-clamp recordings and 100 CsMeSO₃, 5 CsCl, 10 HEPES, 10 BAPTA-4Cs, 4 MgATP, 0.3 Na₃GTP, 10 phosphocreatine-2Na, 4 QX-314, 0.3% biocytin (pH 7.35) for voltage-clamp recordings. Series resistance was typically &#x0003c;20&#x02009;M&#x003a9;. Signals were filtered at 1&#x02013;4&#x02009;kHz, digitized at 5&#x02013;20&#x02009;kHz (USB-6259, National Instruments) and acquired by a custom-made program written in LabVIEW (National Instruments). Miniature EPSCs were recorded at &#x02212;70&#x02009;mV in ACSF containing 1&#x02009;&#x003bc;M tetrodotoxin (TTX), 10&#x02009;&#x003bc;M SR-95531 (gabazine) and 5&#x02009;&#x003bc;M (RS)-CPP, whereas miniature IPSCs were recorded at +15&#x02009;mV in the presence of 1&#x02009;&#x003bc;M TTX, 5&#x02009;&#x003bc;M 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) and 5&#x02009;&#x003bc;M (RS)-CPP (all drugs were purchased from Tocris). Miniature synaptic events were detected based on the amplitude threshold of 5&#x02009;pA and fitting of the events with an alpha function of 1&#x02009;ms rise and 2&#x02009;ms decay time constants for miniature EPSCs and 2&#x02009;ms rise and 20&#x02009;ms decay time constants for miniature IPSCs, followed by visual inspection to exclude inappropriate ones such as overlapped events or events on noisy baseline. Spontaneous synaptic activities without TTX were observed in slightly excitable ACSF in which 2.5&#x02009;mM KCl and 1&#x02009;mM MgCl₂ were replaced with 4&#x02009;mM KCl and 0.5&#x02009;mM MgCl₂, respectively<xref ref-type="bibr" rid="b28">28</xref>. EPSC and IPSC components were recorded at &#x02212;60 and +15&#x02009;mV, respectively, which are close to the reversal potential of the other synaptic component. Because of high occurrences of events, the spontaneous activities were evaluated by calculating the area or charge of the responses. All data analyses were performed by using Igor Pro (WaveMetrics).</p></sec><sec disp-level="2"><title><italic>In vivo</italic> whole-cell patch-clamp recordings</title><p>Animals (P29&#x02013;93) were anaesthetized with urethane (1.5&#x02013;2&#x02009;g per kg body weight, intraperitoneal). After incision of scalp, a metal plate was attached to the skull with dental cement to fix a head and make a chamber, and a craniotomy of about 2&#x02009;mm diameter was made over the barrel cortical region. The dura mater was carefully removed with a needle while the chamber was superfused with ACSF. The exposed surface was then covered with 1.5% agarose. <italic>In vivo</italic> whole-cell patch-clamp was obtained by a &#x02018;blind&#x02019; technique<xref ref-type="bibr" rid="b51">51</xref>. The patch pipette and the internal solution were the same as those used in slice experiments. Initially, LFP recording was performed in layer 2/3 to estimate the primary whisker of the target recording site by stimulating whiskers of the opposite side randomly. Then, a micropipette electrode that has a positive pressure of 30&#x02013;40&#x02009;mbar was inserted into the target region while the current response is continuously monitored in a voltage-clamp mode. Positive pressure was released when the resistance suddenly increased, indicating that the tip of the pipette may have been pushed against a cell membrane. Gentle suction was then applied to achieve a G&#x003a9; seal if needed. The whole-cell recording configuration was established by voltage clamp, with series resistances between 10 and 75&#x02009;M&#x003a9;<xref ref-type="bibr" rid="b51">51</xref>. For current-clamp recordings, bridge balance was adjusted manually to eliminate voltage errors arising from series resistance. Sensory stimulations were done as described<xref ref-type="bibr" rid="b52">52</xref>, by delivering mechanical stimuli (9.5&#x000b0; deflection angle) to each whisker of the opposite side by a capillary attached to a piezoelectric apparatus (Piezo Systems) for 200&#x02009;ms at a frequency of about 1&#x02009;Hz. Recordings were obtained with a MultiClamp 700B patch-clamp amplifier, filtered at 10&#x02009;kHz and digitized at 10&#x02013;20&#x02009;kHz using a similar system to that of slice experiments. After recordings, animals were perfused transcardially with 4% paraformaldehyde in phosphate buffer.</p></sec><sec disp-level="2"><title>LFP recordings in awake animals</title><p>All experiments were performed with the experimenter blind to strain of mice. Adult (over 2 months old) Nkx2-1 mutants and their control litter mates were implanted with custom-built microdrives in the left barrel cortex (1.0&#x02009;mm posterior to bregma and 3.0&#x02009;mm lateral to midline) using stereotaxis as described previously<xref ref-type="bibr" rid="b53">53</xref>. One electrode was placed in the superficial layer (100&#x02009;&#x003bc;m deep from the pia mater) and the other was in the deeper layer (500&#x02013;600&#x02009;&#x003bc;m deep). After 10 days recovery from the surgery, LFPs were obtained using the Cheetah system (Neuralynx) in a novelty-induced exploration condition, where the animals were actively whisking and moving around the novel objects in the arena. Spectral analysis was performed with multitaper technique in Chronux software<xref ref-type="bibr" rid="b54">54</xref> and the better oscillation detection method (BOSC)<xref ref-type="bibr" rid="b33">33</xref> using Matlab (Mathworks).</p></sec><sec disp-level="2"><title>Photostimulation by ChR2</title><p>ChR2 fused to YFP was selectively transfected in L2/3 pyramidal neurons in the somatosensory cortex by a stereotaxic injection of adeno-associated viral vectors based on the &#x003b1;-CaMKII promoter. The viral solution of about 90&#x02009;nl was injected into the barrel cortical region at P13 or 14 (posterior 1.2&#x02009;mm and lateral 3.0&#x02009;mm relative to bregma and depth 0.25&#x02009;mm from the dura mater) and the brain slices were prepared 7&#x02013;12 days after the injection. After whole-cell recording was established from a cell located in the centre of ChR2-expressing area, ChR2 was activated by illumination of a blue light at around 470&#x02009;nm through the objective using an LED (LEDC5, Thorlabs), which is attached to the microscope and controlled by the patch-clamp software. The illumination of ramped intensity for 3&#x02009;s could effectively induce the oscillatory synaptic activities in control neurons. The IPSCs were measured from the trace between 2 and 3&#x02009;s with respect to the onset of light stimulation. The IPSC decay time constants were measured by fitting with a single exponential function. The degree of rhythmicity was evaluated with a highest frequency component and its intensity of the PSD.</p></sec></sec><sec disp-level="1"><title>Author contribution</title><p>N.T. carried out all experiments, except <italic>in vivo</italic> LFP recordings that were done by H.J.P. P.O. and N.T. designed the experiments, P.O., V.H.S., J.W., G.F. and A.K. co-wrote the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Takada, N. <italic>et al.</italic> A developmental cell-type switch in cortical interneurons leads to a selective defect in cortical oscillations. <italic>Nat. Commun.</italic> 5:5333 doi: 10.1038/ncomms6333 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Figures and Tables</title><p>Supplementary Figures 1-3 and Supplementary Tables 1-5</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6333-s1.pdf"/></supplementary-material></sec>

Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by &#x003b3;-TuRC in yeast and human cells

<p>Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the &#x003b3;-tubulin ring complex (&#x003b3;-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to &#x003b3;-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at &#x003b3;-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.</p>

<contrib contrib-type="author"><name><surname>Olmsted</surname><given-names>Zachary T.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Colliver</surname><given-names>Andrew G.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Riehlman</surname><given-names>Timothy D.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Paluh</surname><given-names>Janet L.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>State University of New York Polytechnic Institute, College of Nanoscale Science, Nanobioscience Constellation</institution>, Albany, New York 12203, <country>USA</country></aff>

Nature Communications

<p>The microtubule cytoskeleton is a self-assembling network that underlies specialized, often polarized, cellular functions in eukaryotes. Knowledge of its mechanisms is fundamental to understanding normal development and disease and is expected to assist new technologies through biomimicry. The microtubule-based mitotic spindle apparatus is perhaps the best studied self-assembly platform<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref> and a primary target for cancer therapeutics<xref ref-type="bibr" rid="b3">3</xref>. Spindle pole microtubule-organizing centres (MTOCs) utilize a &#x003b3;-tubulin template within a ring complex (&#x003b3;-tubulin ring complex, &#x003b3;-TuRC) to orchestrate addition of &#x003b1;-/&#x003b2;-tubulin heterodimeric microtubule building blocks into 25&#x02009;nm polarized microtubules<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>. Conserved protein structural features of the &#x003b3;-TuRC MTOC have been identified through crystallography studies from multiple model organisms and include &#x003b1;-/&#x003b2;-tubulin<xref ref-type="bibr" rid="b10">10</xref>, &#x003b3;-tubulin<xref ref-type="bibr" rid="b11">11</xref>, GCP4 (ref. <xref ref-type="bibr" rid="b12">12</xref>) and the &#x003b3;-tubulin small complex (&#x003b3;-TuSC) cryo-EM structure<xref ref-type="bibr" rid="b13">13</xref>. Conserved structural features are additionally supported by cross-species analysis<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. Still unknown is how dynamic control over MTOC functions for microtubule nucleation and organization is achieved. The fission yeast <italic>Schizosaccharomyces pombe</italic> provides an ideal eukaryotic platform to address conserved MTOC mechanisms<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>.</p><p>The coordination of spindle microtubules into a bipolar array requires kinesin-like proteins (Klps), though Klp mitotic functions are not limited to interactions solely on microtubules. Studies of the functionally diverse kinesin-14 Klp family across eukaryotes have indicated an ability by some members to affect microtubule number and organization at spindle poles<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. In fission yeast, kinesin-14 Pkl1 interacts directly with the &#x003b3;-TuRC MTOC to alter its composition and function<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. Conservation of the kinesin-14 &#x003b3;-TuRC regulatory mechanism is expected from yeast to human, as human kinesin-14 HSET replaces fission yeast kinesin-14 Pkl1 (ref. <xref ref-type="bibr" rid="b23">23</xref>) and all human &#x003b3;-TuSC protein components are also compatible<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. Nearly as ubiquitous and complex in eukaryotes as kinesin-14 Klps are members of the kinesin-5 family that oppose kinesin-14 function. In fission yeast, kinesin-5 Cut7 opposes the action of kinesin-14 Pkl1 in mitosis, but the detailed mechanism is not yet characterized. Elucidating this mechanism could be informative for understanding &#x003b3;-TuRC regulation and spindle bipolarity.</p><p>In this study, we expand the mechanism for kinesin-14 regulation of &#x003b3;-TuRC. Studies from our lab and others describe genetic interactions of Pkl1 with &#x003b3;-TuRC proteins<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>, checkpoint pathways<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b26">26</xref> and spindle pole organization<xref ref-type="bibr" rid="b20">20</xref>. More recently we identified key Tail elements in Pkl1 that function along with Motor binding to &#x003b3;-tubulin to regulate &#x003b3;-TuRC<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. Here we demonstrate that kinesin-14 Pkl1 asymmetrically blocks microtubule nucleation <italic>in vivo</italic> in fission yeast and that a kinesin-14 Pkl1 Tail peptide can similarly prevent nucleation and generate mitotic arrest in two human breast cancer cell lines. We reveal that, in fission yeast, kinesin-5 Cut7 counters Pkl1 ability to block nucleation by also associating with &#x003b3;-TuRC and binding similarly to &#x003b3;-tubulin. This counteraction requires the additional conserved kinesin-5 BimC domain. Balanced regulation by kinesin-14 Pkl1 and kinesin-5 Cut7 generates optimal mitotic fidelity, although both proteins are co-dispensable as determined by genetic analysis of single and double mutants, biochemical approaches and timelapse fluorescence microscopy. Analysis of <italic>pkl1&#x00394;</italic> single and <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutants also reveals separate mitotic roles for both kinesin-14 Pkl1 and kinesin-5 Cut7. Our findings identify kinesin-14 Pkl1 as a Klp-negative regulator of microtubule nucleation at &#x003b3;-TuRC and demonstrate conservation of this mechanism in human breast cancer cells, resulting in mitotic arrest. We expect these discoveries to be broadly relevant to the microtubule cytoskeleton field with potential as a novel strategy and target for future development in cancer therapeutics.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Kinesin-5 is dispensable in the absence of kinesin-14 Pkl1</title><p>Spindle bipolarity in fission yeast requires kinesin-5 Cut7 (ref. <xref ref-type="bibr" rid="b27">27</xref>). The mechanism underlying its essential nature remains unclear as another Klp, kinesin-6 Klp9, is capable of crosslinking antiparallel microtubules and is required for spindle elongation<xref ref-type="bibr" rid="b28">28</xref>. In eukaryotes, an opposing relationship between kinesin-5 and kinesin-14 Klps in microtubule regulation is highly conserved. We previously demonstrated that kinesin-14 Pkl1 directly binds and downregulates &#x003b3;-TuRC function<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b23">23</xref>. We tested the hypothesis that a required role of kinesin-5 Cut7 (<italic>cut7</italic> gene), which localizes at spindle poles, is to oppose kinesin-14 Pkl1 (<italic>pkl1</italic> gene). By homologous recombination (<xref ref-type="fig" rid="f1">Fig. 1a</xref>), we simultaneously deleted the <italic>cut7</italic> gene while marking the locus with <italic>ura4</italic> (<italic>cut7&#x00394;::ura4+</italic>) in a strain deleted for Pkl1 (<italic>pkl1&#x00394;::his3+</italic>). The <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant strain exhibits robust viability by serial growth assays, similar to wild-type cells (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). Spindle pole body (SPB) separation is not affected in the double mutant <italic>pkl1&#x00394; cut7&#x00394;</italic> versus wild type (<xref ref-type="fig" rid="f1">Fig. 1c,d</xref>) nor is mitotic progression through anaphase affected as compared with wild type or <italic>pkl1&#x00394;</italic> strains (<xref ref-type="fig" rid="f1">Fig. 1c,e&#x02013;g</xref>). However, spindle breakdown is delayed as indicated by a persistent spindle following anaphase B elongation (<xref ref-type="fig" rid="f1">Fig. 1c,g</xref>). We demonstrate that kinesin-5 Cut7 is dispensable in the absence of Pkl1, indicating that a kinesin-5-independent mechanism for spindle assembly can exist in fission yeast. This also supports a required role for Cut7 that is to counter Pkl1, which is a direct negative regulator of the &#x003b3;-TuRC MTOC.</p></sec><sec disp-level="2"><title>Kinesin-5 Cut7 binds &#x003b3;-TuRC through Motor and Tail domains</title><p>Kinesin-14 Pkl1 negatively regulates &#x003b3;-TuRC through two internal protein domains that include elements of its Motor and Tail regions<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b22">22</xref>. To determine whether Cut7 binds the &#x003b3;-TuRC MTOC by a similar mechanism, we performed Fast Protein Liquid Chromatography (FPLC)<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b17">17</xref> using V5-tagged deletion derivatives of Cut7 previously generated<xref ref-type="bibr" rid="b29">29</xref>, immunocytology using newly generated V5-tagged deletion and site-directed mutagenesis derivatives, and Pkl1 peptide co-immunoprecipitation assays (<xref ref-type="fig" rid="f2">Fig. 2</xref>). Fractionation of whole-cell extracts carrying V5-tagged full-length Cut7 and two Cut7 truncation constructs (Cut7-Head-Stalk or Cut7HS, aa 1&#x02013;88; Cut7-Stalk-Tail or Cut7-ST, aa 443&#x02013;1,085) were examined in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant and in the <italic>gtb1-K5A</italic> strain that inhibits Pkl1 regulation of &#x003b3;-TuRC by blocking its Motor domain binding to &#x003b3;-tubulin (<xref ref-type="fig" rid="f2">Figs 2a,b</xref> and <xref ref-type="fig" rid="f3">3a&#x02013;c</xref>)<xref ref-type="bibr" rid="b22">22</xref>. A FLAG-tagged truncated Pkl1 construct that retains full function and localizes to &#x003b3;-TuRC (<italic>pkl1&#x00394;95</italic>)<xref ref-type="bibr" rid="b23">23</xref> was used as a positive control along with Alp4 (gamma complex protein GCP2 yeast orthologue) and &#x003b3;-tubulin proteins that are core subunits of the &#x003b3;-TuSC and the &#x0003e;2,000&#x02009;kDa high-molecular weight &#x003b3;-TuRC MTOC. The &#x003b3;-TuRC peaks in FPLC fractions 15 and 16 (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). Profiles of the three <italic>cut7</italic> constructs in <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant cells that are pREP81V5/<italic>cut7</italic>, pREP81V5/<italic>cut7HS</italic> and pREP81V5/<italic>cut7ST</italic> are shown (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>). Western blot analysis following FPLC fractionation reveals that Cut7, Cut7HS and Cut7ST exhibit similar high-molecular weight profiles as Pkl1&#x00394;95 and all peak in identical fractions to core &#x003b3;-TuRC proteins &#x003b3;-tubulin and Alp4. To further confirm Cut7ST binding to &#x003b3;-TuRC, we performed co-immunoprecipitations from high-molecular weight FPLC fraction 15 in strain <italic>alp4-HA</italic> pREP81V5<italic>/cut7ST</italic> (<xref ref-type="fig" rid="f2">Fig. 2d,e</xref>). Magnetic beads with histidine affinity were conjugated to small His-tagged Pkl1 Tail peptides (P&#x003b3;T or P&#x003b3;M)<xref ref-type="bibr" rid="b17">17</xref> that bind &#x003b3;-TuRC (P&#x003b3;T, targeting) or cannot interact with fission yeast &#x003b3;-TuRC (P&#x003b3;M, mutated). Using this approach, we detect &#x003b3;-TuRC core subunit proteins &#x003b3;-tubulin and Alp4-HA in addition to Cut7ST (V5 tag) by western blot analysis after elution off of beads (P&#x003b3;T). These proteins were not recovered by the mutated peptide, as expected. We were unable to detect &#x003b1;-/&#x003b2;-tubulin in high-molecular weight fractions following FPLC, which further suggests that the Klps detected were directly bound to &#x003b3;-TuRC.</p><p>We previously demonstrated that mutation of a conserved lysine residue to alanine in &#x003b3;-tubulin helix 11 (<italic>gtb1-K5A</italic>) abolishes Pkl1 Motor domain binding to &#x003b3;-TuRC and blocks its full function <italic>in vivo</italic><xref ref-type="bibr" rid="b22">22</xref>. To determine whether Cut7 similarly binds to &#x003b3;-tubulin through helix 11, we examined its FPLC profile in the <italic>gtb1-K5A</italic> strain (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The mutant &#x003b3;-tubulin-K5A fractionates similarly to wild-type &#x003b3;-tubulin by FPLC. The V5-Cut7 signal in high-molecular weight &#x003b3;-TuRC fractions is significantly reduced in the <italic>gtb1-K5A</italic> strain, whereas steady-state expression levels of V5-Cut7 in <italic>gtb1</italic> wild type and <italic>gtb1-K5A</italic> mutant backgrounds by western blot analysis of whole-cell extracts is similar (<xref ref-type="fig" rid="f3">Fig. 3c</xref>). Fluorescence microscopy of V5-Cut7 in the <italic>gtb1-K5A</italic> mutant reveals that, while pole localization is reduced, Cut7 retains localization to spindle microtubules, which is enhanced. Truncated Cut7HS that contains the Cut7 Motor and Stalk domains and lacks the Tail is completely absent from high-molecular weight &#x003b3;-TuRC fractions in the <italic>gtb1-K5A</italic> background (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). Similarly to V5-Cut7, it retains the ability to bind to spindle microtubules as observed by immunocytology (<xref ref-type="fig" rid="f3">Fig. 3d</xref>). Interestingly, the truncated Cut7ST protein in this strain that does not retain the Motor domain is unaltered in its association with &#x003b3;-TuRC (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The V5-Cut7ST with a fused nuclear localization single (NLS) to allow nuclear localization<xref ref-type="bibr" rid="b29">29</xref> is sufficient to target spindle poles in mitosis (<xref ref-type="fig" rid="f3">Fig. 3d</xref>). This indicates that, similar to Pkl1, Cut7 contains distinct Motor and Tail domain elements that offer independent binding sites to &#x003b3;-TuRC.</p></sec><sec disp-level="2"><title>The kinesin-5 Cut7 BimC Tail element directs pole targeting</title><p>The eukaryotic BimC domain is highly conserved across kinesin-5 members<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>. The domain was first identified in the <italic>Aspergillus nidulans</italic> kinesin-5 BimC protein<xref ref-type="bibr" rid="b32">32</xref>, but its precise role in mitosis has remained unknown for two decades. To examine the consequences of a mutation to the BimC domain of Cut7 on spindle pole localization, we used the loss of function temperature sensitive allele <italic>cut7-22</italic> that contains a single-point mutation within this region (1,021 Pro to Ser)<xref ref-type="bibr" rid="b22">22</xref>. To determine whether the BimC box is the primary spindle pole targeting site in the Cut7 Tail domain, we utilized V5-tagged Cut7 deletion and/or site-directed mutagenesis constructs with a fused NLS (<xref ref-type="fig" rid="f3">Fig. 3e</xref>)<xref ref-type="bibr" rid="b23">23</xref>. These constructs encode the fusion proteins V5-NLS-Cut7ST, V5-NLS-Cut7ST<xref ref-type="bibr" rid="b22">22</xref> (point mutation at 1,021 Pro to Ser resulting in lost bipolarity)<xref ref-type="bibr" rid="b22">22</xref>, V5-NLS-Cut7T (Cut7-Tail, aa 888&#x02013;1,085) and V5-NLS-Cut7T<xref ref-type="bibr" rid="b22">22</xref> and were analysed by immunocytochemistry of <italic>pkl1&#x00394; cut7&#x00394;</italic> cells that were fixed after shifting to 36&#x02009;&#x000b0;C. V5-NLS-Cut7ST and V5-NLS-Cut7T lack the Motor domain and retain spindle pole localization. In contrast, mutated V5-NLS-Cut7ST<xref ref-type="bibr" rid="b22">22</xref> and V5-NLS-Cut7T<xref ref-type="bibr" rid="b22">22</xref> constructs are unable to localize to poles but are expressed and retained within the nucleus, suggesting that the BimC sequence constitutes a key domain in the Cut7/&#x003b3;-TuRC interaction. Together, our data support the model in which Cut7 interacts physically with the &#x003b3;-TuRC MTOC in a manner poised to allow opposition to Pkl1 activity.</p></sec><sec disp-level="2"><title>Pkl1 regulates spindle morphology from &#x003b3;-TuRC</title><p>Mitotic phenotypes in strains carrying single <italic>pkl1&#x00394;</italic> or <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutants versus wild type were evaluated by live cell and timelapse imaging of microtubules (&#x003b1;-tubulin as mCherry-Atb2 or GFP-Atb2), spindle poles (&#x003b3;-TuRC pericentrin protein 1, Pcp1-GFP), antiparallel microtubules and chromatin (anaphase B and chromatin binding kinesin-6 member Klp9-GFP) as well as Hoechst staining of DNA. An increase in spindle thickness is observed in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant strain versus wild type as seen in single-strain imaging (<xref ref-type="fig" rid="f4">Fig. 4a</xref>) or by live cell imaging of a mixed culture with wild-type cells (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). This increased thickness is also present in the <italic>pkl1&#x00394;</italic> strain (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). In <xref ref-type="fig" rid="f4">Fig. 4b</xref>, the ratio of double mutant to wild-type cells at 2:1 resulted in an accordant increase in the ratio of thick:thin spindles. Thick spindles (&#x0003e;0.5&#x02009;&#x003bc;m in a spindle of length 4 to 6&#x02009;&#x003bc;m) are observed in 4&#x000b1;2% of wild-type cells, 63&#x000b1;8% <italic>pkl1&#x00394;</italic> single mutant and 68&#x000b1;8% of <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant cells (mean&#x000b1;s.e.m., <italic>n</italic>=90 cells for each strain). Although the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant background generates a slight increase in the thick spindle phenotype (<italic>P</italic>&#x0003c;0.05 by Student&#x02019;s <italic>t</italic>-test), the change versus <italic>pkl1&#x00394;</italic> single mutant alone is small. This indicates that it is primarily the loss of Pkl1 that induces this phenotype, which is exacerbated by additional loss of Cut7.</p><p>The nature of the morphological change to spindle thickness could be the result of an increase in the number of antiparallel microtubules from both poles, parallel microtubules that emanate from a single pole, or due to unattached microtubules or disorganized arrays<xref ref-type="bibr" rid="b20">20</xref> at a single pole. To distinguish among these possibilities, we used multiple approaches. kinesin-6 Klp9-GFP crosslinks antiparallel microtubule arrays<xref ref-type="bibr" rid="b28">28</xref> and can be used to preferentially mark the extent of microtubule overlap (generally midzone), and is used with &#x003b1;-tubulin (mCherry-Atb2) to visualize spindle microtubules and length of the mitotic spindle. In wild-type cells, the Klp9-GFP signal spans the entire spindle midzone width (<xref ref-type="fig" rid="f4">Fig. 4d</xref>, left images), whereas in the double mutant (<xref ref-type="fig" rid="f4">Fig. 4d</xref>, right images) we observe microtubule staining adjacent to the zone of antiparallel overlap that appears to emanate primarily from a single pole. We do not detect increased resistance to the microtubule-depolymerizing drug Thiabendazole (TBZ) in <italic>pkl1&#x00394;</italic> or <italic>pkl1&#x00394; cut7&#x00394;</italic> strains versus wild type (<xref ref-type="fig" rid="f4">Fig. 4e</xref>) consistent with no or limited changes to microtubule number. We favour the interpretation that increased spindle width is likely due to asymmetric spindle pole effects (lost organization) in the absence of Pkl1 as seen<xref ref-type="bibr" rid="b20">20</xref> and to an increased number of spindle microtubules.</p><p>Morphological changes in spindle thickness do not affect mitotic progression in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant cells through anaphase as seen by timelapse imaging and kymographic analysis versus wild type (<xref ref-type="fig" rid="f5">Fig. 5</xref>). However, following anaphase B, timely spindle breakdown is delayed in a significant percentage of <italic>pkl1&#x00394; cut7&#x00394;</italic> cells. In 78% of double mutant cells (<italic>n</italic>=15 timelapse series) spindles remain intact significantly longer than wild type (<italic>n</italic>=7 timelapse series) for an additional 24&#x000b1;7&#x02009;min, and can persist beyond formation of equatorial MTOC arrays (<xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c</xref>). Three patterns of spindle microtubule density in <italic>pkl1&#x00394; cut7&#x00394;</italic> anaphase cells were observed (<xref ref-type="fig" rid="f5">Fig. 5a&#x02013;c,e</xref>) and are referred to as Type 1, Type 2 and Type 3. In Type 1, central microtubule antiparallel overlap is identical to wild type. In Type 2, microtubule density is highly biased to one pole, and, in Type 3, central microtubules are diminished compared with thicker pole-biased microtubules. The relative frequencies of these patterns averaged over three time points after hydroxyurea synchronization (120, 140 and 160&#x02009;min) are indicated in a stacked histogram (<xref ref-type="fig" rid="f5">Fig. 5f</xref>). Our findings indicate that changes occur to spindle width and organization in both the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant and <italic>pkl1&#x00394;</italic> single mutant strains. Thickness along the spindle length is distributed in three patterns, two that are distinct from wild type. Spindle morphology phenotypes in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant are only modestly altered versus the single <italic>pkl1&#x00394;</italic> mutant strain. These findings indicate that kinesin-14 Pkl1 is the primary kinesin regulating microtubule organization at &#x003b3;-TuRC, a loss that results in broader spindles with asymmetric microtubule density along the spindle length.</p></sec><sec disp-level="2"><title>Daughter pole disorganization persists in the double mutant</title><p>An asymmetric effect on SPB organization with loss of the typical plaque-like appearance at one pole has been observed by TEM analysis of the <italic>pkl1&#x00394;</italic> strain<xref ref-type="bibr" rid="b20">20</xref>. Similarly in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant we observe an asymmetric effect on spindle poles, including altered astral microtubule arrays as previously shown<xref ref-type="bibr" rid="b33">33</xref>. Here we additionally identify the daughter pole as being primarily affected (<xref ref-type="fig" rid="f6">Fig. 6</xref>). Asymmetry in astral microtubule lengths from opposing poles is observed and orientation is parallel to the spindle axis beginning in early mitosis. Asymmetric astral arrays are observed in 35&#x000b1;6% of <italic>pkl1&#x00394;</italic> cells and 33&#x000b1;6% of <italic>pkl1&#x00394; cut7&#x00394;</italic> cells (<xref ref-type="fig" rid="f6">Fig. 6b</xref>; mean&#x000b1;s.e.m., <italic>n</italic>=200 cells per strain). A small percentage of cells in both strains (8&#x000b1;3% and 6&#x000b1;2%, respectively) exhibits parallel arrays that are symmetric, and the remainder of cells examined have normal appearing astral arrays. Cells without astral microtubules were also observed, but excluded from this analysis. We do not observe protrusions in the nuclear envelope as observed by co-imaging with nuclear envelope and SPB markers shown in <xref ref-type="fig" rid="f6">Fig. 6c</xref> (<italic>n</italic>=0/57 cells), suggesting that these arrays are cytoplasmic. The septation-initiation network (SIN) protein Cdc7 loads primarily to the daughter pole in mitosis<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>. We found that the longer abnormal astral microtubules extend from poles that harbour Cdc7-GFP (68&#x000b1;8% of cells with a signal, <italic>n</italic>=29 cells) and that this is also the pole with increased thickness (<xref ref-type="fig" rid="f6">Fig. 6a,d</xref>). These data extend the asymmetric SPB disorganization phenotype observed in <italic>pkl1&#x00394;</italic> cells<xref ref-type="bibr" rid="b20">20</xref>, identifying a role for Pkl1 in maintaining daughter pole organization from &#x003b3;-TuRC that indirectly alters astral microtubule arrays in the cytoplasm. Further, the data are consistent with daughter pole disorganization contributing to the thicker spindle phenotypes we observe in both <italic>pkl1&#x00394;</italic> and <italic>pkl1&#x00394; cut7&#x00394;</italic> mutant cells.</p></sec><sec disp-level="2"><title><italic>pkl1&#x00394;</italic> and <italic>pkl1&#x00394; cut7&#x00394;</italic> share chromosome segregation defects</title><p>We monitored chromosome segregation in the double mutant by live cell timelapse fluorescence microscopy and immunocytology of microtubules and DNA, as well as mini chromosome loss (<xref ref-type="fig" rid="f7">Fig. 7</xref>). Three types of chromosome segregation defects are present in the double mutant that are unequal segregation, lagging chromosomes and lost chromosomes (<xref ref-type="fig" rid="f7">Fig. 7a</xref>). Compared with wild-type cells, the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant strain has increased lagging chromosomes and unequally segregated chromosomes, as well as a minor increase in lost chromosomes<xref ref-type="bibr" rid="b36">36</xref>. However, we along with others<xref ref-type="bibr" rid="b20">20</xref> observe that the efficiency of chromosome segregation is already markedly reduced in the absence of kinesin-14 Pkl1. Compared with the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant strain, no lost chromosomes are observed in <italic>pkl1&#x00394;</italic> and unequal segregation is reduced, though lagging chromosomes are prominent. <xref ref-type="fig" rid="f7">Figure 7b</xref> is a histogram representation of the relative frequencies of these phenotypes in wild type, <italic>pkl1&#x00394;</italic> single mutant and <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant strains. Shown are frequencies (<italic>n</italic>=500 cells) for unequal segregation (Type 1; wild type: 0%, <italic>pkl1&#x00394;</italic>: 7&#x000b1;3% and <italic>pkl1&#x00394; cut7&#x00394;</italic>: 9&#x000b1;3%, mean&#x000b1;s.e.m.), lagging chromosomes (Type 2; 3&#x000b1;2, 23&#x000b1;5 and 30&#x000b1;5%) and chromosome loss from the spindle (Type 3; 0, 0 and 1%). We further quantified chromosome missegregation by monitoring the loss rate of a mini chromosome (<italic>cen1-7L</italic>) <italic>sup3E</italic> in the three strains (<xref ref-type="fig" rid="f7">Fig. 7c</xref>). Mini chromosome loss in the <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant is greater than that in wild type (27% increase) but reduced by 5% with respect to <italic>pkl1&#x00394;</italic> (32%). Our findings on chromosome segregation with <italic>pkl1&#x00394;</italic> are consistent with previous studies<xref ref-type="bibr" rid="b20">20</xref> and reveal both rescue (lagging chromosomes) and exacerbation (unequal segregation) of the <italic>pkl1&#x00394;</italic> phenotype in the double mutant along with the additional lost chromosome phenotype.</p><p>In wild-type cells, Mad2 monitors proper bipolar attachment of spindle microtubules to the kinetochore (KT), then transitions from KTs to both SPBs coincident with anaphase A onset. In anaphase B, Mad2 becomes asymmetric and makes a subsequent transition from the daughter pole to the equatorial MTOC, but remains asymmetrically localized at the mother pole<xref ref-type="bibr" rid="b26">26</xref>. We observe Mad2-GFP associated with the lost chromosome that is attached by an intranuclear microtubule (<xref ref-type="fig" rid="f7">Fig. 7d</xref>), similar to what was observed previously with the lost chromosome phenotype<xref ref-type="bibr" rid="b36">36</xref>. This lost chromosome event occurs in 1% of <italic>pkl1&#x00394; cut7&#x00394;</italic> cells (<italic>n</italic>=3/300 cells) and is not observed in <italic>pkl1&#x00394;</italic> cells. Mad2-GFP was also useful in confirming that the increased spindle microtubule thickness we observe in Type 2 morphologies (<xref ref-type="fig" rid="f5">Fig. 5b,e</xref>) is associated with the daughter pole. By imaging cells with Mad2-GFP and mCherry-Atb2, the increased spindle microtubule thickness extends primarily from the daughter spindle pole that is unmarked or dimly marked by Mad2-GFP in early anaphase B (<xref ref-type="fig" rid="f7">Fig. 7e</xref>), consistent with our Cdc7-GFP data (<xref ref-type="fig" rid="f6">Fig. 6</xref>). Our findings indicate that the chromosome segregation defects in the double mutant strain generally resemble those of <italic>pkl1&#x00394;</italic> except for the additional presence of the rare lost chromosome phenotype.</p></sec><sec disp-level="2"><title>An asymmetric block on &#x003b3;-TuRC nucleation competency by Pkl1</title><p>We previously demonstrated that kinesin-14 Pkl1 physically interacts with &#x003b3;-TuRC in prophase to downregulate its function and oppose bipolar spindle assembly<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b22">22</xref>. We established an <italic>in vivo</italic> assay system to examine the impact of elevated levels of Pkl1 in double mutant cells where kinesin-5 Cut7 is absent (<xref ref-type="fig" rid="f8">Fig. 8a</xref>). Multi copy <italic>pkl1</italic> was expressed using the low strength thiamine repressible nmt promoter (pREP81/<italic>pkl1</italic>). The <italic>pkl1&#x00394; cut7&#x00394;</italic> cells containing pREP81/<italic>pkl1</italic> were grown with or without 5&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 thiamine and were fixed after 17&#x02009;h. Cells were stained for &#x003b1;-tubulin and DNA using TAT1 antibody and Hoechst, respectively (<xref ref-type="fig" rid="f8">Fig. 8a</xref>). After thiamine release we observed that 89&#x000b1;9% of cells were arrested with unformed spindles. This frequency was only 6&#x000b1;2% when cells were grown in the presence of thiamine (<xref ref-type="fig" rid="f8">Fig. 8c</xref>, left plot; mean&#x000b1;s.e.m., <italic>n</italic>=200 cells for both conditions). In cells with unformed spindles, tubulin signal was concentrated near the cell centre. We were unable to obtain <italic>pkl1&#x00394; cut7&#x00394;</italic> cells transformed with <italic>pkl1</italic> expressed under a higher strength promoter (pREP90x/<italic>pkl1</italic>), even under promoter repressing conditions in the presence of thiamine. We additionally performed live cell fluorescence microscopy with GFP-Atb2 (&#x003b1;-tubulin) in the <italic>cut7-22</italic> temperature sensitive strain with or without the native <italic>pkl1</italic> gene (<xref ref-type="fig" rid="f8">Fig. 8b</xref>). Cells were synchronized by hydroxyurea for 4&#x02009;h, released at the 36&#x02009;&#x000b0;C restrictive temperature and imaged 4&#x02009;h after release. We observed similar unformed spindles in 82&#x000b1;9% of cells containing Pkl1. The <italic>pkl1&#x00394; cut7-22</italic> cells grown similarly exhibit robust spindle assembly (<xref ref-type="fig" rid="f8">Fig. 8c</xref>, right plot; <italic>n</italic>=200 cells for both conditions), and these data are consistent with the serial growth assays performed with these two strains at 36&#x02009;&#x000b0;C (<xref ref-type="fig" rid="f1">Fig. 1b</xref>).</p><p>We envision three possible models in which spindle assembly would fail via a spindle pole based mechanism that are based on tubulin seeding at &#x003b3;-TuRC (<xref ref-type="fig" rid="f8">Fig. 8d</xref>). This includes no addition of tubulin to &#x003b3;-TuRC, symmetrical addition but without microtubule elongation, or asymmetric addition of tubulin at a single spindle pole. Our data indicate the presence of short microtubules, ruling out model 1. To distinguish between models 2 and 3, we generated the strain [<italic>pkl1+ cut7-22 pcp1-GFP mCherry-atb2</italic>] to simultaneously visualize poles and microtubules (compare <xref ref-type="fig" rid="f8">Fig. 8b</xref> with <xref ref-type="fig" rid="f8">Fig. 8e</xref>). Our data support model 3. We observe that in 100% of arrested cells where two distinct poles could be identified, the tubulin signal was associated with a single pole (<italic>n</italic>=25 cells). <italic>In vivo</italic> nucleation shown by cold depolymerization and reformation of microtubules in <xref ref-type="fig" rid="f8">Fig. 8f</xref> demonstrates that, in <italic>pkl1&#x00394; cut7&#x00394;</italic> cells, &#x003b3;-TuRC can nucleate both interphase and spindle microtubules, consistent with our other data. Together, the <italic>in vivo</italic> data here are consistent with our model in which a required role for Cut7 in spindle assembly is to bind &#x003b3;-TuRC to oppose Pkl1 activity. Therefore, of these Klps, kinesin-14 Pkl1 in fission yeast is the primary negative regulator of &#x003b3;-TuRC microtubule nucleation.</p></sec><sec disp-level="2"><title>Kinesin-14 P&#x003b3;TR peptide arrests human breast cancer cells</title><p>All human &#x003b3;-TuSC proteins are functional in fission yeast<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. We previously developed biochemical tools in the form of kinesin-14 Tail peptides (<xref ref-type="fig" rid="f2">Fig. 2e</xref>)<xref ref-type="bibr" rid="b17">17</xref> that regulate &#x003b3;-TuRC <italic>in vitro</italic> and here tested the conserved capability of these peptides to block microtubule nucleation in human MCF-7 breast cancer cells (<xref ref-type="fig" rid="f9">Fig. 9</xref>). By immunocytology in fixed MCF-7 cells we demonstrate that 6-His tagged P&#x003b3;T targets the centrosome (<xref ref-type="fig" rid="f9">Fig. 9a</xref>) and co-localizes with &#x003b3;-tubulin (<xref ref-type="fig" rid="f9">Fig. 9b</xref>). By microtubule nucleation assays with or without &#x003b3;-TuRC, we also investigated the effect of kinesin-14 peptides on human &#x003b3;-TuRC nucleation competency <italic>in vitro</italic> (<xref ref-type="fig" rid="f9">Fig. 9c</xref>). The control targeting peptide P&#x003b3;T is insufficient to block &#x003b3;-TuRC microtubule nucleation alone. However, P&#x003b3;TR that contains an additional &#x003b3;-tubulin binding and regulatory sequence<xref ref-type="bibr" rid="b17">17</xref> is a potent inhibitor of nucleation. Peptides P&#x003b3;T and P&#x003b3;R (separated TR elements) isolate human &#x003b3;-TuRC components <italic>in vitro</italic> similar to their action in fission yeast (<xref ref-type="fig" rid="f2">Figs 2e</xref> and <xref ref-type="fig" rid="f9">9d</xref>). That is, P&#x003b3;T co-immunoprecipitates human GCP2, the yeast Alp4 counterpart, in addition to &#x003b3;-tubulin, while P&#x003b3;R binds and removes &#x003b3;-tubulin from the complex<xref ref-type="bibr" rid="b17">17</xref>.</p><p>By live cell transfection of human MCF-7 breast cancer cells that exhibit low aggressiveness or MDA-MB-231 cells that are highly aggressive, we show that P&#x003b3;TR is a potent mitotic spindle protein (MSP) class regulator of mitotic arrest (<xref ref-type="fig" rid="f9">Fig. 9e&#x02013;g</xref>). Cell lines were transfected with 1&#x02009;&#x003bc;g of 6-His tagged P&#x003b3;TR in 2&#x02009;ml (108&#x02009;&#x003bc;M) using the Chariot system (Active Motif) and fixed after 24&#x02009;h. At this time point, 43.3% of MCF-7 cells (681/1,572 cells counted from <italic>n</italic>=12 fields at &#x000d7; 200) and 27.7% of MDA-MB-231 cells (497/1,732 cells counted from <italic>n</italic>=12 fields at &#x000d7; 200) were transfected with peptide based on fluorescence staining using the 6-His tag. Of this, 39% of MCF-7 cells (613/1,572) and 22.6% of MDA-MB-231 cells were arrested in mitosis. Cells lacking peptide signal did not arrest. Breast cancer lines transfected with a control 6-His hexamer also did not arrest, indicating that this tag does not contribute to the anti-mitotic effect of P&#x003b3;TR. In a small percentage of the populations (4.3% MCF-7; 5.1% MDA-MB-231) with very low but detectable levels of peptide at the centrosome, cells appeared normal (MCF-7 shown in <xref ref-type="fig" rid="f9">Fig. 9f</xref>). In arrested cells, residual clumped microtubules are present but do not appear to extend from centrosomes, suggesting that P&#x003b3;TR has an inhibitory effect on &#x003b3;-TuRC microtubule nucleation <italic>in vivo</italic> in human cells. These findings suggest that kinesin-14 action at &#x003b3;-TuRC is conserved from the yeast SPB to the human centrosome. We expect that P&#x003b3;TR will be a powerful mechanistic tool to elucidate &#x003b3;-TuRC function for microtubule nucleation in multiple model organisms and a potential therapeutic tool for preventing cell proliferation in disease.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Microtubule organizing centres play major roles in specialized eukaryotic processes of broad interest such as spindle assembly, neuronal function and immunological synapse formation that involves cell polarization. Understanding the detailed mechanisms for microtubule nucleation requires combined knowledge of the underlying structure along with regulatory insights. In this work we demonstrate that the ability of fission yeast kinesin-14 Pkl1 to bind and alter &#x003b3;-TuRC structure and function<xref ref-type="bibr" rid="b17">17</xref> results in blocked microtubule nucleation <italic>in vivo</italic> generating failed spindle bipolarity and mitotic arrest. Conservation of this mechanism is revealed through use of a kinesin-14 Pkl1 peptide P&#x003b3;TR in human breast cancer cells that localizes to centrosomes and is sufficient to arrest nucleation in the two breast cancer cell lines investigated by preventing bipolar spindle formation. In fission yeast, kinesin-5 Cut7 but not kinesin-14 Pkl1, is an essential mitotic protein<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b27">27</xref>. To better understand kinesin-14 Pkl1 function at &#x003b3;-TuRC, and kinesin-5 counteraction of this Klp, we applied genetic analysis, biochemistry and timelapse imaging. Here we show that kinesin-5 Cut7 is dispensable in the absence of kinesin-14 Pkl1 and that counteraction of Pkl1 by Cut7 requires Cut7 binding to &#x003b3;-TuRC through its Motor and BimC domains. These Klps are the first identified to directly bind and regulate &#x003b3;-TuRC, actions that are sufficient to impact microtubule nucleation capability. These findings are expected to have significant impact in the cytoskeleton field, particularly in understanding MTOC function as well as in potential therapeutic anti-cancer applications that utilize mitotic spindle protein agonists.</p><p>Distinct mitotic phenotypes are present with loss of either Pkl1 or Cut7. The loss of <italic>pkl1</italic> in the presence of <italic>cut7</italic>, although viable, results in an asymmetric effect on daughter spindle pole organization that influences spindle width and impairs chromosome segregation. Compared with the double mutant <italic>pkl1&#x00394; cut7&#x00394;</italic>, no amelioration or exacerbation of the <italic>pkl1&#x00394;</italic> phenotype is observed, indicating that these phenotypes are likely due to loss of Pkl1. The additional loss of Cut7, however, does result in delayed spindle breakdown for mitotic exit. Cut7 localizes to the spindle midzone in anaphase<xref ref-type="bibr" rid="b37">37</xref>, and, although not required for anaphase B spindle elongation<xref ref-type="bibr" rid="b29">29</xref>, our data indicate it may contribute to normal progression through this stage. A primary role for kinesin-5 Cut7 is therefore to counteract kinesin-14 Pkl1 at &#x003b3;-TuRC. Only in the presence of Pkl1 does removal of Cut7 or inactivation of the Cut7 BimC domain (<italic>cut7-22</italic>) allow an asymmetric block on &#x003b3;-TuRC microtubule nucleation to be imposed that results in failed spindle bipolarity (<xref ref-type="fig" rid="f8">Fig. 8</xref>).</p><p>Extensive studies demonstrate both the importance of kinesin-5 motors in spindle assembly along with kinesin-5-independent mechanisms. In the latter, force generation by other microtubule motors such as nuclear envelope-associated dynein and kinesin-12 operate and include microtubule pushing forces on the opposing pole and kinetochore-mediated microtubule interactions in prophase<xref ref-type="bibr" rid="b38">38</xref>. Our ability to remove kinesin-5 Cut7 in the absence of kinesin-14 Pkl1 reveals that in fission yeast kinesin-5-independent mechanisms exist to establish spindle bipolarity.</p><p>Spindle phenotypes in the double mutant and single <italic>pkl1&#x00394;</italic> strains do not include changes to timing for prophase SPB separation or mitotic progression through anaphase B versus wild type. The increase in spindle thickness on loss of Pkl1 that we observe is reminiscent of phenotypes induced by loss of kinesin-14 Kar3 in budding yeast<xref ref-type="bibr" rid="b19">19</xref>. The thick spindle morphology did not result in increased resistance to the microtubule-depolymerizing drug TBZ at 10 or 20&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 concentrations in single or double mutant backgrounds versus wild type, consistent with no change in spindle microtubule number. We favour the model that altered microtubule organization of parallel microtubules emanating from the daughter pole results in increased spindle width at this pole as opposed to an increase in spindle microtubule number. This is consistent with studies by ref. <xref ref-type="bibr" rid="b20">20</xref> in which TEM analysis of <italic>pkl1&#x00394;</italic> cells revealed a decrease in pole organization characterized by loss of the typical plaque-like structure with apparent normal microtubule number. The replication of the SPB and centrosome is semi-conservative with the mother pole used as a template. To identify whether the mother or daughter pole is affected by loss of <italic>pkl1</italic>, we applied live cell fluorescence microscopy with asymmetric pole markers Cdc7-GFP and Mad2-GFP along with mCherry-Atb2 to mark microtubules. Our studies reveal that the daughter pole is affected in both <italic>pkl1&#x00394;</italic> single mutant and <italic>pkl1&#x00394; cut7&#x00394;</italic> double mutant cells. Daughter pole disorganization additionally affects cytoplasmic astral microtubule arrays. Mitotic events can influence licensing and semi-conservative centrosome replication in the succeeding G1/S. In human cells, separase and polo kinase license centrosomes for duplication in the next cell cycle during mitosis<xref ref-type="bibr" rid="b39">39</xref>. Whether loss of kinesin-14 Pkl1 impacts subsequent cell cycle events outside of mitosis is not known. However, the changes to daughter spindle pole integrity without Pkl1 indicate a broader role beyond microtubule nucleation for spindle assembly, such as maturation or integrity of the daughter MTOC. We did not detect a similar role with kinesin-5 at the daughter MTOC and additional loss of Cut7 does not exacerbate these phenotypes. The concept of asymmetric events at spindle poles is well known. In budding yeast, &#x003b3;-tubulin mutants have been isolated that block robust microtubule nucleation from a single pole as seen by transmission electron microscopy<xref ref-type="bibr" rid="b40">40</xref>. In human cells, mother centriole stability is asymmetrically affected by kinesin-13 Kif24 (ref. <xref ref-type="bibr" rid="b41">41</xref>). As well, regulation of poles can be asymmetric and is observed in mitotic checkpoint pathways<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref><xref ref-type="bibr" rid="b46">46</xref><xref ref-type="bibr" rid="b47">47</xref> that monitor spindle assembly, positioning and timing to help ensure the accurate segregation of chromosomes in cell division.</p><p>In this study, we demonstrate that the role of kinesin-14 at &#x003b3;-TuRC is to block microtubule nucleation and that key domains are required for this mechanism. This ability to localize to &#x003b3;-TuRC at spindle pole bodies is conserved with &#x003b3;-TuRC in the mammalian centrosome. We additionally identified the ability of kinesin-5 to bind &#x003b3;-TuRC as a key component in the Klp/&#x003b3;-TuRC regulatory mechanism in fission yeast. Pkl1 interacts with &#x003b3;-TuRC through two domains, a Motor domain that binds to &#x003b3;-tubulin helix 11 and distinct Tail domain binding to the complex. The combined domains provide strongest interactions with &#x003b3;-TuRC<xref ref-type="bibr" rid="b17">17</xref>. Our data indicate that similar to Pkl1, the Tail domain of Cut7 is the primary &#x003b3;-TuRC targeting element. We hypothesize that the Motor domain plays a role in assisted targeting to the &#x003b3;-TuRC site at spindle poles and in competition with Pkl1 binding to this site. Consistent with our previously published findings on Pkl1, we observe that the combined domains of Cut7 provide the strongest interactions with &#x003b3;-TuRC. However, unlike Pkl1 that has low affinity to microtubules<xref ref-type="bibr" rid="b48">48</xref>, Cut7 retains the ability to bind strongly to spindle tubulins when &#x003b3;-tubulin specific binding is prevented. This alternative site of interaction may lower the pool of Cut7 at &#x003b3;-TuRC. Thus, as a consequence of blocked loading due to the helix 11 mutation and retained high microtubule binding affinity, we would expect reduced binding of Cut7 to &#x003b3;-TuRC complexes with &#x003b3;-tubulin-K5A present as compared with Cut7ST, as observed. Interestingly, the <italic>cut7-22</italic> mutation lies within a MAP kinase phosphorylation consensus sequence in the conserved BimC sequence of the Cut7 Tail domain<xref ref-type="bibr" rid="b29">29</xref>, indicating that phosphorylation at this or other kinase sites within this domain may be important in the &#x003b3;-TuRC mechanism. Finally the dual regulatory relationship of kinesin-14 and kinesin-5 at the &#x003b3;-TuRC in fission yeast, along with the ability of P&#x003b3;TR peptide to block nucleation and spindle bipolarity in breast cancer cells, is expected to be impactful in regard to cancer therapy<xref ref-type="bibr" rid="b49">49</xref>. We are interested in exploring development of this new class of mitotic spindle protein (MSP) reagents as an addition to combined cancer therapies, in particular with Ispinesib/Monastrol<xref ref-type="bibr" rid="b50">50</xref><xref ref-type="bibr" rid="b51">51</xref> that targets human kinesin-5 or in response to taxol-resistant cancers.</p><p>Our findings are of particular interest in regard to multiple clinically oriented studies<xref ref-type="bibr" rid="b52">52</xref><xref ref-type="bibr" rid="b53">53</xref><xref ref-type="bibr" rid="b54">54</xref><xref ref-type="bibr" rid="b55">55</xref><xref ref-type="bibr" rid="b56">56</xref><xref ref-type="bibr" rid="b57">57</xref><xref ref-type="bibr" rid="b58">58</xref><xref ref-type="bibr" rid="b59">59</xref><xref ref-type="bibr" rid="b60">60</xref> that demonstrate overexpression of &#x003b3;-tubulin and other centrosomal proteins is characteristic of tumorigenesis and human malignancies in multiple tissues. In this case supernumerary microtubule-nucleating centrosomes are often observed and result in abnormal multipolar mitoses, aneuploidy and ultimately cell death<xref ref-type="bibr" rid="b58">58</xref><xref ref-type="bibr" rid="b59">59</xref><xref ref-type="bibr" rid="b61">61</xref>. Similarly, overexpression of &#x003b3;-tubulin in malignant cells can also produce ectopic microtubule nucleation in the cytoplasm. This is thought to result from &#x003b3;-tubulin-centrosome decoupling as well as subcellular sorting changes to soluble cytoplasmic pools or insoluble centrosomal complexes<xref ref-type="bibr" rid="b52">52</xref><xref ref-type="bibr" rid="b57">57</xref><xref ref-type="bibr" rid="b58">58</xref><xref ref-type="bibr" rid="b60">60</xref> as well as insoluble cytoplasmic aggregates<xref ref-type="bibr" rid="b53">53</xref>. Interestingly, an increase in the percentage of soluble cytoplasmic &#x003b3;-tubulin is associated with cell lines of higher aggressiveness and poorer prognosis versus those of low or moderate aggressiveness<xref ref-type="bibr" rid="b58">58</xref>. Further, &#x003b3;-tubulin can be incorporated within the &#x003b1;-/&#x003b2;-tubulin lattice of cytoplasmic microtubules that may impact drug resistance<xref ref-type="bibr" rid="b58">58</xref>. The ability of the P&#x003b3;TR peptide to target complexed &#x003b3;-tubulin could allow a means to prevent ectopic microtubule nucleation, although currently this is untested. Regardless, the peptide can mitotically arrest both the MCF-7 low and MDA-MB-231 high aggressive cell lines with similar efficiency. The benefit of P&#x003b3;TR versus other anti-mitotic agents that solely target microtubules is yet to be determined but is of interest in particular for malignant cell lines that are difficult to arrest and that often develop resistance.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>General yeast strains and growth conditions</title><p>Standard procedures for genetic manipulation of fission yeast are as described<xref ref-type="bibr" rid="b62">62</xref> (<italic>S. pombe</italic> strains in <xref ref-type="table" rid="t1">Table 1</xref>). Cultures grown in fully supplemented YES-rich medium or minimally supplemented medium are also as described<xref ref-type="bibr" rid="b62">62</xref>. For yeast transformations, we used the EZ-YEAST Transformation Kit (MP Biochemicals). In growth assays, cells were grown to logarithmic phase in 10&#x02009;ml rich YES media at 27&#x02009;&#x000b0;C. Cells were counted by haemocytometer and equalized and spotted at an initial concentration of 2 &#x000d7; 10⁷ cells&#x02009;ml^&#x02212;1 with followed by 1/10 serial dilutions. Plates at 30&#x02009;&#x000b0;C and 36&#x02009;&#x000b0;C were grown for 4&#x02013;5 days (<italic>n</italic>=3 experiments). Plates at 25&#x02009;&#x000b0;C were grown for 7 days. For promoter induction using the pREP81 low strength<xref ref-type="bibr" rid="b63">63</xref> or pREP90x high strength<xref ref-type="bibr" rid="b64">64</xref> nmt plasmids, cells were maintained on plates containing 5&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 thiamine before inoculation in 10&#x02009;ml selective media with (control) or without (test) 5&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 thiamine for 17&#x02009;h. Plates used to assess viability contained 5&#x02009;mg&#x02009;l^&#x02212;1 Phloxine B (Sigma-Aldrich). Mini chromosome loss was measured as described<xref ref-type="bibr" rid="b65">65</xref>. Growth curves were obtained using haemocytometer.</p></sec><sec disp-level="2"><title>Yeast strain constructions</title><p>Integration of the <italic>ura4</italic> gene at the <italic>cut7</italic> locus was done using a PCR-based gene-targeting approach with long tracts of flanking homology as previously described<xref ref-type="bibr" rid="b66">66</xref> (Epicentre MasterAmp Extra-Long PCR Kit). We used 500&#x02009;bp homology upstream and downstream of the <italic>cut7</italic> open reading frame and verified stable integrants by colony PCR (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). Plasmid Integration was done with pREP vectors using homologous recombination at the autonomous replication site (Mlu1, New England Biolabs). All genetic crosses were done on minimal sporulation media, followed by marker selection and colony PCR.</p></sec><sec disp-level="2"><title>Synchronous yeast culture</title><p>Cultures were grown overnight in YES-rich or selective minimal media at 27&#x02009;&#x000b0;C using baffled flasks. Hydroxyurea (11&#x02009;mM) was added to cultures in logarithmic phase and incubated for 4&#x02009;h. Cells were then washed twice with 10&#x02009;ml sterile water before release in fresh media. Depending on the experiment, cells were released at 27&#x02009;&#x000b0;C (permissive temperature), 32&#x02009;&#x000b0;C (microtubule repolymerization) or 36&#x02009;&#x000b0;C (restrictive temperature).</p></sec><sec disp-level="2"><title>FPLC sedimentation and immunoprecipitate analysis</title><p>Yeast whole-cell extracts were prepared using mechanical bead beating (Mini-Beadbeater-16, Biospec) in Buffer P (50&#x02009;mM Na₂PO₄ pH 7.2, 10% glycerol, 150&#x02009;mM NaCl 5&#x02009;mM ATP, 100&#x02009;&#x003bc;M GTP])with protease inhibitors (PMSF-1&#x02009;mM, Leupeptin-50&#x02009;&#x003bc;M, Pepstatin-2&#x02009;&#x003bc;M, Aproptinin-175&#x02009;nM and Pefabloc-200&#x02009;&#x003bc;M). Three centrifugations at 17,000&#x02009;<italic>g</italic> (1&#x02009;min, 5&#x02009;min, 30&#x02009;min) were used to clarify cell extracts. Separose 6 FPLC was performed as described<xref ref-type="bibr" rid="b15">15</xref>. For immunoprecipitation, whole-cell extracts were incubated with anti-V5-tag mAb-Magnetic beads (MBL International) at 4&#x02009;&#x000b0;C for 30&#x02009;min. Beads were washed three times with Buffer P before elution by boiling and immediate analysis by western blotting. Pkl1 peptide co-immunoprecipitation assays were performed as previously described<xref ref-type="bibr" rid="b17">17</xref>. Antibodies used were primary mouse anti-&#x003b3;-tubulin monoclonal (1:10,000; Sigma-Aldrich cat. T5326), primary rabbit anti-HA epitope tag (1:5,000; Rockland cat. 600-401-384), primary rabbit anti-FLAG polyclonal (1:320; Sigma-Aldrich cat. F7425), primary mouse anti-V5 monoclonal (1:5,000; Life Technologies cat. R96025) or mouse anti-V5 IgG HRP conjugated monoclonal (1:5,000; Life Technologies cat. R96125), goat anti-rabbit IgG HRP conjugate (1:20,000; Millipore cat. 12-348) and goat anti-mouse IgG HRP conjugate (1:10,000; Novagen cat. 71045).</p><p>Human lysates were prepared by harvesting confluent cells with 2&#x02009;ml TrypLE (Life Technologies) and centrifuging for 5&#x02009;min at 1,000&#x02009;r.p.m. followed by two washes with 1&#x02009;ml 1X PBS. Cells were lysed by incubation on ice in RIPA+ Buffer (Tris&#x02013;HCl pH 7.5, 50&#x02009;mM, NaCl 150&#x02009;mM, 1% Triton X-100, 1% deoxycholic acid sodium salt, 0.1% SDS; supplemented with Leupeptin 5&#x02009;mM, Pepstatin 2&#x02009;&#x003bc;M, Aprotinin 175&#x02009;nM, PMSF 1&#x02009;mM+GTP 100&#x02009;&#x003bc;M) for 45&#x02009;min, mixing occasionally. Lysates were clarified by centrifugation at 14,000&#x02009;r.p.m. (20,817&#x02009;<italic>g</italic>) at 4&#x02009;&#x000b0;C for 1&#x02009;h. Peptide co-immunoprecipitation assays were performed as above. Antibodies used for western blots were primary mouse anti-&#x003b3;-tubulin monoclonal (1:9,000; Sigma-Aldrich cat. T5326) and primary rabbit anti-GCP2 polyclonal (1:2,000; Thermo Scientific cat. PIPA521433). The secondary antibodies mentioned above were used for detection by HRP. Uncropped scans of western blots are provided in <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1</xref> and <xref ref-type="supplementary-material" rid="S1">2</xref>.</p></sec><sec disp-level="2"><title>Breast cancer cell culture and peptide transfection</title><p>MCF-7 and MDA-MB-231 (ATCC) cells were maintained in 25&#x02009;cm² tissue culture treated Corning flasks (Sigma-Aldrich) in DMEM complete medium with Glutamax-1 and supplemented with 10% fetal bovine serum. MCF-7 DMEM complete medium was additionally supplemented with 0.01&#x02009;mg&#x02009;ml^&#x02212;1 bovine insulin (Sigma-Aldrich). Flasks were maintained at 37&#x02009;&#x000b0;C in 5% CO₂, 95% air, and cells were passaged every 5&#x02013;7 days using 1&#x02009;ml TrypLE (Life Technologies).</p><p>For live cell peptide transfection we used the Chariot system (Active Motif) according to the manufacturer&#x02019;s instructions. Cells were seeded into 35&#x02009;mm tissue culture-treated dishes containing coverslips and grown in complete medium to ~60% confluency. One microgram of kinesin-14 Tail peptide P&#x003b3;TR (GenScript) was diluted in 100&#x02009;&#x003bc;l 1 &#x000d7; PBS on ice. Six microlitres of 1/10 PBS-diluted Chariot reagent was further diluted into 100&#x02009;&#x003bc;l sterile water on ice in a separate tube. Diluted peptide and diluted Chariot were combined and incubated at room temperature for 30&#x02009;min to allow the Chariot-peptide complex to form. Following incubation, media was aspirated from cells, which were then washed once in 1 &#x000d7; PBS. The entire 200&#x02009;&#x003bc;l volume was added to cells in 400&#x02009;&#x003bc;l serum-free media and gently rocked to ensure even delivery. Plates were returned to 37&#x02009;&#x000b0;C for 1&#x02009;h. Next, DMEM complete growth medium was added to 2&#x02009;ml without removing the peptide delivery solution (108&#x02009;&#x003bc;M peptide in 2&#x02009;ml). Cells were further incubated at 37&#x02009;&#x000b0;C overnight and fixed after 24&#x02009;h.</p></sec><sec disp-level="2"><title>Fluorescence microscopy and immunocytochemistry</title><p>Fluorescence microscopy was performed using Zeiss Observer.Z1 inverted microscope with 63X Plan-Apochromat 1.4 NA oil and &#x000d7; 100 oil 1.45 PlanFLUAR DIC objectives. Data were obtained using a Hamamatsu ORCA ER CCD camera with Zeiss Axiovision Rel 4.8 acquisition software. We acquired 20-image 0.1&#x02009;&#x003bc;m Z-stacks. Timelapse series were acquired every 30&#x02009;s to 6&#x02009;min, with a median interval of 2&#x02009;min. The 10-image 0.1-micron Z-stacks were superimposed on each timelapse image in a series. With live cells, GFP-Atb2 was imaged at 50&#x02013;60&#x02009;ms exposure and mCherry-Atb2 was imaged at 500&#x02009;ms exposure. Only GFP-Atb2 was used in timelapse. Average temperature in the imaging room was 23&#x02009;&#x000b0;C. Using &#x02212;20&#x02009;&#x000b0;C methanol fixation, we were able to preserve GFP signal for quantifying phenotypes. In immunocytochemistry, microtubules were stained with a primary TAT1 antibody (1:25)<xref ref-type="bibr" rid="b67">67</xref>, followed by secondary goat anti-mouse Alexa Fluor 488 IgG (1:50; Life Technologies cat. A-11001) and DNA was stained in 1&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 Hoechst. A monoclonal anti-V5 primary antibody conjugated to FITC was used for viewing V5-tagged Cut7 constructs in fixed cells (1:500; Life Technologies cat. R963-25). Cells were imaged immediately using the Zeiss Observer.ZI system. In <xref ref-type="fig" rid="f8">Fig. 8e</xref>, zoomed images were made high contrast. Z-stacks were made into 2D projections using ImageJ. Cold depolymerization and repolymerization of <italic>in vivo</italic> microtubules was performed as previously described<xref ref-type="bibr" rid="b68">68</xref>. Wild type and <italic>pkl1&#x00394; cut7&#x00394;</italic> cells containing the integrated <italic>GFP-atb2</italic> (&#x003b1;-tubulin) plasmid were fixed to preserve GFP signal and analysed using the Zeiss system.</p><p>Human cells were fixed on glass coverslips in &#x02212;20&#x02009;&#x000b0;C methanol for 10&#x02009;min, washed with 1 &#x000d7; PBS and permeabilized in 0.5% Triton X-100 for 20&#x02009;min. Following further washes, cells were blocked for 30&#x02009;min in 1% bovine serum albumin/knock out serum replacement (BSA/KOSR). For peptide localization to centrosomes, 1&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 of peptide was applied after blocking and before primary antibody application. Antibodies used were primary mouse anti-&#x003b3;-tubulin monoclonal (1:5,000; Sigma-Aldrich cat. T5326) or primary mouse anti-&#x003b1;-tubulin monoclonal DM1A (1:1,000; Santa Cruz Biotech cat. sc-32293), primary His-tag polyclonal antibody (1:1,000; Cell Signaling cat. 2365), secondary goat anti-mouse Alexa Fluor 488 IgG (1:1,000; Life Technologies cat. A-11001) and secondary goat anti-rabbit Texas Red (1:1,000; Life Technologies cat. T-6391). Antibodies were diluted in 1% BSA/KOSR antibody dilution buffer. After secondary antibody application and washes, DNA was stained with 1&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 Hoechst solution in 1 &#x000d7; PBS for 10&#x02009;min followed by three times final PBS washes and mounted on slides with ProLong Gold anti-fade (Life Technologies). The 40-image 0.1&#x02009;&#x003bc;m Z-stacks were made into maximum intensity 2D projections using ImageJ.</p></sec><sec disp-level="2"><title><italic>In vitro</italic> microtubule nucleation assays</title><p><italic>In vitro</italic> microtubule nucleation assays were performed in a total volume of 5&#x02009;&#x003bc;l. That is, 3&#x02009;&#x003bc;l for the sample and 2&#x02009;&#x003bc;l of tubulin at a 1:5 ratio of Rhodamine:unlabelled tubulin (Cytoskeleton). Total tubulin concentration was 3.75&#x02009;&#x003bc;g&#x02009;&#x003bc;l^&#x02212;1 in 2.5 &#x000d7; tubulin working buffer (2.5 &#x000d7; BRB80: 200&#x02009;mM PIPES, 2.5&#x02009;mM MgCl₂, 2.5&#x02009;mM EGTA at pH 6.8 and 2.5&#x02009;mM GTP). For whole-cell extract nucleation analysis with peptide, 2&#x02009;&#x003bc;l of whole-cell extract was added with 1&#x02009;&#x003bc;l of peptide at 300&#x02009;nM for a final peptide concentration of 60&#x02009;nM. This 3&#x02009;&#x003bc;l combination was added first followed by tubulin working buffer. For samples with no peptide, the 5&#x02009;&#x003bc;l final volume comprised 3&#x02009;&#x003bc;l RIPA buffer and 2&#x02009;&#x003bc;l of whole-cell extract. The 5&#x02009;&#x003bc;l reaction was combined on ice, quickly spun and returned to ice before incubating in a 37&#x02009;&#x000b0;C water bath for 4&#x02009;min. Sample incubation was staggered at 20&#x02009;s intervals to allow for pipetting. After 4&#x02009;min, 50&#x02009;&#x003bc;l of 1% glutaraldehyde fixing solution was added and tubes were incubated at room temperature for 3&#x02009;min. Samples were completed by addition of 1&#x02009;ml 1 &#x000d7; BRB80, inverting multiple times to mix. For analysis, 50&#x02009;&#x003bc;l of this mixture per sample was sedimented by ultracentrifugation at 173,000 &#x000d7; <italic>g</italic> through a 15% glycerol cushion onto glass coverslips and imaged by Rhodamine epifluorescence using the Zeiss system at &#x000d7; 630. Images of multiple fields were collected and the average microtubule number per field was determined.</p></sec><sec disp-level="2"><title>Structural analysis</title><p>PyMol molecular visualization software (V1.5) was used for structural analysis of the conserved &#x003b1;-&#x003b2;-tubulin heterodimer 1TUB<xref ref-type="bibr" rid="b10">10</xref> and conserved &#x003b3;-tubulin monomer 1Z5V<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b69">69</xref> in <xref ref-type="fig" rid="f3">Fig. 3</xref>.</p></sec><sec disp-level="2"><title>Statistical analysis</title><p>For statistical analysis of phenotypes, <italic>n</italic> values were chosen as number of cells per strain needed to ensure adequate power to detect significant outcomes. <italic>P</italic>-values were generated using Student&#x02019;s <italic>t</italic>-test and statistical significance was considered for <italic>P</italic>&#x0003c;0.05 as appropriate. All statistical data in this study are reported as mean&#x000b1;s.d. or &#x000b1;s.e.m., as indicated. For cell cycle arrest by 1&#x02009;&#x003bc;g transfection of P&#x003b3;TR, 12 fields at &#x000d7; 200 were counted. Arrested cells with positive peptide signal were taken as a percentage of the entire population. Cells that were negative for peptide signal did not arrest.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>J.L.P. and Z.T.O. designed experiments and wrote the manuscript. Z.T.O. performed genetics, strain construction, cell biology microscopy, cell culture and constructed figures. A.G.C. assisted in microscopy and performed fission yeast biochemistry. T.D.R. performed <italic>in vitro</italic> microtubule nucleation, human biochemistry, yeast tetrad dissection and serial growth assays.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Olmsted, Z. T. <italic>et al</italic>. Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by &#x003b3;-TuRC in yeast and human cells. <italic>Nat. Commun.</italic> 5:5339 doi: 10.1038/ncomms6339 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-2</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6339-s1.pdf"/></supplementary-material></sec>

Satellite telemetry and social modeling offer new insights into the origin of primate multilevel societies

<p>Multilevel societies (MLS), in which polygynous reproductive units are nested in a larger social matrix, represent a highly complex social system documented only in a small number of mammalian species. Using long-term behavioural data, satellite telemetry and social network analysis, we present a new framework for understanding the function and social dynamics of the golden snub-nosed monkey MLS. Here we show that several one-male units form a cohesive breeding band that associates with one or more all-male units to form a herd. Herds seasonally aggregate and exchange members, thus facilitating gene flow and inbreeding avoidance. This MLS evolved from the aggregation of independent one-male, multifemale units that characterize ancestral Asian colobines; the evolutionary pathway leading to this MLS contrasts with that proposed for African papionins, which appear to have undergone internal fissioning of multimale&#x02013;multifemale groups. The results suggest that both environmental and phylogenetic factors are important in the evolution of a primate MLS.</p>

<contrib contrib-type="author"><name><surname>Qi</surname><given-names>Xiao-Guang</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Garber</surname><given-names>Paul A.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ji</surname><given-names>Weihong</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Zhi-Pang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Kang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Peng</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Song-Tao</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Xiao-Wei</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Gang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Pei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Bao-Guo</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a5">5</xref></contrib><aff id="a1"><label>1</label><institution>College of Life Sciences, Northwest University</institution>, Xi&#x02019;an 710069, <country>China</country></aff><aff id="a2"><label>2</label><institution>Anthropology Department, University of Illinois</institution>, Urbana, Illinois 61801, <country>USA</country></aff><aff id="a3"><label>3</label><institution>Institute of Natural Resource, Massey University</institution>, Albany 0632, <country>New Zealand</country></aff><aff id="a4"><label>4</label><institution>School of Sociology and Anthropology, Sun Yat-Sen University</institution>, Guangzhou 510275, <country>China</country></aff><aff id="a5"><label>5</label><institution>Institute of Zoology, Shaanxi Academy of Sciences</institution>, Xi'an 710032, <country>China</country></aff>

Nature Communications

<p>The diversity of animal social systems represents an adaptive response to environmental and social pressures experienced during their evolutionary history<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref>. Multilevel or modular societies (MLS) represent a complex form of group organization composed of several independent social units nested within a larger social matrix of several hundred individuals<xref ref-type="bibr" rid="b4">4</xref>. Although rare among mammals, MLS are found in elephants, equids, whales and a small number of primate species<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>. Primates are noteworthy in exhibiting enhanced cognitive abilities, which appear to have evolved in response challenges associated with living in a complex and changing social environment<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>. Most primate species live in social groups containing 4&#x02013;30 individuals that occupy partially exclusive home ranges or are territorial and generally avoid each other<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b13">13</xref>. However, primate MLS differ from this pattern in that they are comprised of several socially and spatially distinct one-male, multifemale units (OMUs) that coordinate their activities and feed, forage, rest and travel together to form a single large band. This social system has only been reported in geladas (<italic>Theropithecus gelada</italic>), hamadryas baboons (<italic>Papio hamadryas</italic>), some Asian colobines (in particular snub-nosed monkeys of the genus <italic>Rhinopithecus</italic> spp.) and humans<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>.</p><p>Despite certain similarities in the structural properties of primate MLS, studies conducted over several decades on geladas and hamadryas baboons revealed that the underlying social dynamics of their MLS are fundamentally different<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b17">17</xref>. In geladas, several OMUs aggregate to form a band, characterized by male dispersal and female philopatry within their natal OMU<xref ref-type="bibr" rid="b18">18</xref>. Strong female kin-bonds within a matrilineal harem play an important role in social cohesion<xref ref-type="bibr" rid="b19">19</xref>. Bands may merge to form a large community of up to 600 individuals<xref ref-type="bibr" rid="b20">20</xref>. In the case of hamadryas baboons, however, two or three OMUs spatially associate to form a patrilineal clan<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>. Males commonly remain within their natal clans and females transfer between OMUs and clans<xref ref-type="bibr" rid="b23">23</xref>. Several clans aggregate to form a band, OMUs rarely transfer between clans or bands<xref ref-type="bibr" rid="b22">22</xref>. In contrast to geladas, female hamadryas baboons develop stronger social relationships with their residential male than with other harem females<xref ref-type="bibr" rid="b24">24</xref>.</p><p>Although there is no consensus as to the set of factors that selected for primate MLS, two evolutionary pathways for MLS have been proposed<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>. In gelada and hamadryas baboon, ancestral mixed-sex multimale multifemale groups of African papionins appear to have undergone a process of internal fissioning resulting in the formation of harem-based mating units nested within the band (Fission Model). Enhanced sexual dimorphism in body mass, hair patterns and pelage colour in African papionins resulting from intrasexual competition may have enabled individual males to improve their breeding success by maintaining exclusive access to several female mating partners<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>. This Fission model assumes that a change in the spatial dispersion of feeding sites into small and scattered patches coupled with male monopolization of females<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b31">31</xref> are the primary drivers of a MLS.</p><p>More recently, evidence of the existence of a MLS in Asian colobines of the genus <italic>Rhinopithecus</italic> has offered new insights into the origin and evolutionary history of modular societies. In snub-nosed monkeys, band size ranges from 50 to several hundred individuals distributed into four to &#x0003e;25 OMUs, one or more all-male units (AMUs) composed of sub-adult and adult males and solitary males<xref ref-type="bibr" rid="b32">32</xref>. This is in marked contrast to the size and social organization found in other Asian colobines, in which single OMUs of 7&#x02013;20 individuals living in separate home ranges are common<xref ref-type="bibr" rid="b33">33</xref>. Researchers have proposed that social pressures associated with reproductive competition by invading bachelor males<xref ref-type="bibr" rid="b34">34</xref> and ecological pressures associated with efficiently locating and harvesting widely scattered and seasonally limited resources<xref ref-type="bibr" rid="b35">35</xref> have selected for the semi-permanent aggregation of several OMUs into a large MLS (Fusion Model).</p><p>Snub-nosed monkeys (genus <italic>Rhinopithecus</italic> spp.) include five species of endangered leaf monkeys that inhabit the northern-most distribution of Asian colobines. These primates exploit seasonal mountainous temperate and subtropical forests at an altitude of up to 4,500&#x02009;m in China, Vietnam and Myanmar<xref ref-type="bibr" rid="b36">36</xref><xref ref-type="bibr" rid="b37">37</xref>. Although early accounts of snub-nosed monkeys suggested that they lived in aggregation of extremely large number of social units<xref ref-type="bibr" rid="b38">38</xref>, difficulties in habituating and following identified individuals across their large and rugged mountainous terrain made direct observations almost impossible<xref ref-type="bibr" rid="b39">39</xref>, leading to a limited understanding of the dynamics of their MLS.</p><p>Long-term observations of golden snub-nosed monkeys (<italic>Rhinopithecus roxellana</italic>) at one site in the Qinling Mountains of north central China, have resulted in detailed data on the social organization of a breeding band. Initial research revealed that each OMU represents a socially close-knit and cohesive unit that engages in few direct social interactions with other OMUs that are embedded in the same band<xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref>. Studies of affiliation patterns between individuals within OMUs showed that both intersexual bonds and female-female kin-bonds contribute to the maintenance and cohesion of an OMU<xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref>. There also is evidence that adult and sub-adult females transfer between OMUs of the same band, whereas males tend to disperse between bands<xref ref-type="bibr" rid="b41">41</xref><xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref>. In this regard patterns of individual transfer and social affiliation in golden snub-nosed monkeys differ from that reported in the matrilineal social units of geladas or the patrilineal clans of hamadryas baboons<xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b46">46</xref>.</p><p>Given the fact that snub-nosed monkey breeding bands utilize extremely large home ranges (20&#x02009;km²) and that neighbouring bands rarely come into contact<xref ref-type="bibr" rid="b47">47</xref>, a critical set of questions in understanding the evolution and dynamics of the snub-nosed monkey MLS concerns how do neighbouring bands partition the landscape, whether inter-band relations are best characterized by attraction or avoidance, and how do AMUs and solitary males move in relation to each other and neighbouring breeding bands. In previous years, we have noted several cases in which individuals from outside the band have migrated into the study band. This includes individual adult males who replaced the residential male, fully formed OMUs that entered the band and unidentified females who entered the band and joined existing OMUs (<xref ref-type="fig" rid="f4">Fig. 4</xref>). The origin of these immigrants was unknown. Beginning in 2003, we encountered a neighbouring breeding band (DJF) as well as their satellite AMUs that occasionally merged with our study band into a single large troop of over 300 individuals. Limited evidence indicated tolerance between the two bands when they merged.</p><p>Over the course of several years, we observed neighbouring bands at our site to occasionally fuse and we recorded unknown individuals entering our primary study band. This led us to hypothesize that in golden snub-nosed monkeys two or more breeding bands, their satellite AMUs and solitary males temporally aggregate to form the West Ridge Troop (WRT). The function of this troop-level social grouping appears to facilitate the exchange or transfer of individuals between otherwise isolated breeding bands. We refer to this as the &#x02018;Gene Flow Hypothesis&#x02019;. Due to logistical constraints associated with following multiple bands of golden snub-nosed monkeys simultaneously across steep mountainous terrain, we fitted global positioning system (GPS) collars on five adult males, each from a different component of the snub-nosed monkey MLS: (1) the GNG-breeding band, composed of 13 OMUs with 138 individuals of both sexes, (2) the GNG-all-male band, composed of 3 AMUs with 28 bachelor males, (3) the DJF-breeding band, composed of 7 OMUs with 87 individuals of both sexes, (4) the DJF-AMU, composed of 16 bachelor males and (5) a solitary adult male that followed the GNG-breeding band. We integrated 14 years of monitoring the social dynamics of golden snub-nosed monkeys in the Qinling Mountains, Zhouzhi, China with satellite telemetry and social network analysis (SNA) to record the spatial associations and movement patterns of these five social components, to identify the underlying structure and function of the multilevel society of the golden snub-nosed monkey.</p><p>The results demonstrate that the golden snub-nosed monkey MLS is organized into a nested series of expanding social relationships including the OMU, AMU, band, herd and troop. Each level is characterized by different patterns of temporal and spatial association, individual and OMU exchange and differential access to reproductive partners. We hypothesize that the golden snub-nosed monkey MLS evolved from the aggregation of several independent OMUs, into a large and stable band of over 100 individuals that functions to decrease predation risk, promote inbreeding avoidance and enhance gene flow exploiting extremely large home ranges and highly fragmented high altitude mountainous landscapes. We conclude that both phylogenetic and ecological factors play an important role in the origin and evolution of primate MLS.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Association pattern among social components of MLS</title><p>Based on a Kernel density analysis of location data obtained from the five GPS collars, we found that the home ranges of the GNG-breeding band, the GNG-all-male band and the solitary male overlapped by 72.9% (<xref ref-type="fig" rid="f1">Fig. 1</xref>). The average distance between the GNG-breeding band and the GNG-all-male band was 0.52&#x000b1;0.21&#x02009;km (0.13&#x02013;1.53&#x02009;km), which was significantly smaller than the distance between either social component and the solitary male (One-way analysis of variance: <italic>F</italic>_{2, 5109}=240.59, <italic>P</italic>&#x0003c;0.001) (<xref ref-type="fig" rid="f1">Fig. 1</xref>). For example, the distance from the GNG-breeding band to the solitary male was 1.37&#x000b1;0.57&#x02009;km (0.21&#x02013;4.41), and the distance from the GNG-all-male band to the solitary male was 1.34&#x000b1;0.58&#x02009;km (0.03&#x02013;4.37&#x02009;km).</p><p>In addition, the half-weighted index-based SNA with a 2&#x02009;h sampling interval confirmed that members of the GNG-breeding band, the GNG-all-male band and the solitary male (<xref ref-type="fig" rid="f2">Fig. 2</xref>) maintained in closer spatial association than each had with the DJF-breeding band and the DJF-AMU. These data indicate that the three GNG-social components consistently coordinated their patterns of movements to form an integrated level of social affiliation, the &#x02018;herd&#x02019;.</p><p>In contrast the DJF-AMU often travelled apart from the DJF-breeding band (mean distance 2.06&#x000b1;1.04&#x02009;km; range 0.84&#x02013;4.11&#x02009;km). Home range overlap between the DJF-breeding band and the DJF-AMU was 12.9% (<xref ref-type="fig" rid="f1">Fig. 1</xref>), and the average distance between the DJF-breeding band and the DJF-AMU was significantly greater than that between the GNG-breeding band and the GNG-all-male band (independent <italic>t</italic>-test, <italic>t</italic>=&#x02212;53.87; <italic>P</italic>&#x0003c;0.001; degree of freedom (<italic>df</italic>)=3407).</p><p>Although the ranging pattern of the DJF-breeding band and DJF-AMU indicates weak social affinity, the demographic evidence shows that most individuals of DJF-AMU were originally members from the DJF-breeding band. SNA also demonstrates that the DJF-AMU and the DJF-breeding band were less closely associated to each other compared with the social components of the GNG-herd (<xref ref-type="fig" rid="f2">Fig. 2</xref>).</p></sec><sec disp-level="2"><title>Fission&#x02013;fusion dynamics of the snub-nosed monkey MLS</title><p>Importantly, the telemetry data (<xref ref-type="fig" rid="f3">Fig. 3a</xref>) revealed that during a 3-week period in February and March, the social components represented by the five collared males merged to form a single large troop. Based on the spatial affinity coefficient (SAC) analyses (<xref ref-type="fig" rid="f3">Fig. 3a,b</xref>), we identified four stages of social affiliation that characterized the golden snub-nosed monkey multilevel society: the fission stage, the reform stage, the fusion stage and the separation stage. Our results indicate that these periods accounted for 20.9, 24.7, 18.4 and 36.0% of the study period, respectively) (<xref ref-type="fig" rid="f3">Fig. 3b</xref>). The SACs of the troop during each time segment indicated a lower SAC value during the fusion stage, which means a higher level of troop cohesion, compared with all other stages (Wilcoxon rank sum test, <italic>Z</italic>=4.736, <italic>P</italic>&#x0003c;0.001; <italic>df</italic>=1703).</p></sec><sec disp-level="2"><title>Individual exchange between social components of MLS</title><p>Although the telemetry data do not provide details of individual movements, we have accumulated evidence of individual dispersal patterns between the social components of the WRT based on 14 years of field observations. During this period, we identified 127 individuals (73 emigration events and 54 immigration events) and 20 OMU transfers that occurred between the GNG-breeding band and the DJF-breeding band. In 14 of 20 cases in which an entire OMU from the DJF-breeding band immigrated into the GNG-breeding band, it remained in the band for a period of at least 3 months (<xref ref-type="fig" rid="f4">Fig. 4</xref>). We also identified nine bachelor adult males from the GNG-all-male band and the DJF-AMU who successfully immigrated into the GNG-breeding band on their own, and formed an OMU, either by attracting females or by taking over an existing OMU. We recorded 14 resident males who lost their tenure as the leader male of an OMU in the GNG-breeding band. Seven of these cases involved a takeover by an immigrant male, and in these instances, the ex-harem leader joined the GNG-all-male band on four occasions, one male transferred into the DJF-AMU, one became a solitary male and one died soon after he was replaced. We also recorded a male from the GNG-all-male band becoming a harem leader in the GNG-breeding band, losing his harem after 4 months and then transferring back into the GNG-all-male band. This male disappeared from the GNG-all-male band and 6 months later was observed as a member of the DJF-AMU (<xref ref-type="fig" rid="f4">Fig. 4</xref>).</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>These data reveal, for the first time, the complex social dynamics of the golden snub-nosed monkey multilevel society (<xref ref-type="fig" rid="f5">Fig. 5</xref>) and confirm the process of the fusion of two herds to form a troop. During troop fusion, members of different breeding bands have the opportunity to exchange individuals across social components that were previously regarded as socially and reproductively separate or distinct. Thus, the multilevel society of the golden snub-nosed monkeys is best described as a system of nested social, spatial and reproductive relationships among individuals forming distinct OMUs that merge into a cohesive breeding band, an associated or satellite all-male band (or AMUs) and solitary males. The breeding band and associate satellite AMUs and solitary male form a herd-level of social and spatial organization. Neighbouring herds periodically fuse to form a troop. The formation of this highly inclusive multilevel social organization appears to facilitate gene exchange among otherwise relatively isolated golden snub-nosed monkey populations.</p><p>The results of this study offer new insights into the evolution and maintenance of this primate MLS and serve to differentiate the set of social and ecological factors that selected for the formation of the <italic>Rhinopithecus</italic> breeding band and troop. Phylogenetic reconstructions based on mitochondrial DNA data and paleobiogeography indicate that <italic>Rhinopithecus</italic> diverged from the other genera of odd-nosed monkeys during the late Miocene, and then differentiated into five species<xref ref-type="bibr" rid="b48">48</xref>. This appears to have begun ~2 million years ago in response to oscillations in paleoclimate associated with the uplifting of the Tibetan Plateau, a decrease in temperature and rainfall, a reduction in broadleaf and evergreen forest habitats, glacial expansion and geographical barriers that restricted gene flow across populations<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref><xref ref-type="bibr" rid="b51">51</xref>. Although the precise factors that led to the formation of a multilevel society remain unclear, increased environmental heterogeneity and forest fragmentation during periods of glacial maximum may have resulted in feeding sites that were hyper-dispersed across the landscape<xref ref-type="bibr" rid="b52">52</xref>. This may have favoured the year-round aggregation of separate snub-nosed monkey OMUs, as is found in most Asian colobines, into a larger, more cohesive and socially and spatially complex breeding band<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref>. We term this the fusion model of breeding band formation to distinguish it from the MLS of gelada and hamadryas baboons that is argued to have formed by the internal fissioning of a multimale multifemale social organization into several nested harem reproductive units within a larger band<xref ref-type="bibr" rid="b31">31</xref>.</p><p>Several theories have been proposed to explain the aggregation of individual OMUs to form a band-level social organization in snub-nosed monkeys. These include the bachelor threat hypothesis<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b53">53</xref>, the predator defence hypothesis<xref ref-type="bibr" rid="b54">54</xref><xref ref-type="bibr" rid="b55">55</xref> and the inbreeding avoidance hypothesis<xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b56">56</xref>.</p><p>The bachelor threat hypothesis argues that the formation of a breeding band favoured several harem males acting collectively to form a coalition and defend OMUs against invading bachelor males who threaten to take over a harem<xref ref-type="bibr" rid="b34">34</xref>. It also had been reported that the bachelor males of geladas monitor the interactions of females and their harem leaders and may use this information to evaluate the strength and quality of harem holders and the cohesiveness of the OMU<xref ref-type="bibr" rid="b57">57</xref>. In the black-and-white snub-nosed monkey, an AMU was regularly found 200&#x02013;500&#x02009;m from the OMUs and occasionally located in the centre of the band within the reach of the reproductive units<xref ref-type="bibr" rid="b32">32</xref>. Our behavioural observations and satellite telemetry provide the first quantitative and detailed account of the movement patterns of breeding bands, AMUs and solitary males. The GNG-all-male band travelled independently from the GNG-breeding band, but maintained a close spatial association and coordinated their movement patterns, resulting in a high degree of social affinity and spatial overlap. The distance between the all-male band and the breeding band consistently ranged from 100&#x02013;1,500&#x02009;m. This distance could be travelled by a bachelor male within the span of several minutes to several hours. Moreover, we observed leader males to form coalitions and aggressively chase members of the all-male band when individuals were in close proximity to the breeding band. This same behavioural pattern has been reported in a study of golden snub-nosed monkeys in Hubei Province, China<xref ref-type="bibr" rid="b58">58</xref>.</p><p>Similar to the threat of bachelor males, some have argued that predation risk posed by large ground predators may have also played a critical role in the evolution of the <italic>Rhinopithecus</italic> breeding band<xref ref-type="bibr" rid="b54">54</xref><xref ref-type="bibr" rid="b55">55</xref>. If multiple individuals engage in predator detection, this might provide advantages to all members of a cohesive breeding band. Sperm whales (<italic>Physeter macrocephalus</italic>) represent another mammal that lives in a complex nested social organization. Eastern Pacific populations of sperm whales live in small units composed of several unrelated matrilines. These units temporarily merge to form a clan in response to high density of killer whales<xref ref-type="bibr" rid="b59">59</xref>. In contrast, the same species of sperm whales in the North Atlantic rarely group with other social units and do not form clans due to reduced predator pressure. The results argue that protection against predation by killer whales in the eastern Pacific is the primary reason for the formation of a MLS<xref ref-type="bibr" rid="b59">59</xref>.</p><p>Another factor proposed to select for the aggregation of snub-nosed monkey OMUs into a breeding band is inbreeding avoidance. It had been reported that under conditions in which the breeding tenure of a harem male exceeds 4&#x02013;5 years, female golden snub-nosed monkeys transfer from their natal OMU into another OMU in the breeding band to avoid mating with their father<xref ref-type="bibr" rid="b43">43</xref>. An advantage of nested OMUs within a breeding band is that it reduces the cost of female transfer by allowing sub-adult and young adult females to move directly from one OMU in the breeding band to another<xref ref-type="bibr" rid="b43">43</xref>. Finally, food distribution may also have contributed to the evolution of large multilevel groupings<xref ref-type="bibr" rid="b34">34</xref>. In the temperate forests of snub-nosed monkey habitat, evenly distributed food resources allow a larger number of OMUs remain in close proximity within a modular society<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b40">40</xref>. In contrast, most Asian colobines that live in habitats characterized by patchy food distribution form small territorial social units, which has shown agonistic inter-unit interactions and food defence<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b60">60</xref><xref ref-type="bibr" rid="b61">61</xref>.</p><p>We also provide quantitative evidence for a new level of social organization within the MLS of golden snub-nosed monkeys, the troop. Based on spatial analysis of GPS monitoring data, we documented the temporary fusion of two herds during a limited period of the year (February and March). The fact that both individuals and entire OMUs transfer between herds provides an opportunity for gene flow across social components living under conditions of low population density (&#x0003c;10 individual per km²) and distributed across large home ranges in mountainous habitats<xref ref-type="bibr" rid="b33">33</xref>. However this troop-level social organization is not stable, likely in response to increased feeding and reproductive competition among the 20 OMUs and the 44 bachelor males that compose the troop. In our study the fusion stage of troop formation coincided with a period of local food abundance and the flushing of buds and young leaves, which are high in protein<xref ref-type="bibr" rid="b47">47</xref><xref ref-type="bibr" rid="b62">62</xref>. The formation of a large troop has also been reported in the Grey snub-nosed monkey (<italic>Rhinopithecus brelichi</italic>), which are reported to contain as many as 600 individuals who aggregate in spring/early summer but break down into smaller groups during most of the year<xref ref-type="bibr" rid="b63">63</xref>.</p><p>In summary, our results using satellite telemetry, SNA and long-term behavioural observations demonstrate that snub-nosed monkeys live in a modular society organized into several levels of association. In this system the basic elements are solitary males, OMUs and AMUs. Breeding bands consist of an aggregation of several OMUs and all-male bands consist of an aggregation of several AMUs. These bands represent socially and spatially distinct components of the MLS. A herd is defined as the spatial aggregation of the breeding band, an all-male band (or single AMU), and solitary males. Previous studies have used the term band to describe the social and spatial association of several OMUs and AMU<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b36">36</xref>. Using long-term observations and satellite telemetry, we found that an all-male band or single AMU, as well as solitary males travel independently from the breeding band during most part of the year. Therefore, we use the term herd to document the association between different breeding bands and all-male bands. The classification we use to distinguish the band-level and the herd-level is based on analysis of SNA (<xref ref-type="fig" rid="f2">Fig. 2a</xref>), which indicated a statistical threshold to highlight patterns of social and spatial affinity among these different social elements. During a brief period of the year, two neighbouring herds fuse to form a troop in which individuals and OMUs transfer between otherwise isolated reproductive units.</p><p>We documented that each structural component of the snub-nosed monkey MLS appears to serve a distinct function: OMUs represent the main reproductive units; band members experience mutual benefits from leader males collectively acting against challenges from bachelor males, a reduction in predation risk and inbreeding avoidance by the ability of females to transfer directly between OMUs within the breeding band; and the fusion of two herds to form a troop enables individuals from different breeding bands to exchange membership and promotes gene flow between otherwise isolated breeding bands that travel across extremely large home ranges and inhabit highly fragmented and heterogeneous high altitude temperate forest habitats.</p><p>In line with Grueter and van Schaik<xref ref-type="bibr" rid="b34">34</xref>, we suggest the MLS of <italic>Rhinopithecus</italic> evolved by the aggregation of separate OMUs from an ancestral Asian colobine into a larger, more cohesive breeding band, which is an alternative evolution pathway compared with the MLS of African papionins from the internal fissioning of a multimale multifemale social organization. We conclude that both phylogenetic and ecological factors have played an important role in the origin and evolution of a primate MLS. Additional field-based observational data on individual interactions among different tiers of the MLS are needed to augment our understanding of the MLS of <italic>R. roxellana</italic>.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Study subject and social dynamics</title><p>Since 1999, we have been investigating the social structure of WRT of Golden snub-nosed monkeys in the Zhouzhi National Reserve in central China<xref ref-type="bibr" rid="b43">43</xref>. The WRT is composed of the GNG-breeding band, the GNG-all-male band, the DJF-breeding band, the DJF-AMU and several solitary males. We habituated the GNG-breeding band in 2001 using a method of semi-provisioning<xref ref-type="bibr" rid="b44">44</xref>. The home range of the GNG-breeding band encompasses 2250, ha of mountainous forest, ranging from deciduous broadleaf to coniferous forest (1,380&#x02013;2,974&#x02009;m; ref. <xref ref-type="bibr" rid="b47">47</xref>). Each individual was fitted with a RFID identification tag (TX1411SSL) subcutaneously in the arm, and tattooed with an individually unique sequence of numbers and colours (red, blue) on the upper or lower area of its lips. This enabled us to unambiguously identify each individual and monitor both short-term and long-term dispersal events. We also used micro-satellite DNA markers to confirm the kinship and individual identification.</p></sec><sec disp-level="2"><title>GPS location and collars configuration</title><p>To more accurately monitor band movements, the location and duration of troop fusion and fission dynamics, and patterns of female, male and OMU inter-band transfer in August 2012 we fitted five fully adult males with GPS collars (7000SLU series, LOTEK Wireless Inc., Canada). Based on observations conducted over several years, each male was selected to represent one of the independent social components of the WRT.</p><p>GPS Collars were configured to automatically locate every 2&#x02009;h from 0500 to 1900, h for a period of 104 weeks. After that time, the collars were programmed to unlock and release from the animal&#x02019;s neck using the Time Drop-off feature of the collar.</p><p>The collars recorded the location (in universal transverse mercator coordinates) and elevation of the target golden snub-nosed using a system of seven satellites. The data were stored in the collar&#x02019;s memory chip. We used a BIOTRACKER receiver (LOTEK Wireless Inc.) to track each individual male by VHF beacon signals. Once we were in the vicinity of the band, we used a hand-control wireless unit (LOTEK Wireless Inc.) to download previous location data via a UHF radio signal. Location data from each GPS collar were collected during 291 days (September 2012 to May 2013). Within this study period, each of the social components of the MLS travelled freely across their home range without any interference by the researchers.</p></sec><sec disp-level="2"><title>Range pattern data analysis</title><p>We used the dilution of precision algorithm to determine the level of accuracy for each location data point. We eliminated location data for which dilution of precision&#x02265;8 (2.0% of all). In an additional 2.7% of the records, prevailing weather conditions or other factors resulted in the collars failing to record the location for a given time interval. In these cases we used the piecewise cubic spline Interpolation method to reconstruct the data for the empty time segment.</p><p>Location data obtained were analyzed using the Kernel density analysis of home range utilization and spatial overlap estimation tools from ArcGIS 10.0 (Esri, USA) to determine the ranging patterns of the golden snub-nosed monkey.</p></sec><sec disp-level="2"><title>Fission&#x02013;fusion dynamics and satellite telemetry modelling</title><p>To reduce the random error of GPS locations, we calibrated the GPS coordinate data using a smoothing function for each of the five individuals, representing a different social component of the snub-nosed monkey MLS,</p><p><disp-formula id="eq1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e543" xlink:href="ncomms6296-m1.jpg"/></disp-formula></p><p>Where <inline-formula id="d33e546"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e547" xlink:href="ncomms6296-m2.jpg"/></inline-formula> is the smoothed location calibrated from an average of 15 GPS points per collar; <inline-formula id="d33e549"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e550" xlink:href="ncomms6296-m3.jpg"/></inline-formula> is the uncorrected location data downloaded from the GPS collars, with <italic>i</italic> as the time variable and <italic>j</italic> as the loop variable.</p><p>To estimate the spatial dispersion pattern and fission&#x02013;fusion dynamics of the five social components, we used the SAC as a measure of social attraction.</p><p>With,</p><p><disp-formula id="eq4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e564" xlink:href="ncomms6296-m4.jpg"/></disp-formula></p><p>the value <italic>&#x003c3;</italic>² represents the variance of the GPS coordinates among the <italic>n</italic> social components in one dimension (longitude or latitude) within the same time segment. <italic>L</italic>_{<italic>i,j</italic>} denotes the <italic>j</italic>th spatial coordinate in one dimension at time=<italic>i</italic>, and <inline-formula id="d33e588"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e589" xlink:href="ncomms6296-m5.jpg"/></inline-formula> is the mean value of <italic>n</italic> spatial coordinate points at time=<italic>i</italic>.</p><p>By taking the modular of the s.d. vector <inline-formula id="d33e599"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e600" xlink:href="ncomms6296-m6.jpg"/></inline-formula></p><p><disp-formula id="eq7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e603" xlink:href="ncomms6296-m7.jpg"/></disp-formula></p><p>we are able to measure social attraction in space of <italic>n</italic> social components at a given time segment. Then we standardize the <italic>A</italic>_{<italic>i</italic>} by</p><p><disp-formula id="eq8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e618" xlink:href="ncomms6296-m8.jpg"/></disp-formula></p><p>Where SAC_{<italic>i</italic>} represents the standardized <italic>A</italic>_{<italic>i</italic>} and ranges from 0 to 1, with SAC approaching zero, indicating a tendency for greater spatial fusion, and SAC approaching 1, indicating a tendency for greater spatial fissioning. The SAC values are also presented as the width of the vertical and horizontal projections of each time segment in <xref ref-type="fig" rid="f3">Fig. 3a</xref>.</p><p>To measure quantitatively fission and fusion events across the study period, we calculated the threshold of dispersion based on the SAC values. We assume that the golden snub-nosed monkeys could continue to effectively communicate within a maximum spatial range of 513.12&#x02009;m. This distance was determined based on a calculation of the average maximum distance recorded between OMUs within the GNG-breeding band (1,726&#x02009;h of data collected using instantaneous scan sampling that recorded the distance between each OMU dyad within the GNG-breeding band from 2003&#x02013;2007). We conservatively used the maximum distance between any two OMUs (OMU dyad) for each scan to calculate the average distance. We also assumed that the spatial pattern of all social components within a multilevel society at time segment=<italic>i</italic> fits the shape of a geometric polygon with each social component represented as a node. Therefore, the fission and fusion threshold values of SAC were calculated based on the assumption that the length of each side of the polygon is equal to the distance of effective communication.</p><p>With</p><p><disp-formula id="eq9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e643" xlink:href="ncomms6296-m9.jpg"/></disp-formula></p><p>Where <italic>d</italic> is the length of the sides of an isogon (equilateral polygon) with <italic>n</italic> sides (here <italic>n</italic>=5 as there were five social components within the studied troop). The <italic>A</italic>^fu/fi is the radius of the circumcircle that fits the isogon. This value is equal to the s.d. of the spatial coordinate value of <italic>n</italic> nodes in two dimensions. Where <italic>t</italic>=the radius of earth (<italic>r</italic>) multiplied by 2<italic>&#x003c0;</italic>/360&#x000b0;. By substituting <italic>A</italic>^fu/fi into <xref ref-type="disp-formula" rid="eq8">equation (4)</xref>, thefission or fusion thresholds of SAC were calculated.</p><p>The threshold of SAC^fission=0.1139 was based on the calculation of an isogon with sides of 513.12&#x02009;m (distance of effective communication). We defined the fusion stage as occurring when the SAC_{<italic>i</italic>} values for all five social components&#x02264;SAC^fusion and this spatial association continued for more than three time segments (&#x02265;4&#x02009;h). In contrast, the threshold of SAC^fission=0.4571 was identified when the sides of the isogon were equal to twice the distance of effective communication (2 &#x000d7; 513.12&#x02009;m, which we assume is beyond visual and vocal communication). We defined the fission stage when the SAC_{<italic>i</italic>} was greater than the SAC^fission threshold and continued for more than three time segments (&#x02265;4&#x02009;h). The reform stage occurs after a fission stage when SAC^fusion&#x02264;SAC_{<italic>i</italic>}&#x02264;SAC^fission and before the next fusion stage. Finally, the separation stage was defined as SAC^fusion&#x02264;SAC&#x02264;SAC^fission and followed the fusion stage and continued until the next fission stage.</p><p>Calculation of the smoothing function, distance function and SACs were performed using Matlab 7.0 (MathWorks, USA).</p></sec><sec disp-level="2"><title>Multilevel association estimate and SNA</title><p>To better understand the dynamics of troop formation, stability and their social relationships among different social components, we used SNA to measure affiliative relationships based on patterns of spatial/temporal association (<xref ref-type="fig" rid="f2">Fig. 2</xref>).</p><p>Affiliation using SNA is divided into association and interaction, where association is defined in terms of spatial proximity<xref ref-type="bibr" rid="b65">65</xref><xref ref-type="bibr" rid="b66">66</xref>. For this analysis, we calculated the distances between each dyadic social components to identify the nearest neighbour in time segments of 2-h intervals using the distance function,</p><p><disp-formula id="eq10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e736" xlink:href="ncomms6296-m10.jpg"/></disp-formula></p><p><italic>D</italic>_{<italic>&#x003b1;,&#x003b2;</italic>} is the distance between locations <italic>&#x003b1;</italic> and <italic>&#x003b2;,</italic> La and Lo denote the latitude and longitude of the spatial coordinate of each location, and the radius of earth (<italic>r</italic>) was set to 6378.137&#x02009;km in all calculations. The sampling period was October 2012 to May 2013.</p><p>We used the half-weighted Index (HWI)<xref ref-type="bibr" rid="b67">67</xref><xref ref-type="bibr" rid="b68">68</xref> to measure the association between each individual OMU member using SNA.</p><p><disp-formula id="eq11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e759" xlink:href="ncomms6296-m11.jpg"/></disp-formula></p><p>Where <italic>X</italic> is the number of the observation samplings in which social component <italic>i</italic> and <italic>j</italic> were each other&#x02019;s nearest neighbour; <italic>Y</italic>_{<italic>i</italic>} or <italic>Y</italic>_{<italic>j</italic>} is the number of observations that social component <italic>i</italic> or <italic>j</italic> was the nearest neighbour of social component <italic>j</italic> or <italic>i</italic>, but social component <italic>j</italic> or <italic>i</italic> was not the nearest neighbour of social component <italic>i</italic> or <italic>j</italic>; <italic>Y</italic>_{<italic>ij</italic>} is the number of simultaneous observations that social components <italic>i</italic> and <italic>j</italic> had other nearest neighbours.</p><p>Hierarchical cluster analysis<xref ref-type="bibr" rid="b65">65</xref><xref ref-type="bibr" rid="b66">66</xref> presented by the dendrogram shows the degree of associations between each social component. The cutoff value<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b68">68</xref><xref ref-type="bibr" rid="b69">69</xref> in the dendrogram provide quantitative threshold to identify the herd-level social grouping in the study troop. To estimate the stability of troop cohesion of the two estimated herd during sampling periods, we used temporal analysis<xref ref-type="bibr" rid="b67">67</xref> presented by lagged association rate to predict the probability of decay in the association rate; null association rate proposed value of the lagged association rate if there is no preferred association. We also used principal coordinate analysis to identify hierarchies of different social components coordinated with each other. All statistical and graphical analyses of SNA were performed using SOCPROG 2.1 (ref. <xref ref-type="bibr" rid="b6">6</xref>).</p></sec><sec disp-level="2"><title>Standardized statistic analyses</title><p>Standardized statistic analyses were performed in SPSS 16.0 (SPSS Inc., USA). In all analyses, significance level was set at <italic>&#x003b1;</italic>=0.05, average values are expressed as mean&#x000b1;s.e. Sampling in each test were independent.</p></sec><sec disp-level="2"><title>Research protocols</title><p>All research protocols reported here adhered to the regulatory requirements of and approved by the animal care committee of the Wildlife Protection Society of China (SL-2012-42). The comfort and security of collars devices received clearance from and complied with the protocols approved by the specialist committee of the State Forestry Administration of China (SFA: LHXZ-2012-2788), Chinese Academy of Science and China Duty.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>X.-G.Q. and B.-G.L. conceived and designed the research; X.-G.Q. and Z.-P.H. performed the fieldwork; X.-G.Q., K.H. and Z.-P.H. analyzed the data; all authors contributed to the writing and editing of this manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Qi, X. G. <italic>et al.</italic> Satellite telemetry and social modelling offer new insights into the origin of primate multilevel societies. <italic>Nat. Commun.</italic> 5:5296 doi: 10.1038/ncomms6296 (2014).</p></sec>

Critical behaviours of contact near phase transitions

<p>A central quantity of importance for ultracold atoms is contact, which measures two-body correlations at short distances in dilute systems. It appears in universal relations among thermodynamic quantities, such as large momentum tails, energy and dynamic structure factors, through the renowned Tan relations. However, a conceptual question remains open as to whether or not contact can signify phase transitions that are insensitive to short-range physics. Here we show that, near a continuous classical or quantum phase transition, contact exhibits a variety of critical behaviours, including scaling laws and critical exponents that are uniquely determined by the universality class of the phase transition, and a constant contact per particle. We also use a prototypical exactly solvable model to demonstrate these critical behaviours in one-dimensional strongly interacting fermions. Our work establishes an intrinsic connection between the universality of dilute many-body systems and universal critical phenomena near a phase transition.</p>

<contrib contrib-type="author"><name><surname>Chen</surname><given-names>Y.-Y.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jiang</surname><given-names>Y.-Z.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Guan</surname><given-names>X.-W.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Qi</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a4">4</xref></contrib><aff id="a1"><label>1</label><institution>State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences</institution>, Wuhan 430071, <country>China</country></aff><aff id="a2"><label>2</label><institution>Center for Cold Atom Physics, Chinese Academy of Sciences</institution>, Wuhan 430071, <country>China</country></aff><aff id="a3"><label>3</label><institution>Department of Theoretical Physics, Research School of Physics and Engineering, Australian National University</institution>, Canberra, Australian Capital Territory 0200, <country>Australia</country></aff><aff id="a4"><label>4</label><institution>Department of Physics, The Chinese University of Hong Kong</institution>, Shatin, New Territories, <country>Hong Kong</country></aff>

Nature Communications

<p>The notion of contact <inline-formula id="d33e159"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e160" xlink:href="ncomms6140-m1.jpg"/></inline-formula> strikingly captures the universality of ultracold atoms. As revealed by the Tan relations<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref> and their expressions in other forms<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>, regardless of the choice of microscopic parameters, a wide range of quantities in dilute systems is governed by <inline-formula id="d33e166"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e167" xlink:href="ncomms6140-m2.jpg"/></inline-formula> that characterizes the probability that two particles may be separated by a short distance less than <italic>d</italic>. For instance, when <italic>d</italic> approaches zero, the two-body correlation function of two-component fermions in three dimensions follows <inline-formula id="d33e176"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e177" xlink:href="ncomms6140-m3.jpg"/></inline-formula>, where <inline-formula id="d33e179"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e180" xlink:href="ncomms6140-m4.jpg"/></inline-formula> is the density operator at position <bold>x</bold> for spin-up(down) particles. While the definition of contact is apparently independent of the many-body phase the system is exhibiting, there is much interest in exploring the behaviour of contact near a phase transition<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. The success of such efforts will significantly deepen our understanding of the connection between short-ranged two-body correlations and phase transitions, which are generally believed to be disentangled from each other, since the latter is insensitive to the details of short-range physics.</p><p>Even though the contact of strongly interacting fermions remains finite in both the normal and the superfluid phase, experimental studies have provided evidence indicating that it gets enhanced near the superfluid transition temperature. However, owing to a lack of resolution, it is unclear whether contact exhibits any critical features near the transition point. On the theoretical side, it is extremely difficult to exactly calculate the contact of strongly interacting fermions near the transition temperature, and therefore certain approximations have to be made. Theoretical approaches based on different techniques lead to contradictory results<xref ref-type="bibr" rid="b10">10</xref>, ranging from a kink to a discontinuous jump of the contact near the transition point. Therefore, it is of fundamental importance to provide a concrete answer for the relation between contact and phase transitions.</p><p>In this work, our approach is to derive exact results on the behaviour of contact near a classical or quantum phase transition based on a fundamental thermodynamic relation that is free from any approximations. These results unambiguously show that contact must display critical behaviours near the transitions, and that the corresponding critical behaviours are uniquely determined by the universality class of the phase transition. We use a one-dimensional (1D) exactly solvable model of strongly interacting fermions exhibiting exotic quantum phase transitions to demonstrate these critical phenomena. Our exact result for contact is obtained from the Bethe ansatz for a 1D Fermi gas that provides a precise understanding of critical phenomena beyond the Tomonaga&#x02013;Luttinger liquid (TLL) physics. Whereas our general results apply to all dimensions, this 1D example sheds light on the universal features of contact near a phase transition.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Critical behaviours of contact in three dimensions</title><p>We consider the fundamental thermodynamic relation,</p><p><disp-formula id="eq5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e203" xlink:href="ncomms6140-m5.jpg"/></disp-formula></p><p>where <italic>P</italic> is the pressure, <italic>n</italic>, <italic>s</italic>, <italic>M</italic>, <italic>&#x003c1;</italic>_s and <inline-formula id="d33e224"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e225" xlink:href="ncomms6140-m6.jpg"/></inline-formula> are the densities of the particles, the entropy, the magnetization, the superfluid and the contact, respectively, <inline-formula id="d33e227"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e228" xlink:href="ncomms6140-m7.jpg"/></inline-formula> is the volume of the system, and <italic>&#x003bc;</italic>, <italic>T</italic>, <italic>H</italic> and <italic>a</italic>_{3<italic>D</italic>} are the chemical potential, temperature, magnetic field and scattering length, respectively. Compared with the usual definition of contact, here the prefactor <italic>&#x0210f;</italic>²/(4<italic>&#x003c0;m</italic>) (with <italic>m</italic> denoting the mass) has been absorbed into <italic>c</italic>. In this relation, <italic>w</italic>=<italic>v</italic>_s&#x02212;<italic>v</italic>_n is the difference between the velocity of the superfluid and normal components, which can be generated by slowly rotating the atomic cloud so that the critical velocity of the superfluid is not reached anywhere in the trap. <xref ref-type="disp-formula" rid="eq5">Equation (1)</xref> has been used to measure thermodynamic quantities such as the pressure, the equation of state and density&#x02013;density response function<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>.</p><p>Compared with the original Tan relation <inline-formula id="d33e282"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e283" xlink:href="ncomms6140-m8.jpg"/></inline-formula>, where <italic>E</italic> and <italic>S</italic> are the total energy and entropy of the system, respectively, <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> has the advantage of allowing one to directly correlate the contact with phase transitions for both classical and quantum ones. First, as <italic>c</italic> is a partial derivative of the pressure, that is, <inline-formula id="d33e298"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e299" xlink:href="ncomms6140-m9.jpg"/></inline-formula>, as are <italic>n</italic>, <italic>s M</italic> and <italic>&#x003c1;</italic>_s, <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> tells one that contact near the critical point must exhibit critical behaviour determined by the universality class of the phase transition. In particular, the contact should vary continuously across a continuous phase transition. For instance, across the superfluid phase transition of strongly interacting fermions in three dimensions, <italic>c</italic> is continuous. Second, the Maxwell relations derived from <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> show that the derivatives of the contact with respect to <italic>&#x003bc;</italic>, <italic>T</italic>, <italic>H</italic> and <italic>w</italic> exhibit critical behaviour. These Maxwell relations can be written as</p><p><disp-formula id="eq10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e336" xlink:href="ncomms6140-m10.jpg"/></disp-formula></p><p><disp-formula id="eq11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e339" xlink:href="ncomms6140-m11.jpg"/></disp-formula></p><p><disp-formula id="eq12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e342" xlink:href="ncomms6140-m12.jpg"/></disp-formula></p><p><disp-formula id="eq13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e345" xlink:href="ncomms6140-m13.jpg"/></disp-formula></p><p>The exact relations (<xref ref-type="disp-formula" rid="eq10">equations (2)&#x02013;(5)</xref>) bring new physical insight into the correlations between the contact and other physical quantities, including the magnetization and superfluid density that characterizes magnetic and transport properties, respectively, which have not been explored in the literature. Among these exact relations, <xref ref-type="disp-formula" rid="eq13">equation (5)</xref> is of particular interest. It directly correlates the contact with <italic>&#x003c1;</italic>_s characterizing superfluid phase transitions. Despite <italic>c</italic> being finite in both the normal and superfluid phases, there is a difference. <xref ref-type="disp-formula" rid="eq13">Equation (5)</xref> shows that in the normal phase, <inline-formula id="d33e366"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e367" xlink:href="ncomms6140-m14.jpg"/></inline-formula>, and the contact remains unchanged after a slow rotation is turned on, since <italic>&#x003c1;</italic>_s&#x02261;0. In the superfluid phase, <italic>&#x003c1;</italic>_s in general depends on the scattering length, and therefore <inline-formula id="d33e379"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e380" xlink:href="ncomms6140-m15.jpg"/></inline-formula> is finite. In particular, in a stationary system with <italic>w</italic>=0, <italic>&#x003c1;</italic>_s follows the standard scaling law near the transition point, <inline-formula id="d33e391"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e392" xlink:href="ncomms6140-m16.jpg"/></inline-formula>, where the tuning parameter <italic>&#x003b4;</italic> can be <italic>T</italic> and <italic>&#x003bc;</italic> (or <italic>H</italic>) for classical and quantum phase transitions, respectively. Here, <italic>A</italic> is independent of <italic>&#x003b4;</italic>, and <italic>&#x003b6;</italic> is the corresponding critical exponent. One then obtains the scaling law for <inline-formula id="d33e416"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e417" xlink:href="ncomms6140-m17.jpg"/></inline-formula> near the transition point,</p><p><disp-formula id="eq18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e421" xlink:href="ncomms6140-m18.jpg"/></disp-formula></p><p><xref ref-type="disp-formula" rid="eq18">Equation (6)</xref> shows that the exponent of <inline-formula id="d33e427"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e428" xlink:href="ncomms6140-m19.jpg"/></inline-formula> is entirely determined by the universality class of the superfluid phase transition. The above properties of the contact can be easily tested in experiments on trapped atoms, where the superfluid and normal phases are distributed in different regions of the trap. With the high-resolution images available in current experiments, the local contact density <italic>c</italic> can be extracted as a function of <italic>&#x003bc;</italic> using <inline-formula id="d33e436"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e437" xlink:href="ncomms6140-m20.jpg"/></inline-formula>. One then could examine the distinct responses of <italic>c</italic> to rotation, <inline-formula id="d33e443"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e444" xlink:href="ncomms6140-m21.jpg"/></inline-formula>, in both the superfluid and normal phases.</p><p>Equations (2)&#x02013;(4) can also be experimentally tested. Near the phase transition point, the scaling law in a system with <italic>w</italic>=0 for a quantity <italic>O</italic> takes the form <inline-formula id="d33e454"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e455" xlink:href="ncomms6140-m22.jpg"/></inline-formula>, where <italic>O</italic>=<italic>n</italic>, <italic>M</italic> or <italic>s</italic>, <italic>O</italic>_r is the regular part of <italic>O</italic> and <italic>&#x003b7;</italic>_{<italic>O</italic>} is the corresponding critical exponent. One then obtains:</p><p><disp-formula id="eq23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e486" xlink:href="ncomms6140-m23.jpg"/></disp-formula></p><p><disp-formula id="eq24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e489" xlink:href="ncomms6140-m24.jpg"/></disp-formula></p><p><disp-formula id="eq25"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e492" xlink:href="ncomms6140-m25.jpg"/></disp-formula></p><p>where the subscripts on the derivatives of <italic>O</italic>_r have been suppressed. The above differential forms also indicate scaling laws for <italic>c</italic>. For instance, if one chooses <italic>&#x003b4;</italic>=<italic>&#x003bc;</italic>, then from <xref ref-type="disp-formula" rid="eq23">equation (7)</xref> one obtains <inline-formula id="d33e513"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e514" xlink:href="ncomms6140-m26.jpg"/></inline-formula>, where <inline-formula id="d33e516"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e517" xlink:href="ncomms6140-m27.jpg"/></inline-formula>. Note that the dependences of <italic>c</italic> and <italic>n</italic> on <italic>&#x003bc;</italic> have the same exponent, so that one concludes that</p><p><disp-formula id="eq28"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e530" xlink:href="ncomms6140-m28.jpg"/></disp-formula></p><p>In particular, if <italic>c</italic>_r=<italic>n</italic>_r=0, one sees that the contact per particle in the critical region becomes a constant that is entirely determined by <inline-formula id="d33e543"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e544" xlink:href="ncomms6140-m29.jpg"/></inline-formula>. As the non-uniform distribution of trapped atoms allows experimentalists to trace the dependence of the contact on the chemical potential<xref ref-type="bibr" rid="b18">18</xref>, <xref ref-type="disp-formula" rid="eq23">equations (7)</xref> and <xref ref-type="disp-formula" rid="eq28">(10)</xref> can be directly tested in experiments.</p></sec><sec disp-level="2"><title>Contact in an exactly solvable 1D Fermi gas</title><p>Whereas the above discussion applies to all ultracold atomic systems, it is particularly interesting to use an exactly solvable model to demonstrate some of the critical behaviour of contact. Here, we consider <xref ref-type="disp-formula" rid="eq23">equations (7)</xref> and <xref ref-type="disp-formula" rid="eq28">(10</xref>), since they can be implemented in experiments easily without a rotation. In one dimension, <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> becomes</p><p><disp-formula id="eq30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e571" xlink:href="ncomms6140-m30.jpg"/></disp-formula></p><p>where <italic>a</italic>_1D is the 1D scattering length and <italic>c</italic> differs from the ordinary definition by a trivial prefactor <italic>&#x0210f;</italic>²/(2<italic>m</italic>). Each of the equations (1)&#x02013;(10) has a direct analogue in one dimension that is obtained by the simple replacement <inline-formula id="d33e591"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e592" xlink:href="ncomms6140-m31.jpg"/></inline-formula>. We study a 1D Fermi gas with <italic>&#x003b4;</italic>-function interactions, described by the Yang&#x02013;Gaudin Hamiltonian<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref></p><p><disp-formula id="eq32"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e600" xlink:href="ncomms6140-m32.jpg"/></disp-formula></p><p>where <inline-formula id="d33e603"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e604" xlink:href="ncomms6140-m33.jpg"/></inline-formula> is the Zeeman energy induced by a magnetic field <italic>H</italic>, <inline-formula id="d33e609"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e610" xlink:href="ncomms6140-m34.jpg"/></inline-formula> characterizes the interaction strength determined by the effective 1D scattering length <inline-formula id="d33e612"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e613" xlink:href="ncomms6140-m35.jpg"/></inline-formula><xref ref-type="bibr" rid="b26">26</xref>, <italic>a</italic>_&#x022a5; is the transverse oscillation length and <italic>A</italic>&#x02248;1.0326. We introduce the polarization <italic>P</italic>=(<italic>n</italic>_&#x02191;&#x02212;<italic>n</italic>_&#x02193;)/<italic>n</italic>, and define a dimensionless interaction parameter &#x003b3;=<italic>mg</italic>_1D/(<italic>n&#x00127;</italic>²)=&#x02212;2(<italic>na</italic>_1D)^&#x02212;1 for our analysis, choosing natural units 2<italic>m</italic>=<italic>&#x0210f;</italic>=<italic>k</italic>_B=1.</p><p>The model described by <xref ref-type="disp-formula" rid="eq32">equation (12)</xref> has been solved using the Bethe ansatz<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref> and has had a tremendous impact in statistical mechanics. The experimental developments in studying 1D fermions<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref> have inspired significant interest in relating theoretical results to experimental observables<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b32">32</xref>. It was found<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref> that although the thermodynamic Bethe ansatz (TBA) equations involve non-trivial collective behaviour of the particles, that is, the motion of one particle depends on all others, the total effect of the complex behaviours of all the individual particles leads to qualitatively new forms of simplicity in many-body phenomena<xref ref-type="bibr" rid="b36">36</xref><xref ref-type="bibr" rid="b37">37</xref>.</p><p>Contact of the ground state, in the extremely polarized limit with a single spin-down atom, has been studied in refs <xref ref-type="bibr" rid="b38">38</xref>, <xref ref-type="bibr" rid="b39">39</xref>. However, reaching the goal of finding critical behaviours of contact requires a theoretical framework, beyond mean-field theory, capable of analytically deriving the thermodynamic properties of such gases at finite temperatures (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>). This has been a fundamental challenge in theoretical physics owing to the strong interaction between the atoms. Here we compute the contact by numerically solving the TBA equations and obtaining analytic expressions in the physical regime <inline-formula id="d33e699"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e700" xlink:href="ncomms6140-m36.jpg"/></inline-formula> and <inline-formula id="d33e702"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e703" xlink:href="ncomms6140-m37.jpg"/></inline-formula>, where <inline-formula id="d33e706"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e707" xlink:href="ncomms6140-m38.jpg"/></inline-formula> is the binding energy of the pairs, and explore its behaviour near the phase transition. Even though there is no finite-temperature phase transition in one dimension, there does exist a universal finite-temperature crossover that remarkably separates the low-energy critical TLL with relativistic dispersion from the collective matter of free Fermi criticality with non-relativistic dispersion. Moreover, quantum phase transitions between two of the following phases in this model, the vacuum phase (V), the fully paired phase (P), the fully polarized phase (F) and the partially polarized phase (PP)<xref ref-type="bibr" rid="b36">36</xref><xref ref-type="bibr" rid="b37">37</xref>, provide a precise description of the critical behaviours exhibited by contact in many-body systems.</p><p>The phase diagram <xref ref-type="fig" rid="f1">Fig. 1</xref> shows numerical results for the dimensionless contact density <inline-formula id="d33e716"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e717" xlink:href="ncomms6140-m39.jpg"/></inline-formula> at zero temperature as a function of the dimensionless chemical potential <inline-formula id="d33e719"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e720" xlink:href="ncomms6140-m40.jpg"/></inline-formula> and magnetic field <italic>h</italic>&#x02261;<italic>H</italic>/&#x003b5;_b, where <italic>c</italic> is obtained from the TBA equations (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>) through <italic>c</italic>=&#x02212;(<italic>&#x02202;P</italic>/<italic>&#x02202;a</italic>_1D)_{<italic>&#x003bc;</italic>,<italic>H</italic>,<italic>T</italic>}, and <italic>w</italic> has been set to be zero. Here we have chosen &#x003b5;_b as the energy scale. Alternatively, one may choose the Fermi energy, <italic>E</italic>_F, which will not change the later discussion and results. Since we have chosen natural units by setting <italic>&#x0210f;</italic> and 2<italic>m</italic> to be 1, <italic>c</italic> has the same dimension as <inline-formula id="d33e781"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e782" xlink:href="ncomms6140-m41.jpg"/></inline-formula> so that <inline-formula id="d33e784"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e785" xlink:href="ncomms6140-m42.jpg"/></inline-formula> as defined is dimensionless. Across the transition from V to P, the regular part <inline-formula id="d33e787"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e788" xlink:href="ncomms6140-m43.jpg"/></inline-formula>, since <inline-formula id="d33e791"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e792" xlink:href="ncomms6140-m44.jpg"/></inline-formula> in V, and past the critical point <inline-formula id="d33e794"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e795" xlink:href="ncomms6140-m45.jpg"/></inline-formula> continuously increases from zero as <inline-formula id="d33e797"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e798" xlink:href="ncomms6140-m46.jpg"/></inline-formula>. Correspondingly, in P, <inline-formula id="d33e800"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e801" xlink:href="ncomms6140-m47.jpg"/></inline-formula> diverges as <inline-formula id="d33e803"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e804" xlink:href="ncomms6140-m48.jpg"/></inline-formula> at this transition point. The aforementioned scaling laws for <inline-formula id="d33e806"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e807" xlink:href="ncomms6140-m49.jpg"/></inline-formula> and <inline-formula id="d33e810"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e811" xlink:href="ncomms6140-m50.jpg"/></inline-formula> are derived directly from the zero temperature scaling law for density near this critical point, <inline-formula id="d33e813"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e814" xlink:href="ncomms6140-m51.jpg"/></inline-formula>. By taking the derivative of <italic>n</italic> with respect to <italic>a</italic>_1D, one sees that the critical exponents for <inline-formula id="d33e824"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e825" xlink:href="ncomms6140-m52.jpg"/></inline-formula> and <inline-formula id="d33e828"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e829" xlink:href="ncomms6140-m53.jpg"/></inline-formula> are indeed 1/2 and &#x02212;1/2, respectively. Near the other transition point from P to PP, <italic>c</italic> also changes continuously with a kink <inline-formula id="d33e834"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e835" xlink:href="ncomms6140-m54.jpg"/></inline-formula> and <inline-formula id="d33e837"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e838" xlink:href="ncomms6140-m55.jpg"/></inline-formula> has the same <inline-formula id="d33e840"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e841" xlink:href="ncomms6140-m56.jpg"/></inline-formula> divergence.</p><p>At finite temperatures, <inline-formula id="d33e845"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e846" xlink:href="ncomms6140-m57.jpg"/></inline-formula> no longer diverges (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). Nevertheless, critical phenomena exist for both <inline-formula id="d33e851"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e852" xlink:href="ncomms6140-m58.jpg"/></inline-formula> and <inline-formula id="d33e854"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e855" xlink:href="ncomms6140-m59.jpg"/></inline-formula> in a region expanded to finite temperatures, as is typical for quantum criticality. We work out the analytic expressions for <inline-formula id="d33e857"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e858" xlink:href="ncomms6140-m60.jpg"/></inline-formula> and derive the scaling form for <inline-formula id="d33e861"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e862" xlink:href="ncomms6140-m61.jpg"/></inline-formula> and its derivatives in the quantum critical region. For the physical regime, <inline-formula id="d33e864"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e865" xlink:href="ncomms6140-m62.jpg"/></inline-formula> and <inline-formula id="d33e867"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e868" xlink:href="ncomms6140-m63.jpg"/></inline-formula>, <inline-formula id="d33e870"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e871" xlink:href="ncomms6140-m64.jpg"/></inline-formula> is given explicitly by</p><p><disp-formula id="eq65"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e875" xlink:href="ncomms6140-m65.jpg"/></disp-formula></p><p>where</p><p><disp-formula id="eq66"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e881" xlink:href="ncomms6140-m66.jpg"/></disp-formula></p><p><disp-formula id="eq67"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e884" xlink:href="ncomms6140-m67.jpg"/></disp-formula></p><p>Here, we use the notation <inline-formula id="d33e887"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e888" xlink:href="ncomms6140-m68.jpg"/></inline-formula>, <inline-formula id="d33e890"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e891" xlink:href="ncomms6140-m69.jpg"/></inline-formula>, <inline-formula id="d33e893"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e894" xlink:href="ncomms6140-m70.jpg"/></inline-formula>, and <inline-formula id="d33e896"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e897" xlink:href="ncomms6140-m71.jpg"/></inline-formula> is the polylog function. We have defined <italic>t</italic>&#x02261;<italic>T</italic>/<italic>&#x003b5;</italic>_b, <inline-formula id="d33e911"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e912" xlink:href="ncomms6140-m72.jpg"/></inline-formula>, and <inline-formula id="d33e914"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e915" xlink:href="ncomms6140-m73.jpg"/></inline-formula>, where the labels b and u indicate if a quantity describes a property of bound states or unpaired particles. The result (<xref ref-type="disp-formula" rid="eq65">equation (13)</xref>) is valid for both the TLL phase and the critical region (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). Physically, <inline-formula id="d33e924"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e925" xlink:href="ncomms6140-m74.jpg"/></inline-formula> and <inline-formula id="d33e927"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e928" xlink:href="ncomms6140-m75.jpg"/></inline-formula> represent the dimensionless pressure and chemical potential of unpaired fermions and bound pairs, respectively. Owing to the residual interaction between them, <inline-formula id="d33e930"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e931" xlink:href="ncomms6140-m76.jpg"/></inline-formula> and <inline-formula id="d33e933"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e934" xlink:href="ncomms6140-m77.jpg"/></inline-formula> are correlated through the above coupled equations.</p><p>It is interesting to note that, apart from a small correction <inline-formula id="d33e938"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e939" xlink:href="ncomms6140-m78.jpg"/></inline-formula>, the terms within the square brackets of (<xref ref-type="disp-formula" rid="eq65">equation (13)</xref>) give the pressure of the interacting system after subtracting that of an ideal gas consisting of single fermionic atoms with mass <italic>m</italic> and composite atoms with mass 2<italic>m</italic>, namely</p><p><disp-formula id="eq79"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e952" xlink:href="ncomms6140-m79.jpg"/></disp-formula></p><p>where up to the order of <inline-formula id="d33e955"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e956" xlink:href="ncomms6140-m80.jpg"/></inline-formula>, the pressure is <inline-formula id="d33e958"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e959" xlink:href="ncomms6140-m81.jpg"/></inline-formula>. In these equations, <inline-formula id="d33e961"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e962" xlink:href="ncomms6140-m82.jpg"/></inline-formula> and <inline-formula id="d33e964"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e965" xlink:href="ncomms6140-m83.jpg"/></inline-formula>, with <inline-formula id="d33e967"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e968" xlink:href="ncomms6140-m84.jpg"/></inline-formula> and <inline-formula id="d33e971"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e972" xlink:href="ncomms6140-m85.jpg"/></inline-formula> (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>). Physically, <inline-formula id="d33e977"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e978" xlink:href="ncomms6140-m86.jpg"/></inline-formula> represents the pressures of free unpaired fermions or bound pairs, as one sees clearly that their expressions are identical to those for non-interacting particles. The term in the parentheses of <xref ref-type="disp-formula" rid="eq79">equation (14)</xref> reveals an important characteristic of contact in the strongly interacting region: it accounts for the interaction between bound pairs, and that between pairs and unpaired fermions, in addition to the contribution from each pair itself. The high-order corrections to contact from multibody interaction effects, that is, scattering involving three pairs, are relatively small in the strong-coupling regime. In this regard, the two-body interaction, including both pair&#x02013;pair and pair&#x02013;unparied fermions scattering, are important for determining the critical behaviours of contact in a strongly interacting Fermi gas. On the other hand, in order to capture proper thermal and quantum fluctuations in the quantum critical region, the universal scaling behaviour of the contact requests such marginal contributions from those higher order corrections (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>).</p><p>Using <xref ref-type="disp-formula" rid="eq65">equation (13)</xref>, we find the universal scaling form of <italic>c</italic> in the quantum critical region,</p><p><disp-formula id="eq87"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e996" xlink:href="ncomms6140-m87.jpg"/></disp-formula></p><p>where <italic>c</italic>_r is a temperature-independent regular part, the constant <italic>&#x003bb;</italic>_G depends on <italic>&#x003bc;</italic>_c, and <inline-formula id="d33e1014"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1015" xlink:href="ncomms6140-m88.jpg"/></inline-formula> is a dimensionless scaling function that can be determined by the TBA equations (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref>). The dynamic and correlation length exponents have been found to be <italic>z</italic>=2 and <italic>&#x003bd;</italic>=1/2, see the data collapse after use of scaling law (<xref ref-type="disp-formula" rid="eq87">equation (15)</xref>) in <xref ref-type="fig" rid="f2">Fig. 2</xref>. From <xref ref-type="disp-formula" rid="eq87">equation (15)</xref>, we obtain:</p><p><disp-formula id="eq89"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1038" xlink:href="ncomms6140-m89.jpg"/></disp-formula></p><p><xref ref-type="fig" rid="f3">Figure 3</xref> shows the scaling behaviour of this derivative of contact. Similar results can be obtained if one chooses <italic>H</italic> as the tuning parameter (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 3 and 4</xref>). Comparing <xref ref-type="disp-formula" rid="eq87">equation (15)</xref> with the standard scaling form of the density in the quantum critical region, <inline-formula id="d33e1053"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1054" xlink:href="ncomms6140-m90.jpg"/></inline-formula>, we find that <inline-formula id="d33e1056"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1057" xlink:href="ncomms6140-m91.jpg"/></inline-formula> and</p><p><disp-formula id="eq92"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1061" xlink:href="ncomms6140-m92.jpg"/></disp-formula></p><p>in analogy to <xref ref-type="disp-formula" rid="eq28">equation (10)</xref> in three dimensions. For the phase transition V&#x02013;P, <inline-formula id="d33e1067"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1068" xlink:href="ncomms6140-m93.jpg"/></inline-formula> and <inline-formula id="d33e1070"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1071" xlink:href="ncomms6140-m94.jpg"/></inline-formula>, so that <xref ref-type="disp-formula" rid="eq92">equation (17)</xref> can be rewritten as <inline-formula id="d33e1076"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1077" xlink:href="ncomms6140-m95.jpg"/></inline-formula>. For other phase transitions, such as P&#x02013;PP and F&#x02013;PP, this ratio has different constant values. The scaling forms, <xref ref-type="disp-formula" rid="eq87">equations (15)</xref> and <xref ref-type="disp-formula" rid="eq89">(16)</xref>, lead to the intersection of the scaled quantities <inline-formula id="d33e1086"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1087" xlink:href="ncomms6140-m96.jpg"/></inline-formula> and <inline-formula id="d33e1089"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1090" xlink:href="ncomms6140-m97.jpg"/></inline-formula> for different temperatures at <italic>&#x003bc;</italic>_c in our system. If one further plots these quantities as functions of (<italic>&#x003bc;</italic>&#x02212;<italic>&#x003bc;</italic>_c)/<italic>T</italic>, different curves collapse to a single one. Such intersections and data collapses are characteristic for the quantum critical behaviour of contact. We have numerically confirmed the validity of these scaling forms for all interaction strengths.</p><p>We now turn to the contact per particle in the quantum critical region. To highlight the quantum critical region and other ones in the <inline-formula id="d33e1111"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1112" xlink:href="ncomms6140-m98.jpg"/></inline-formula> plane, <xref ref-type="fig" rid="f4">Fig. 4</xref> shows a density plot of the entropy. The regions are separated by a crossover temperature <italic>T</italic>*, shown by the white dashed line in <xref ref-type="fig" rid="f4">Fig. 4</xref>. The crossover from the quantum critical region to the TLL region, where the density is finite for <inline-formula id="d33e1123"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1124" xlink:href="ncomms6140-m99.jpg"/></inline-formula> at zero temperature, is obtained from the deviation of the entropy from the linear form of TLL<xref ref-type="bibr" rid="b25">25</xref>. On the other side of the transition point, the crossover temperature from the quantum critical region to the semiclassical region, where the density is exponentially small, is obtained by setting the thermal wavelength equal to the interparticle spacing. In <xref ref-type="fig" rid="f4">Fig. 4a</xref>, three curves are shown for the rather small fixed values of the density <inline-formula id="d33e1132"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1133" xlink:href="ncomms6140-m100.jpg"/></inline-formula> listed in <xref ref-type="fig" rid="f4">Fig. 4b</xref>. One can see that a very large portion of the trajectory at such constant densities remains in the quantum critical region. As a result, <inline-formula id="d33e1138"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1139" xlink:href="ncomms6140-m101.jpg"/></inline-formula> becomes 1. In <xref ref-type="fig" rid="f4">Fig. 4b</xref>, numerical results for the scaled contact per particle for these three densities are shown to satisfy <inline-formula id="d33e1145"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1146" xlink:href="ncomms6140-m102.jpg"/></inline-formula> up to the temperature scale <italic>t</italic>=10^&#x02212;2, which corresponds to a ratio of the temperature to the chemical potential <inline-formula id="d33e1154"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1155" xlink:href="ncomms6140-m103.jpg"/></inline-formula>. These results directly confirm <xref ref-type="disp-formula" rid="eq92">equation (17)</xref>.</p><p>At higher densities and with increasing temperature, the trajectory at constant density first enters the TLL region, quickly passes the quantum critical region and eventually enters the high temperature region with <inline-formula id="d33e1162"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1163" xlink:href="ncomms6140-m104.jpg"/></inline-formula>, where the entropy density becomes large and the universal scaling laws of contact fail, as shown in <xref ref-type="fig" rid="f4">Fig. 4a</xref>. Below <italic>T</italic>*, and in the TLL phase of the paired fermions, referred to as phase TLL_p, the contact in the strong-coupling regime is given by</p><p><disp-formula id="eq105"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1176" xlink:href="ncomms6140-m105.jpg"/></disp-formula></p><p><xref ref-type="fig" rid="f4">Figure 4c</xref> shows both the numerical results of <inline-formula id="d33e1181"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1182" xlink:href="ncomms6140-m106.jpg"/></inline-formula> at large densities and the result of the TLL theory based on <xref ref-type="disp-formula" rid="eq105">equation (18)</xref>. It is clear that the growth of <inline-formula id="d33e1187"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1188" xlink:href="ncomms6140-m107.jpg"/></inline-formula> at low temperatures is described well by <xref ref-type="disp-formula" rid="eq105">equation (18)</xref>. The deviation from the TLL result shows a breakdown of the TLL at crossover temperature <italic>T</italic>*. More interestingly, one sees that before <inline-formula id="d33e1197"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1198" xlink:href="ncomms6140-m108.jpg"/></inline-formula> eventually decreases at high temperatures, a maximum occurs around <italic>t</italic>&#x02248;0.01 (about 0.1&#x02013;0.5&#x02009;<italic>T</italic>_F for strong attractive regime<xref ref-type="bibr" rid="b28">28</xref>), which corresponds to a quantum degenerate temperature <inline-formula id="d33e1210"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1211" xlink:href="ncomms6140-m109.jpg"/></inline-formula>. Such a maximum indicates that the contact per particle gets enlarged in the quantum degenerate region, similar to the possible enhancement of the contact near the transition temperature of three-dimensional (3D) fermions<xref ref-type="bibr" rid="b10">10</xref>.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Whereas the Tan relations have revealed how contact controls various thermodynamic quantities, it is in general difficult to make quantitative predictions as to how contact depends on the many-body physics of the system. Our results have shown that in the critical region near a phase transition point, contact and its derivatives are uniquely determined by the universality class of the phase transition. The exact thermodynamic relations shown in equations (1)&#x02013;(5) lead to both new insights into fundamental physics and profound applications for connecting contact and macroscopic quantum phenomena. Whereas these relations are exact for any microscopic parameters, they are particularly useful in the critical region for establishing exact relations between the universal scaling behaviours of contact and those of other thermal, magnetic and transport quantities. In particular, we have proved that contact in one dimension not only provides an unambiguous determination of the TLL phases and but also identifies in a novel fashion the universality class of quantum critical interacting many-body systems.</p><p>Moreover, equations (1)&#x02013;(5) can be used to ultimately settle the aforementioned controversy over the contact of the 3D unitary Fermi gas near the superfluid phase transition point. On the experimental side, our results suggest that high-resolution <italic>in situ</italic> images may be used to obtain precision measurements of the local pressure and contact as a function of temperature and other microscopic parameters, so that an average in the trap is not necessary. Such experiments will also be useful for exploring the size of the critical region, which is predicted to be of the order unity in the unitary limit<xref ref-type="bibr" rid="b40">40</xref>. On the theoretical side, whereas a number of approaches have obtained a continuous contact across the transition point, consistent with the prediction of our exact thermodynamic relations, one needs to examine whether the results produced by a theory indeed satisfy the exact thermodynamic relations in equations (1)&#x02013;(5).</p><p>In this Article, we have focused on continuous phase transitions, where all physical quantities, including both superfluid density and contact, are continuous across the transition point. It is worth pointing out that a unique phase transition occurs in two-dimensional (2D) superfluids, where the superfluid density has a finite jump, and meanwhile other thermodynamic quantities remain continuous, at the Berezinskii&#x02013;Kosterlitz&#x02013;Thouless transition point. It would be interesting to explore whether contact could signify such a finite jump of superfluid density controlled by the deconfinement of topological excitations, that is, vortices in 2D superfluids.</p><p>Highly controllable ultracold atoms are ideal platforms for exploring both universality of dilute systems governed by contact and universal critical phenomena near a phase transition point in many-body systems. In particular, current experiments with ultracold atoms are capable of measuring the critical behaviours of contact in all dimensions. We hope that our work will stimulate more studies on the intrinsic connection between these two types of fundamental phenomena on universality in physics.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>The model</title><p>For the attractive spin-1/2 Fermi gas at finite temperatures, the thermodynamics of the homogeneous system is described by two coupled Fermi gases of bound pairs and excess fermions in the charge sector and ferromagnetic spin&#x02013;spin interaction in the spin sector, namely the TBA equations read<xref ref-type="bibr" rid="b35">35</xref></p><p><disp-formula id="eq110"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1243" xlink:href="ncomms6140-m110.jpg"/></disp-formula></p><p>with <italic>m</italic>=1,&#x02026;&#x0221e;. In the above equations, * denotes the convolution integral, <inline-formula id="d33e1249"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1250" xlink:href="ncomms6140-m111.jpg"/></inline-formula> and <inline-formula id="d33e1252"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1253" xlink:href="ncomms6140-m112.jpg"/></inline-formula>. Here <italic>&#x003b5;</italic>^{b,u,<italic>m</italic>} are the dressed energies for bound pairs, excess single fermions and <italic>m</italic>-strings of spin wave-bound states, respectively. These dressed energies account for excitation energies above Fermi surfaces. In the above equations the function <inline-formula id="d33e1266"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1267" xlink:href="ncomms6140-m113.jpg"/></inline-formula> is given by <italic>T</italic>_{<italic>nm</italic>} (<italic>k</italic>)=<italic>A</italic>_{<italic>nm</italic>} (<italic>k</italic>)&#x02212;<italic>&#x003b4;</italic>_{<italic>nm</italic>} <italic>&#x003b4;</italic>(<italic>k</italic>) with <italic>A</italic>_{<italic>nm</italic>}=<italic>a</italic>_{|<italic>n</italic>&#x02212;<italic>m</italic>|}+2<italic>a</italic>_{(|<italic>n</italic>&#x02212;<italic>m</italic>|+2)}+&#x022ef;+2<italic>a</italic>_{(<italic>n</italic>+<italic>m</italic>&#x02212;2)}+<italic>a</italic>_{(<italic>n</italic>+<italic>m</italic>)}; see ref. <xref ref-type="bibr" rid="b35">35</xref>.</p><p>The effective chemical potentials of unpaired fermions and pairs were defined by <italic>&#x003bc;</italic>_u=<italic>&#x003bc;</italic>+<italic>H</italic>/2 and <italic>&#x003bc;</italic>_b=<italic>&#x003bc;</italic>+<italic>&#x003b5;</italic>_b/2. The thermal potential per unit length <italic>P</italic>=<italic>p</italic>^u+<italic>p</italic>^b is given in terms of the effective pressures <inline-formula id="d33e1396"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1397" xlink:href="ncomms6140-m114.jpg"/></inline-formula> with <italic>r</italic>=1 and 2 for the unpaired fermions and bound pairs.</p><p>The strategy for working out scaling form of contact near the critical points is to, at first, perform analytical calculation of contact near different phase transition points in the physical regime <inline-formula id="d33e1404"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1405" xlink:href="ncomms6140-m115.jpg"/></inline-formula> and <inline-formula id="d33e1407"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1408" xlink:href="ncomms6140-m116.jpg"/></inline-formula>. Then we confirm the analytical result of the universal scaling forms by numerically solving the TBA equations of the model for all interacting strengths. To this end, we first present the analytical expression of the total pressure <italic>P</italic>=<italic>p</italic>^b+<italic>p</italic>^u for the regime <inline-formula id="d33e1424"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1425" xlink:href="ncomms6140-m117.jpg"/></inline-formula> and <inline-formula id="d33e1427"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1428" xlink:href="ncomms6140-m118.jpg"/></inline-formula> (ref. <xref ref-type="bibr" rid="b37">37</xref>).</p><p><disp-formula id="eq119"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1435" xlink:href="ncomms6140-m119.jpg"/></disp-formula></p><p><disp-formula id="eq120"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1438" xlink:href="ncomms6140-m120.jpg"/></disp-formula></p><p>with the functions</p><p><disp-formula id="eq121"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1443" xlink:href="ncomms6140-m121.jpg"/></disp-formula></p><p><disp-formula id="eq122"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1446" xlink:href="ncomms6140-m122.jpg"/></disp-formula></p><p>In this model, the <italic>SU</italic>(2) spin degree of freedom ferromagnetically couples to the unpaired Fermi sea. Thus, the spin wave contributions to the function <italic>A</italic>_u is negligible owing to an exponentially small contributions at low temperatures<xref ref-type="bibr" rid="b37">37</xref>. By iteration, these effective pressures of bound pairs and unpaired fermions <italic>p</italic>^b,u can be presented in close forms. Here a significant observation from <xref ref-type="disp-formula" rid="eq119">equations (20)</xref> and <xref ref-type="disp-formula" rid="eq120">(21)</xref> is that the pressure <italic>P</italic> can be written in term of a universal scaling form near the critical fields, that is,</p><p><disp-formula id="eq123"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1477" xlink:href="ncomms6140-m123.jpg"/></disp-formula></p><p>where the dimensionless pressure <inline-formula id="d33e1480"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1481" xlink:href="ncomms6140-m124.jpg"/></inline-formula>, ₀ is the background pressure and is the dimensionless scaling function. The dimensionless critical chemical potential <inline-formula id="d33e1486"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1487" xlink:href="ncomms6140-m125.jpg"/></inline-formula> and critical field <italic>h</italic>_c=<italic>H</italic>_c/<italic>&#x003b5;</italic>_b depend on the interaction strength <italic>g</italic>_1D. Therefore, contact would essentially possesses universal scaling form near each critical point.</p></sec><sec disp-level="2"><title>Numerical method</title><p>In principle, the TBA <xref ref-type="disp-formula" rid="eq110">equation (19)</xref> in the paper present full thermodynamical properties of the model for all temperature regimes and interaction strength. Analytical result obtained above are useful for carrying out full thermodynamics of the model throughout all interaction regimes. In the present paper, the numerical calculations have been performed basing on the TBA equations of the spin-1/2 Fermi gas with attractive interaction (<xref ref-type="disp-formula" rid="eq110">equation (19)</xref>). The TBA (<xref ref-type="disp-formula" rid="eq110">equation (19)</xref>) involve infinite number of nonlinear integral equations accounting different lengths of spin strings (spin wave-bound states). This renders one to access the thermodynamics of the model analytically and numerically. The key observation is that for very large <italic>n</italic>, the function <italic>a</italic>_{<italic>n</italic>} (<italic>x</italic>)&#x02192;0. For the string number <italic>n</italic> is greater than a critical cutoff value of the <italic>n</italic>_c-length spin strings, the value <inline-formula id="d33e1545"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1546" xlink:href="ncomms6140-m126.jpg"/></inline-formula> is independent of the interaction. Consequently, the contributions to the <italic>&#x003b5;</italic>^u from higher spin strings, that is, <italic>n</italic> &#x0003e;<italic>n</italic>_c, can be calculated analytically. By iteration, one finds that the value of <italic>&#x003b5;</italic>^{<italic>n</italic>} for <italic>n</italic> &#x0003e;<italic>n</italic>_c is the same as the solution of the TBA (<xref ref-type="disp-formula" rid="eq132">equation (26)</xref>) with <italic>g</italic>_1D&#x02192;&#x0221e;; see ref. <xref ref-type="bibr" rid="b35">35</xref></p><p><disp-formula id="eq127"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1589" xlink:href="ncomms6140-m127.jpg"/></disp-formula></p><p>In our numerical programme, we fixed the value of <italic>n</italic>_c until the iteration error is small enough. In order to make a proper discretization in the variable space <italic>k</italic>, we need to find a cutoff <italic>k</italic>_c for the dressed energies <italic>&#x003b5;</italic>^{<italic>n</italic>}(<italic>k</italic>) in spin sector. For |<italic>k</italic>|&#x02192;&#x0221e;, we see <italic>&#x003b5;</italic>^{<italic>n</italic>}(<italic>k</italic>)&#x02192;<italic>&#x003b5;</italic>^{<italic>n</italic>,&#x0221e;}, which is given in <xref ref-type="disp-formula" rid="eq127">equation (25)</xref>, while for the charge sector <italic>&#x003b5;</italic>^u,&#x0221e;=<italic>&#x003b5;</italic>^b,&#x0221e;=&#x0221e;. Therefore, for |<italic>k</italic>|&#x0003e;<italic>k</italic>_c, we use this constant dressed energy <italic>&#x003b5;</italic>^{<italic>n</italic>,&#x0221e;} for numerical calculation. There exists an error in comparison with the real-dressed energies that is not flat in this region |<italic>k</italic>|&#x0003e;<italic>k</italic>_c. In our programme, we also fix the value <italic>kc</italic> until the iteration error is negilible.</p><p>For an arbitrary interaction strength, we are able to truncate infinite number of strings TBA equations to finite number of TBA equations in terms of the variables <italic>&#x003b5;</italic>^b,u=<italic>T</italic> ln <italic>&#x003be;</italic>^b,u and <italic>&#x003b5;</italic>^{<italic>n</italic>}=<italic>T</italic> ln<italic>&#x003b7;</italic>_{<italic>n</italic>}</p><p><disp-formula id="eq128"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1707" xlink:href="ncomms6140-m128.jpg"/></disp-formula></p><p><disp-formula id="eq129"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1710" xlink:href="ncomms6140-m129.jpg"/></disp-formula></p><p><disp-formula id="eq130"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1713" xlink:href="ncomms6140-m130.jpg"/></disp-formula></p><p><disp-formula id="eq131"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1716" xlink:href="ncomms6140-m131.jpg"/></disp-formula></p><p>&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02003;&#x02026;</p><p><disp-formula id="eq132"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1721" xlink:href="ncomms6140-m132.jpg"/></disp-formula></p><p>Here the functions <inline-formula id="d33e1725"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1726" xlink:href="ncomms6140-m133.jpg"/></inline-formula>. From the parameters <italic>&#x003be;</italic>^b(<italic>k</italic>) and <italic>&#x003be;</italic>^u(<italic>k</italic>), we can get the pressures <italic>p</italic>^b,u. This new set of the TBA equations provide numerical access to the full thermodynamics of the model, including the TLL physics, quantum criticality, thermodynamics and zero temperature phase diagram.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>Q.Z. and X.-W.G. conceived the project. Y.-Y.C. and Y.-Z.J. performed the numerical and analytical studies on the one-dimensional model. Q.Z and X.-W.G. wrote the paper.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article</bold>: Chen, Y.-Y. <italic>et al.</italic> Critical behaviours of contact near phase transitions. <italic>Nat. Commun.</italic> 5:5140 doi: 10.1038/ncomms6140 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-4, Supplementary Note 1 and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6140-s1.pdf"/></supplementary-material></sec>

LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling

<p>The receptor tyrosine kinase Flt3 and its ligand are crucial for dendritic cell (DC) homeostasis by activating downstream effectors including mammalian target of Rapamycin (mTOR) signalling. LAMTOR2 is a member of the Ragulator/LAMTOR complex known to regulate mTOR and extracellular signal-regulated kinase activation on the late endosome as well as endosomal biogenesis. Here we show in mice that conditional ablation of <italic>LAMTOR2</italic> in DCs results in a severe disturbance of the DC compartment caused by accumulation of Flt3 on the cell surface. This results in an increased downstream activation of the AKT/mTOR signalling pathway and subsequently to a massive expansion of conventional DCs and plasmacytoid DCs in ageing mice. Finally, we can revert the symptoms <italic>in vivo</italic> by inhibiting the activation of Flt3 and its downstream target mTOR.</p>

<contrib contrib-type="author"><name><surname>Scheffler</surname><given-names>Julia M.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sparber</surname><given-names>Florian</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tripp</surname><given-names>Christoph H.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Herrmann</surname><given-names>Caroline</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Humenberger</surname><given-names>Alexandra</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Blitz</surname><given-names>Johanna</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Romani</surname><given-names>Nikolaus</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Stoitzner</surname><given-names>Patrizia</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Huber</surname><given-names>Lukas A.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref></contrib><aff id="a1"><label>1</label><institution>Biocenter, Division of Cell Biology, Innsbruck Medical University, Innrain 80-82</institution>, Innsbruck A6020, <country>Austria</country></aff><aff id="a2"><label>2</label><institution>Department of Dermatology and Venereology, Innsbruck Medical University</institution>, Innsbruck A6020, <country>Austria</country></aff><aff id="a3"><label>3</label><institution>Austrian Drug Screening Institute (ADSI)</institution>, Innsbruck A6020, <country>Austria</country></aff>

Nature Communications

<p>Cells use signalling cascades to translate extracellular information into specific downstream responses. A major signalling pathway regulating cell growth, differentiation and survival is the extracellular signal-regulated kinase (ERK) cascade, which belongs to the family of mitogen-activated protein kinase (MAPK) pathways. To ensure specific signalling, a spatial and temporal segregation must be achieved. After activation, receptors are endocytosed and transferred to late endosomes for degradation. However, it was shown that late endosomes also function as signalling platforms. There, the late endosomal/lysosomal adaptor and MAPK and mammalian target of Rapamycin (mTOR) activator (LAMTOR) complex serves as a convergence point for ERK and mTOR complex 1 (mTORC1) signalling. It consists of LAMTOR1 (p18), LAMTOR2 (p14), LAMTOR3 (MP1), LAMTOR4 (HBXIP) and LAMTOR5 (C7orf59)<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>. Deletion of <italic>LAMTOR2</italic> results in a destabilization and cytosolic mislocalization of the remaining complex components<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b9">9</xref>. In addition, conditional gene ablation of <italic>LAMTOR2</italic> in keratinocytes in the epidermis of mice revealed its importance for tissue homeostasis, cellular proliferation and endosomal traffic<xref ref-type="bibr" rid="b10">10</xref>. A previously identified human primary immunodeficiency syndrome was ascribed to a point mutation in the <italic>LAMTOR2</italic> gene causing a hypomorph allele and reduced protein levels of LAMTOR2. Those patients have severe immunological defects affecting the innate and adaptive immunity, which can be related to a disturbed endosomal- and lysosomal biogenesis. They suffer from neutropenia, defects in T-cell function and B-cell maturation and subsequently have recurrent broncho-pulmonary infections<xref ref-type="bibr" rid="b11">11</xref>. In correlation with these observations, we could recently show in a mouse model that LAMTOR2 is crucial for macrophages to fight <italic>Salmonella</italic> infection by controlling replication in the phagosome<xref ref-type="bibr" rid="b12">12</xref>. Based on these findings, we were interested in the role of LAMTOR2 for adaptive immunity.</p><p>Dendritic cells (DCs) are the initiators of adaptive immunity. Their ability to take up, process and finally present pathogenic as well as self-antigens to T cells, is strictly dependent on efficient late endosomal-biogenesis<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. DCs originate from haematopoietic stem cells and differentiate via common progenitors to so-called pre-DCs, which finally seed various organs to become fully differentiated DCs. Specific cytokine signals are indispensable throughout this development as well as for the homeostasis of DCs<xref ref-type="bibr" rid="b15">15</xref>. Originally, it was thought that granulocyte-macrophage colony-stimulating factor (GM-CSF) is the major cytokine promoting DC differentiation, as it allowed for the first time the <italic>in vitro</italic> generation of DCs from human blood and mouse bone marrow (BM)<xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. However, the discovery that mice lacking GM-CSF or its receptor still develop normal DC populations in the spleen and lymph nodes (LNs)<xref ref-type="bibr" rid="b19">19</xref> led to the conclusion that GM-CSF is dispensable for steady-state DC development. As shown recently, this also holds true for differentiation of inflammatory DCs. In contrast, deletion or inhibition of another cytokine receptor, named Fms-like tyrosine kinase 3 ligand receptor (Flt3) and its ligand (Flt3-L), resulted in a tenfold reduction of plasmacytoid DCs (pDCs) and tissue resident DCs<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. Conversely, injection of Flt3-L in mice increased DC numbers of various subtypes in many organs<xref ref-type="bibr" rid="b22">22</xref>. These findings together with the fact that Flt3 is expressed on common DC progenitors (CDPs), pre-DCs and their progeny<xref ref-type="bibr" rid="b23">23</xref> underline the importance of Flt3 receptor signalling for DC differentiation. However, little was known about the downstream Flt3 signalling controlling DC development until recent findings showed that the mammalian target of Rapamycin (mTOR) plays a major role in this signalling cascade. It was shown that the phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling cascade downstream of Flt3 controls DC development and expansion<xref ref-type="bibr" rid="b24">24</xref>. Inhibiting this signalling pathway by Rapamycin resulted in an impairment of steady-state DC generation <italic>in vivo</italic><xref ref-type="bibr" rid="b25">25</xref>.</p><p>As LAMTOR2, as member of the Ragulator/LAMTOR complex, is crucial not only for ERK but also for mTOR activation and influences endosomal biogenesis and receptor trafficking, we decided to investigate its function specifically in DCs. Therefore, we made use of a conditional knockout mouse model in which <italic>LAMTOR2</italic> can be specifically deleted in CD11c⁺ DCs<xref ref-type="bibr" rid="b26">26</xref>. Here we show that genetic ablation of <italic>LAMTOR2</italic> in DCs results in the accumulation of the Flt3-receptor on the plasma membrane accompanied by a deregulation of LAMTOR complex-mediated downstream signalling. As a consequence, late endosomal ERK signalling is abolished. However, despite the loss of the LAMTOR complex, ligand-induced AKT/mTORC1 signalling downstream of the Flt3 receptor is unexpectedly increased. The outcome of this enhanced mTOR signalling is an expansion of pDCs and conventional DCs (cDCs), which finally cause a myeloid proliferative syndrome in ageing mice.</p><p>Thus, we present evidence that LAMTOR2 is crucial for Flt3-dependent DC homeostasis and additionally describe new aspects of LAMTOR complex-mediated late endosomal signalling in immunity.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Deletion of <italic>LAMTOR2</italic> in DCs alters DC homeostasis</title><p>To specifically delete <italic>LAMTOR2</italic> in DCs of mice, we used a conditional mouse model in which the Cre recombinase is expressed under the promotor of the <italic>CD11c</italic> gene<xref ref-type="bibr" rid="b26">26</xref>. These mice were crossed with the <italic>LAMTOR2</italic>_f/f mice<xref ref-type="bibr" rid="b10">10</xref> to obtain CD11c <italic>LAMTOR2</italic>_del mice harbouring the homozygous deletion of <italic>LAMTOR2</italic>. This could be confirmed via genotyping (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1a</xref>), as well as on protein (<xref ref-type="fig" rid="f1">Fig. 1a</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1b</xref>) and mRNA levels (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1c</xref>). In addition, a reduction of the protein levels of all other known LAMTOR components was observed (<xref ref-type="fig" rid="f1">Fig. 1a</xref>), as described before<xref ref-type="bibr" rid="b9">9</xref>. To exclude an effect of background expression of Cre in other major immune cell compartments<xref ref-type="bibr" rid="b26">26</xref>, mRNA levels of LAMTOR2 in sorted splenic B and T cells were assessed (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1c</xref>). No significant reduction was measured. Heterozygous CD11c <italic>LAMTOR</italic>2_del/+ mice, possessing one functional <italic>LAMTOR2</italic> allele, were always used as control mice. CD11c <italic>LAMTOR2</italic>_del mice developed normally and were born in the expected Mendelian frequencies. Body weight (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1d</xref>) and appearance of CD11c <italic>LAMTOR2</italic>_del mice were similar to control mice. However, at 3 months of age, the deficient mice displayed significantly enlarged spleens and LNs (<xref ref-type="fig" rid="f1">Fig. 1b,c</xref>). Further analysis of these two organs revealed severe morphological alterations in tissue architecture. Histological staining with haematoxylin and eosin (H&#x00026;E) of the spleen revealed a disturbed segregation of red and white pulpa, with increased germinal centres size and accumulations of infiltrating leukocytes (<xref ref-type="fig" rid="f1">Fig. 1b</xref>, upper panel, arrow heads). The LNs of the CD11c <italic>LAMTOR2</italic>_del animals showed a similar morphology. B-cell areas also showed enlarged germinal centres and the in the T-cell zone massive infiltrates were found (<xref ref-type="fig" rid="f1">Fig. 1b</xref>, lower panel, arrow heads). Immunohistochemistry for the expression of the DC marker CD11c showed that the cellular infiltrates largely consisted of CD11c⁺ DCs (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). Further histological investigation revealed infiltrates also in other organs of CD11c <italic>LAMTOR2</italic>_del mice. Conditional <italic>LAMTOR2</italic>-deficient mice developed massive infiltrates in the perivascular region of the liver starting at the age of 3 months (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1e</xref>). To further characterize the infiltrating cells in the liver, immunofluorescence staining of cryosections was performed. The infiltrates were positive for the DC marker CD11c (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1e</xref>) but negative for Gr-1 and F4-80 (data not shown).</p><p>As disturbances in cellular populations leading to enlargements of the spleen and LNs of CD11c <italic>LAMTOR2</italic>_del animals were detected, flow cytometry analysis was performed. No significant alterations of cell numbers in major immune cell compartments including CD19⁺ B cells, CD3⁺ T cells and CD3^&#x02212; Nkp46⁺ NK cells were observed (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2a&#x02013;d</xref>). However, major shifts in the DC compartment were revealed. In both lymphoid organs, a significant increase in numbers of PDCA-1^high pDCs and CD11c^high MHCII^high cDCs were measured (<xref ref-type="fig" rid="f1">Fig. 1d,e</xref>). The reduction of migratory DCs in the skin draining LN of the CD11c <italic>LAMTOR2</italic>_del mice was described before<xref ref-type="bibr" rid="b27">27</xref>. To further confirm the DC expansion in those organs, immunofluorescence staining for CD11c and PDCA-1 was performed and underlined the described results (<xref ref-type="fig" rid="f1">Fig. 1f</xref>). Interestingly, especially in the spleen, a population of CD11c⁺ MHCII^int and PDCA-1^int cells expanded significantly (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>). Those cells exhibited a similar morphology as pDCs (<xref ref-type="fig" rid="f2">Fig. 2c</xref>) and were also deficient for <italic>LAMTOR2</italic> (<xref ref-type="fig" rid="f2">Fig. 2d</xref>).</p></sec><sec disp-level="2"><title><italic>LAMTOR2</italic> deletion influences DC-dependent immunity in the spleen</title><p>In a next step, we further characterized the activation status of the expanded DC populations. The expression of co-stimulatory molecules on cDC, pDC and on the PDCA-1^int cells was analysed. We observed increased expression of CD86 and PD-L1 on cDCs and the PDCA-1^int cells of CD11c <italic>LAMTOR2</italic>_del mice, whereas CD40 levels were not significantly changed. In general, pDCs showed a low expression of all three activation markers and no differences between control and <italic>LAMTOR2</italic>-deficient cells were detected (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). As this enhanced DC activation could also lead to T-cell stimulation, we analysed the T-cell compartment for common activation markers. In the CD8⁺ T-cell compartment of CD11c <italic>LAMTOR2</italic>_del mice, a reduction of na&#x000ef;ve and central memory T cells was observed; however, effector memory T cells and the overall CD69 expression were unchanged. PD-1 expression was not detected on the CD8⁺ T cells. In the CD4⁺ T-cell compartment of CD11c <italic>LAMTOR2</italic>_del mice, a shift towards effector memory cells and increased expression of activation markers like CD69 and PD-1 was measured (<xref ref-type="fig" rid="f3">Fig. 3b</xref>). These higher numbers of activated T cells in combination with increased CD86 and PD-L1 levels on DCs indicated induction of inflammation. To confirm these findings, we analysed the serum of control and CD11c <italic>LAMTOR2</italic>_del mice for inflammatory cytokines. There were no significant increases in interleukin (IL)-1&#x003b2;, IL-4 and IL-6, except for tumour-necrosis factor-&#x003b1; levels in the serum of CD11c <italic>LAMTOR2</italic>_del mice (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3a</xref>). However, when we analysed the culture supernatant of total spleen cells, more pro-inflammatory cytokines, like IL-1&#x003b2;, IL-6 and tumour-necrosis factor-&#x003b1;, were secreted by CD11c <italic>LAMTOR2</italic>_del splenic cells. In addition, we also measured an elevation of IL-4, IL-10, interferon (IFN)-&#x003b3; and IL-12p70, cytokines released by DCs and T cells (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3b</xref>).</p><p>Next, we wondered if <italic>LAMTOR2-</italic>deficient DCs were more efficient T-cell stimulators. However, DCs from CD11c <italic>LAMTOR2</italic>_del mice, pulsed with ovalbumin protein, were equally potent in stimulating proliferation of ovalbumin-specific CD4⁺ and CD8⁺T cells in <italic>in vitro</italic> T-cell assays (data not shown).</p><p>Finally, we wanted to know how <italic>LAMTOR2</italic>-deficient DCs behave after Toll-like receptor stimulation with CpG <italic>in vitro</italic>. <italic>LAMTOR2</italic>-deleted cDCs and pDCs secreted increased amounts of IFN-&#x003b1;, IL-12p70, IL-4, IL-6 and IL1-&#x003b2; in response to Toll-like receptor stimulation. Interestingly, IL-10 levels were reduced (<xref ref-type="fig" rid="f3">Fig. 3c</xref>).</p><p>Together, these findings indicated that <italic>LAMTOR2</italic>-deleted DCs showed some signs of activation leading to a local inflammatory process that also affected the T-cell compartment.</p></sec><sec disp-level="2"><title>DC expansion is due to increased proliferation</title><p>To assess whether DC homeostasis in CD11c <italic>LAMTOR2</italic>_del mice was already affected during differentiation, we analysed the macrophage and DC progenitors (MDPs), the common DC progenitors (CDPs) and the pre-DCs<xref ref-type="bibr" rid="b28">28</xref> in the BM. We could not observe any differences for MDP or CDP numbers of both genotypes (<xref ref-type="fig" rid="f4">Fig. 4b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a</xref>). The pre-DC numbers, however, were significantly increased in the CD11c <italic>LAMTOR2</italic>_del mice (<xref ref-type="fig" rid="f4">Fig. 4a,b</xref>).</p><p>In a next step, we analysed the proliferation of DCs in the spleen by Ki67 staining of splenic DCs of control and CD11c <italic>LAMTOR2</italic>_del mice. All DC populations, including the PDCA-1^int one, contained increased numbers of proliferating <italic>LAMTOR2</italic>-deficient DCs (<xref ref-type="fig" rid="f5">Fig. 5a</xref>, <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>). In addition, we analysed the infiltrates in the liver for Ki67 expression and found Ki67-positive CD11c+elevated DCs in the infiltrates (<xref ref-type="fig" rid="f5">Fig. 5b</xref>, white arrow).</p><p>Taken together, DC-specific deletion of <italic>LAMTOR2</italic> severely affected DC homeostasis resulting in an increase of pre-DCs in the BM and proliferation of cDC, pDCs and CD11c⁺MHCII⁺PDCA-1^int cells in the spleen and liver.</p></sec><sec disp-level="2"><title>Altered Flt3-receptor trafficking in DCs depleted of <italic>LAMTOR2</italic></title><p>The Flt3-ligand (Flt3-L) together with its corresponding receptor Flt3, represents an essential cytokine signal for DC homeostasis, regulating development and differentiation of DC progenitors as well as differentiated DCs<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b29">29</xref>. In mice, the deletion or inhibition of Flt3-L and its receptor leads to a severe reduction of cDCs and pDCs. On the contrary, injection or overexpression of Flt3-L or constitutively active Flt3 signalling leads to the expansion of the DC compartment and subsequently to a myeloproliferative disorder (MPD)<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>. As not only differentiated DCs but also their pre-DCs express Flt3, we wondered whether the expansion of the DC compartment might be due to an alteration in the Flt3-L availability. The levels of soluble Flt3-L in serum samples of CD11c <italic>LAMTOR2</italic>_del mice ranging from 1 to 4 months of age were significantly increased compared with control mice (<xref ref-type="fig" rid="f6">Fig. 6a</xref>). Furthermore, we observed alterations concerning the receptor itself. Surface expression analyses by flow cytometry for Flt3 on isolated splenic DCs (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6a</xref>) revealed a significantly increased mean fluorescence intensity (<xref ref-type="fig" rid="f6">Fig. 6b</xref>), indicating an accumulation of the receptor on the surface of DCs of CD11c <italic>LAMTOR</italic>_del mice. As it is known that LAMTOR2 plays a crucial part in receptor tyrosine kinase trafficking by controlling endosomal biogenesis (for example, epidermal growth factor (EGF) receptor<xref ref-type="bibr" rid="b10">10</xref>), we performed pulse chase experiments for Flt3 on isolated splenic DCs. After ligand binding, receptor tyrosine kinases get internalized and are transported through the endocytic pathway to be finally degraded in the lysosome. However, similar to the defect for EGF receptor trafficking seen in <italic>LAMTOR2</italic>-depleted keratinocytes<xref ref-type="bibr" rid="b10">10</xref>, in which the receptor transfer to the late endosome is delayed, also the Flt3 receptor did not reach the late endosomal compartment. Confocal imaging revealed that 20&#x02009;min after stimulation with the ligand, the Flt3 receptor was already localizing to late endosomes in control cells. In contrast, no co-localization of Flt3- and Lamp1-positive vesicles could be observed (<xref ref-type="fig" rid="f6">Fig. 6c</xref>).</p><p>In summary, we could show that DC-specific deletion of <italic>LAMTOR2</italic> in mice led to increased Flt3-ligand serum levels and an accumulation of the receptor on the plasma membrane because of impaired receptor trafficking towards the late endosome.</p></sec><sec disp-level="2"><title>Flt3 surface accumulation leads to increased mTOR activation</title><p>As it was shown that the activation of PI3K-mTOR signalling, downstream of Flt3, is crucial for DC development and inhibition of this pathway reduces DC numbers<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>, we decided to investigate the effect of Flt3 receptor accumulation on cell surface in downstream signalling. Previous studies have revealed that loss of LAMTOR2 affects ERK activation as well as mTOR signalling on the late endosome<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b5">5</xref>. Deletion of <italic>LAMTOR2</italic> in the DC compartment led to a reduction of phosphorylation of ERK in splenic DCs (<xref ref-type="fig" rid="f7">Fig. 7a</xref>). Interestingly, we, however, observed an increased activation of phospho-AKT and phospho-p70 S6 kinase in isolated splenic DC from CD11c <italic>LAMTOR2</italic>_del animals as compared with the controls (<xref ref-type="fig" rid="f7">Fig. 7b</xref>). The increased activation of mTOR was observed at steady state as well as after Flt3-L stimulation. For further analysis, we decided to use BM-derived DCs (BMDCs), differentiated in the presence of Flt3-L<xref ref-type="bibr" rid="b32">32</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6b</xref>). Titration experiments using different concentrations of Flt3-L revealed that low concentrations of Flt3-L significantly increased numbers of DCs in the BM cultures of the CD11c <italic>LAMTOR2</italic>_del mice as compared with the control cultures (<xref ref-type="fig" rid="f8">Fig. 8a</xref>). This result suggests a higher susceptibility of <italic>LAMTOR2-</italic>deficient BMDCs towards Flt3-L. Hence, our <italic>in vitro</italic> BMDC model seems to mimic the DC expansion observed <italic>in vivo</italic> at least partially. In the next step, we analysed the downstream signalling. As expected, BMDC cultures of CD11c <italic>LAMTOR2</italic>_del mice showed a decrease in phospho ERK activation (<xref ref-type="fig" rid="f8">Fig. 8b</xref>), however, again an increased activation of AKT and mTOR at steady state and after stimulation with Flt3-L was detected (<xref ref-type="fig" rid="f8">Fig. 8c,d</xref>, lanes 1 to 4). In addition, we added the inhibitors Rapamycin (RAPA; <xref ref-type="fig" rid="f8">Fig. 8c</xref>) and AC220 (<xref ref-type="fig" rid="f8">Fig. 8d</xref>) to the BMDC cultures in order to inhibit mTOR-activation and Flt3 kinase activity, respectively. AC220 (Quizartinib) is a second-generation Flt3 inhibitor developed to treat patients with acute myeloid leukaemia. In approximately 30% of those patients, Flt3 signalling is constitutively active due to mutations in the receptor. It was shown that AC220 has excellent potency, selectivity and pharmacokinetic properties<xref ref-type="bibr" rid="b33">33</xref> and is used in clinical trials to treat acute myeloid leukaemia<xref ref-type="bibr" rid="b34">34</xref>. Both inhibitors caused a decrease in mTOR activation (<xref ref-type="fig" rid="f8">Fig. 8c,d</xref>, lanes 5 to 8) at steady state but, in particular, after Flt3-L stimulation.</p><p>In summary, we could show that the increase of plasma membrane-associated Flt3 led to the enhanced activation of the downstream AKT-mTOR signalling pathway in <italic>LAMTOR2</italic>-deficient isolated splenic DCs as well as in BMDCs derived under the influence of Flt3-L. However, this enhanced activation of AKT-mTOR signalling could be abrogated by addition of Rapamycin or AC220. Importantly, the enhanced activation of the Flt3 signalling pathway was further reflected by an increase of DC numbers in <italic>LAMTOR2</italic>-deficient BMDC cultures with limiting concentrations of Flt3-L. This might be an explanation for the DC-related MPD observed <italic>in vivo</italic> in DC-specific <italic>LAMTOR2</italic>-deficient mice.</p></sec><sec disp-level="2"><title>Rapamycin reverts the phenotype in CD11c <italic>LAMTOR2</italic> _del mice</title><p>To confirm our hypothesis that increased activation of the AKT-mTOR signalling pathway caused the MPD symptoms in CD11c <italic>LAMTOR2</italic>_del mice, we treated the knockout and control animals with 30&#x02009;&#x003bc;g Rapamycin for 10 days<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>. By doing so, we could rescue the phenotype of conditional <italic>LAMTOR2</italic>-deficient mice, almost comparable to the one of healthy control mice. Spleen and LN size decreased significantly under the treatment compared with the vehicle-treated CD11c <italic>LAMTOR2</italic>_del mice. The size of the organs was almost comparable to that of the control animals (<xref ref-type="fig" rid="f9">Fig. 9a,b</xref>). We investigated the effect of mTOR inhibition on the DC population of the spleen. Therefore, we performed flow cytometry analysis on spleen cell suspensions, where we observed a significant decrease of the DC compartment of CD11c <italic>LAMTOR2</italic>_del mice treated with Rapamycin in comparison to the untreated animals. However, we did not observe an effect of Rapamycin treatment in control animals (<xref ref-type="fig" rid="f9">Fig. 9c</xref>). The reduction of DC-related infiltrates in Rapamycin-treated conditional <italic>LAMTOR2-</italic>deficient animals was also observed in histological sections of LNs and spleens. In addition to the regression of DC-infiltrates, we noticed an obvious recovery of the organ architecture of the spleen and LNs in Rapamycin-treated mice (H&#x00026;E staining, <xref ref-type="fig" rid="f9">Fig. 9d</xref>, first and third row). Finally, immunohistochemistry for the expression of CD11c on frozen organ sections also showed a decrease in infiltrating DCs after mTOR inhibition (<xref ref-type="fig" rid="f9">Fig. 9d</xref>, second and fourth row).</p><p>Taken together, inhibition of mTOR signalling by the administration of Rapamycin rescued the phenotype of CD11c <italic>LAMTOR2</italic>_del mice and restored the size of various organs by suppressing the expansion of DCs.</p></sec><sec disp-level="2"><title>Flt3 inhibition by AC220 treats MPD in CD11c <italic>LAMTOR2</italic> _del mice</title><p>In a second approach, we aimed to suppress DC expansion in the CD11c <italic>LAMTOR2</italic>_del mice by directly blocking Flt3 kinase activity. In another mouse model developing MPD due to enhanced wild-type Flt3 signalling, AC220 could hold the disease in remission during the therapy period<xref ref-type="bibr" rid="b35">35</xref>. The mice were treated daily for 28 days with 10&#x02009;mg&#x02009;kg^&#x02212;1 AC220 or vehicle by oral gavage and then killed to investigate the effect on the DC compartment. AC220-treated conditional <italic>LAMTOR2</italic>-deficient mice revealed a significant reduction in size of spleen and LNs as compared with untreated mice. Also a decrease of the LN size, almost back to control size, could be observed (<xref ref-type="fig" rid="f10">Fig. 10a&#x02013;c</xref>). Flow cytometry analyses of spleen suspension cells from those animals marked a significant drop of the DC numbers. Furthermore, analysis of spleen and LN morphology by H&#x00026;E staining (<xref ref-type="fig" rid="f10">Fig. 10d</xref>, first and third row) was performed. Both organs of CD11c <italic>LAMTOR2</italic>_del mice displayed a partial restoration of the distinct lymphocytic compartments upon treatment, which was more prominent in the LN. Moreover, immunohistochemistry revealed less infiltrating DCs (<xref ref-type="fig" rid="f10">Fig. 10d</xref>, second and fourth row).</p><p>Thus, treatment of the CD11c <italic>LAMTOR2</italic>_del mice with a specific Flt3 inhibitor, AC220, led to the remission of the MPD symptoms. DC expansion in the spleen and LNs was reduced, leading to a normalization of organ morphology and size.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>LAMTOR2 plays a critical role in tissue homeostasis and immunity<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b27">27</xref>. Here, we contribute insights into the function of LAMTOR2, as part of the Ragulator/LAMTOR complex, which operates as molecular switch on the late endosome to differentially regulate mTOR and MAPK-signalling downstream of the Flt3-receptor. As a consequence of DC-specific deletion of <italic>LAMTOR2</italic>, a disruption of the entire complex is observed, followed by a deregulation of Flt3 sorting and signalling. This severely affects DC homeostasis and culminates in a MPD.</p><p>LAMTOR2 was shown to play a crucial role for innate and adaptive immunity, as described in patients harbouring a hypomorph <italic>LAMTOR2</italic> allele and as consequence suffering from recurrent broncho-pulmonary infections. One explanation for their immunodeficiency syndrome are defects in their endo- and lysosomal biogenesis resulting in insufficient digestion of bacteria by neutrophiles<xref ref-type="bibr" rid="b11">11</xref>. Along those lines go observations that mice with <italic>LAMTOR2</italic>-deficient macrophages cannot properly fight <italic>Salmonella</italic> infections due to defects in their phagosomal compartment<xref ref-type="bibr" rid="b12">12</xref>. Recent findings strengthened the assumption that LAMTOR2 also plays a crucial role in DCs as major inducers of the adaptive immunity. Preceding investigations of the same conditional CD11c <italic>LAMTOR2</italic>_del mouse model revealed a permanent loss of epidermal Langerhans cells, a subpopulation of skin DCs, shortly after birth because of reduced proliferation and increased apoptosis. Further investigations of LN resident DC populations showed no alterations at the age of 6&#x02013;8 weeks<xref ref-type="bibr" rid="b27">27</xref>. The massive expansion of cDCs and pDCs we describe here is observed only after 10&#x02013;12 weeks, suggesting that the myeloproliferative syndrome develops later on in ageing mice. Interestingly, unlike Langerhans cells, cDC and pDCs depend strongly on Flt3 receptor signalling through the AKT/mTORC1 pathway for their differentiation and development<xref ref-type="bibr" rid="b36">36</xref>. Hackstein and colleagues observed that the <italic>in vivo</italic> administration of Rapamycin, a classical inhibitor of mTOR, inhibits the effect of Flt3-L as an important DC growth factor<xref ref-type="bibr" rid="b25">25</xref>. Also, the <italic>in vitro</italic> DC development of pDCs and steady-state DCs is impaired after Rapamycin treatment and conversely, deletion of the PI3K inhibitor PTEN, facilitates Flt3-L-induced DC expansion<xref ref-type="bibr" rid="b24">24</xref>.</p><p>mTOR signalling is an essential pathway to promote cell growth and proliferation under nutrient-rich conditions by, for example, protein synthesis and repression of autophagy via amino-acid sensing. Amino acid-induced mTORC1 activation takes place at the membrane of late endosomes. The Ragulator/LAMTOR complex, including LAMTOR2, was shown to be essential for recruiting the Rag proteins to the lysosome, where those, in response to amino-acid stimuli, interact with and activate mTORC1 (ref. <xref ref-type="bibr" rid="b3">3</xref>). In contrast to the intrinsic amino acid-dependent mTOR activation, extrinsic stimuli like cytokines and growth factors induce mTOR signalling via receptor tyrosine kinases. This is followed by the phosphorylation of the tuberous sclerosis complex, which then releases its inhibiting effect on Ras homolog enriched in brain (RHEB), the activator of mTORC1 (ref. <xref ref-type="bibr" rid="b37">37</xref>).</p><p>As the LAMTOR complex is crucial for the activation of MEK/ERK and mTORC1 on late endosomes and deletion of <italic>LAMTOR2</italic> destabilizes the so far known components, we investigated how both signalling pathways would be affected. As expected, we observed differences of the activation state of ERK1/2 in isolated splenic DCs as well as BMDCs derived under the influence of Flt3-L. Upon deletion of <italic>LAMTOR2,</italic> ERK activation was reduced as was also shown previously for <italic>LAMTOR1</italic> (ref. <xref ref-type="bibr" rid="b4">4</xref>), <italic>LAMTOR2</italic> (ref. <xref ref-type="bibr" rid="b10">10</xref>) and LAMTOR3 (ref. <xref ref-type="bibr" rid="b38">38</xref>) depletion. Interestingly, stimulation with Flt3-L did not increase the activation of ERK, confirming a distinct Flt3 downstream signalling pathway inducing the MPD.</p><p>Next, we analysed the AKT/mTOR pathway in DCs of CD11c <italic>LAMTOR2</italic>_del mice. Unexpectedly, we observed there a hyper-activation of AKT, p70 S6K1 and S6 protein in splenic DCs and Flt3-L-BMDCs depleted of <italic>LAMTOR2</italic>. This could be measured under steady state as well as under stimulation with Flt3-L. Thus, we assume that extrinsic stimulation of mTORC1 via the Flt3 receptor must bypass LAMTOR complex-dependent mTORC1 signalling on endosomes and most likely takes place at a different subcellular localization, for example, as recently discussed for the cytosolic compartment<xref ref-type="bibr" rid="b39">39</xref>.</p><p>The consequences of such uncoupled signalling, involving two of the major signalling pathways regulating cell growth and proliferation, are severe. Although ERK signalling is abolished, the elevated mTOR signalling induces a massive expansion of pre-DCs and DCs resulting in a MPD. As a possible mechanism, we could identify a transport defect of the Flt3 receptor to the lysosome followed by an increase of the receptor on the surface of DCs. This finding underlines the role of LAMTOR2 in receptor trafficking and endosomal biogenesis, as it goes in line with previous results, showing slower transport of the EGF-receptor in LAMTOR2-depleted mouse embryonic fibroblasts (MEF) as well as in keratinocytes to the lysosome<xref ref-type="bibr" rid="b10">10</xref>.</p><p>In addition, we also measured increased levels of Flt3-L in the serum of CD11c <italic>LAMTOR2</italic>_del mice. Flt3-L is a crucial cytokine during haematopoiesis and elevated Flt3-L levels, through <italic>in vivo</italic> administration, not only affect stem cells and myeloid progenitors but also lead to an elevation of immature B cell, NK cell and DC numbers in lymphoid organs and can finally result in a MPD<xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref>. Although most cell types express a certain amount of Flt3-L mRNA, stromal cells and activated, proliferating T cells are the major producers<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref>. As we observed an increased expression of maturation markers on splenic DCs in the CD11c <italic>LAMTOR2</italic>_del mice, we speculate that this might be also the result of a failure in LAMTOR2-dependent receptor sorting and consequently as shown, T-cell activation takes place that by itself might lead to elevated Flt3-L secretion. Another possibility could be a reduced consumption of ligand by DCs. This might be explained by the stuck receptor on the cell surface. The bound ligand would not be disassociated and a constitutive active receptor signalling, as observed, would be the consequence.</p><p>Interestingly, the observed increased Flt3-L levels did neither affect MDP and CDP numbers in the BM nor numbers of immature B cells and NK cells in the spleen. However, a significant elevation of CD11c⁺ pre-DCs in the BM and increased proliferation of DCs in the spleen and liver were assessed. This observation led to the conclusion that not the elevated ligand levels alone but their combination with the increased presence of Flt3 on the cell surface causes the pronounced expansion of DCs as well as CD11c⁺ pre-DCs. The impact of this scenario leads to the above-described paradox in the uncoupled Flt3 receptor downstream signalling.</p><p>To further underline our finding, we confirmed that <italic>in vitro</italic> as well as <italic>in vivo</italic> inhibition of Flt3-receptor-induced mTOR activation can be achieved by Rapamycin and the second-generation Flt3-receptor kinase inhibitor AC220 and that this leads to a remission of the MPD symptoms in CD11c <italic>LAMTOR2</italic>_del animals. Furthermore, we observed in AC220-treated control animals already a significant reduction of DC numbers in the spleen emphasizing the specific effect of the Flt3-inhibitor on DC development. However, treatment of control animals with the allosteric mTOR inhibitor Rapamycin did not show a DC reduction. Here we speculate that other signalling pathways downstream of the Flt3 receptor, for example, STAT3 (ref. <xref ref-type="bibr" rid="b44">44</xref>), might compensate for mTORC1 inhibition.</p><p>This leads to the conclusion that LAMTOR-complex-mediated mTOR signalling quality might respond differently to intrinsic, amino acid-induced, versus extrinsic, Flt3-L-induced, stimuli. Therefore, our results support a model in which a molecular switch triggers mTOR activation but ERK downregulation dependent on growth factor, Flt3-ligand, signalling in the absence of LAMTOR2.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Mice</title><p>CD11c Cre mice<xref ref-type="bibr" rid="b26">26</xref> were provided by B. Reizis (Columbia University, New York, NY, USA). The <italic>LAMTOR2</italic>_f/f mice<xref ref-type="bibr" rid="b10">10</xref> were generated in our laboratory. Both mouse strains were backcrossed onto a C57BL/6 background for at least ten generations. To obtain CD11c Cre <italic>LAMTOR2</italic>_del/+ and CD11c Cre <italic>LAMTOR2</italic>_del/del mice, the CD11c Cre mice were crossed with the <italic>LAMTOR2</italic>_f/f mice. The CD11c Cre was kept hemizygous. Genomic DNA was isolated from ear biopsies with Viagen DirectPCR DNA Extraction System overnight and the genotype was determined by PCR<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b26">26</xref>. If not stated differently in the figure legend, sex-matched mice at the age of 3 months were used for the experiments. No randomization strategy was used.</p></sec><sec disp-level="2"><title><italic>In vivo</italic> experiments</title><p>Animal experiments were performed in accordance with the Austrian legislation (TVG BGBl no. 501/1989, i.d.F. BGBl I no. 162/2005) and were granted by the Austrian Federal Ministry of Science and Research (code: 66011/205-II/3b/2011).</p><p><italic>Rapamycin</italic>. Mice at the age of 3 months were injected i.p. for 10 days with 30&#x02009;&#x003bc;g Rapamycin (LC Laboratories) in PBS with 5% dimethylsulphoxide and 10% ethanol or vehicle alone<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>.</p><p><italic>AC220 (Quizartinib)</italic>. AC220 (LC Laboratories) at 10&#x02009;mg&#x02009;kg^&#x02212;1 was administered to female mice (10 weeks old) daily for 4 weeks by gavage. AC220 was reconstituted in 22% hydroxypropyl-&#x003b2;-cyclodextrin (Sigma-Aldrich). A corresponding control group was given only the vehicle<xref ref-type="bibr" rid="b33">33</xref>.</p><p>For both experiments, animals were killed 24&#x02009;h after the last treatment and organs were analysed by flow cytometry and histology.</p><p><italic>BMDCs</italic>. BMDCs were generated in the presence of Flt3-L but in the absence of GM-CSF, as described before<xref ref-type="bibr" rid="b32">32</xref>. In brief, BM cells were isolated and after erythrocyte lysis, 1.5 &#x000d7; 10⁶ cells were plated per well in a 24-well plate in IMDM (Gibco) supplemented with 10% FCS (Lonza) and 50&#x02009;nM mercaptoethanol. 200&#x02009;ng&#x02009;ml^&#x02212;1 Flt3-ligand (Shenandoah Laboratories) was added, and at day 8, the DCs in the supernatant were harvested. Purity was determined by flow cytometry analyses.</p><p><italic>Splenic DCs</italic>. Cells were isolated by MACS Cell Separation (Miltenyi Biotec) according to the manufacturer&#x02019;s protocol. For indicated experiments either the CD11c or the pan-DC microbeads were used. Purity was controlled by fluorescence-activated cell sorting (FACS) analysis.</p><p><italic>T cells, B cells, pDCs and PDCA-1^int cells</italic>. Cells were analysed and sorted by a BD Bioscience FACS-Canto or Aria (BD Bioscience).</p></sec><sec disp-level="2"><title>Histology, immunohistochemistry and immunofluorescence</title><p>For histological analyses, organs were fixed in Bouin&#x02019;s solution and embedded in paraffin. Sections of 5&#x02009;&#x003bc;m were cut and stained with H&#x00026;E. For immunohistochemistry and immunofluorescence, organs were embedded in Tissue-Tek O.C.T. compound, 5&#x02009;&#x003bc;m sections were cut and fixed for 8&#x02009;min in 100% acetone at &#x02212;20&#x02009;&#x000b0;C. For immunofluorescence, sections were blocked for 30&#x02009;min in 5% goat serum in PBS and incubated for 1&#x02009;h with the following antibodies: CD11c (1:100, clone N418, eBioscience), PDCA-1 (1:100, clone 120G8.04, Dendritics) and Ki67 (1:200, clone K-2, Novacastra), followed by a 30&#x02009;min incubation with the secondary antibody (Alexa Fluor 488 goat anti-rat, Alexa Fluor 568 goat anti-hamster, 1:1,000, Invitrogen) at room temperature. Sections were embedded in Mowiol (Sigma-Aldrich). For immunohistochemistry of CD11c, fixed sections were blocked for 5&#x02009;min with 0.03% H₂O₂ and 0.3% goat serum in PBS, followed by a blocking with the Avidin-Biotin blocking kit (VectorLabs). Sections were then incubated for 1&#x02009;h with the CD11c antibody (eBioscience), washed and incubated for 30&#x02009;min with the biotinylated goat anti-hamster secondary antibody (VectorLabs). After washing, sections were incubated for 5&#x02009;min in Vectastain ABC reagent (VectorLabs), followed by incubation with AEC (3-amino-9-ethylcarbazole), the peroxidase substrate (VectorLabs). Pictures were taken on a Zeiss Axio Imager M.1 equipped with a CoolSnapHQ2 CCD camera (Photometrics) or an AxioCam HRc (Carl Zeiss MicroImaging, Inc.). The acquisition software AxioVs40V4.5.0.0 (Carl Zeiss MicroImaging, Inc.) or VisiView (Visitron) were used.</p></sec><sec disp-level="2"><title>Pulse chase experiments</title><p>Splenic DCs were pre-cooled to 4&#x02009;&#x000b0;C on Superfrost slides (Menzel) and incubated for 10&#x02009;min with 100&#x02009;ng&#x02009;ml^&#x02212;1 Flt3-ligand. Cells were then washed with ice-cold medium and incubated for 20&#x02009;min at 37&#x02009;&#x000b0;C, immediately fixed for 1&#x02013;2&#x02009;min in 4% formaldehyde (FA) and blocked for 30&#x02009;min in 5% goat serum and 0.05% Saponin (Sigma-Aldrich) in PBS. Cells were then incubated for 2&#x02009;h with antibodies for Flt3 (1:100, H-300, Santa Cruz) and Lamp1 (1:200, 1D4B, Pharmingen) in blocking buffer, washed and incubated for 30&#x02009;min with the secondary antibodies (Alexa Fluor 488 goat anti-rat, Alexa Fluor 568 goat anti-hamster, 1:1,000, Invitrogen) at room temperature. Confocal pictures were taken at a confocal laser-scanning microscope (LSM510 Meta; Carl Zeiss MicroImaging, Inc.) with a &#x000d7; 63 plan-Apochromat NA 1.4 oil objective (Carl Zeiss MicroImaging, Inc.). For the acquisition of images, LSM Image Examiner software (version 3.1.0.117; Carl Zeiss MicroImaging, Inc.) was used.</p></sec><sec disp-level="2"><title>ELISA</title><p>Blood samples were collected and centrifuged for 20&#x02009;min for 1,500&#x02009;r.p.m. after coagulation. Serum was stored at &#x02212;80&#x02009;&#x000b0;C. Mouse Flt3- ligand Quantakine ELISA was performed according to the manufacturer&#x02019;s assay procedure (R&#x00026;D Systems).</p></sec><sec disp-level="2"><title>Multiplexing immunoassays</title><p>Serum was retrieved as described above. Spleen cell supernatants were prepared from 5 &#x000d7; 10⁶ spleen cells, which were cultured overnight in R10 medium. For the stimulation assays with CpG, splenic DCs were isolated with the pan-DC microbeads (Milteny) and 5 &#x000d7; 10⁵ cells were plated in R10 medium containing 20&#x02009;ng&#x02009;ml^&#x02212;1 GM-CSF and 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 GpG (1,668, TIB Molbiol). The next day, the culture supernatants were collected. The multiplexing immunoassays ProcartaPlex Mouse Th1/Th2/Th9/Th17/Th22/Treg Cytokine Panel (17 plex), with added Simplex IL-1&#x003b1; and the 2-plex IFN-&#x003b1; and IFN-&#x003b2;, were purchased from eBioscience and measured in a FlexMAP3D instrument.</p></sec><sec disp-level="2"><title>Flow cytometry analyses</title><p>Flow cytometry analyses were performed on BMDCs, BM, spleen and LN single-cell suspensions cells. Cell suspension of the spleen and LNs were prepared by cutting the tissue into small pieces and digestion for 25&#x02009;min at 37&#x02009;&#x000b0;C, in a shaking waterbath with 0.5&#x02009;mg&#x02009;ml^&#x02212;1 Collagenase D (Roche) and 120&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 DNAse (Merck) in Hanks buffered salt solution (PAA). After adding 200&#x02009;&#x003bc;l of 0.5&#x02009;M EDTA, pH 8 (AppliChem), cell suspension was pressed through a 70-&#x003bc;m cell strainer, washed and erythrocyte lysis was carried out. For FACS analysis, cells were washed with 1% BSA in PBS, blocked for 20&#x02009;min with Fc&#x003b3;RIII/II- block (1:10,000, clone 2.4G2, BD Bioscience) and then incubated for 1&#x02009;h with the specific fluorophore-conjugated antibodies (APC-CD11c (1:400, clone N418), APC-CD8a (1:400, clone 53-6.7), APC-CD4 (1:400, clone RM4-5), PE-CD8a (1:200, clone 53-6.7), FITC-Gr-1 (1:100, clone RB6-8C5), PE-c-Kit (1:100, clone 2B8), FITC-CD3e (1:100, clone 145-2C11), PE-IgM (1:100, clone r6-60.2), FITC-IgD (1:100, clone 11-26c.2a), CD40-PE (1:100, clone 323), CD44-APC (1:100, clone IM7) from BD Bioscience; PE-CD11b (1:100, clone M1/70), PE-Cy7-CD115 (1:200, clone A2F10), APC-Flt3 (1:100, clone A2F10), PerCP-Cy5.5-MHCII (1:100, clone M5), PE-CD11c (1:100, Clone N418), Brilliant Violett 510-CD3 (1:100, clone 17A2), PE-Cy7-NKp46 (1:100, clone 29A1.4), Brilliant Violett 510-CD4 (1:100, clone RM4-5), Brilliant Violett 421-CD8a (1:100, clone 53-6.7), APC-CD86 (1:100, clone GL1), PE-Cy7-CD62L (1:100, clone MEL-14), PerCPCy5.5- CD69 (1:100, clone H1.2F3), PE-PD-1 (1:100, clone MH5A), APC-PD-L1 (1:100, clone 10F.9G2) from BioLegend; FITC-PDCA-1 (1:100, clone 927), PE-CD4 (1:100, clone GK1.5), CD19-PerCP-Cy5.5 (1:100, clone 6D5), FITC-lineage Mix from eBioscience) and respective isotype controls. For Ki67-PE (1:200, clone 16.A8, BioLegend) staining, surface markers were stained as described before. Then cells were fixed, permeabilized and Ki67-stained with the Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Solution kit (eBioscience) according to the manufacturer&#x02019;s protocol. Cells were analysed by a Calibur (BD Bioscience) flow cytometer and data analysis was performed with FlowJo software.</p></sec><sec disp-level="2"><title>Western blot analyses</title><p>For phosphorylation analysis of AKT, mTOR, p70 S6 Kinase 1, S6 ribosomal protein and Erk1/2, cells were stimulated for 20&#x02009;min with Flt3 ligand. In addition, where stated, cells were treated with Rapamycin (50&#x02009;ng&#x02009;ml^&#x02212;1, LC Laboratories) or AC220 (100&#x02009;ng&#x02009;ml^&#x02212;1, LC Laboratories) for 1&#x02009;h, before stimulation. Cell lysates of BMDCs and splenic DCs were prepared<xref ref-type="bibr" rid="b2">2</xref> and separated on a SDS&#x02013;polyacrylamide gel electrophoresis. Membranes were probed after blotting and blocking against primary antibodies (LAMTOR2, LAMTOR3 (ref. <xref ref-type="bibr" rid="b10">10</xref>), pAKT (1:500, clone D9E), AKT (1:500&#x02013;1,000), pp70S6K1 (1:1,000, clone 1A5), pERK1/2 (1:500, clone E10), ERK1/2 (1:1,000, clone 137F5), pS6 (1:1,000, clone D57.2.2E) from Cell Signaling; Actin (1:5,000, clone C4) from Millipore; LAMTOR1 (1:1,000), LAMTOR4 (1:1,000) from Atlas; LAMTOR5 (1:500, clone C16) from Santa Cruz) overnight at 4&#x02009;&#x000b0;C in blocking buffer (3% BSA, 1&#x02009;mM EDTA, pH8, 0.05% Tween 20, 6&#x02009;mM Sodium Azide, pH 5.2). Membranes were washed with Tris -buffered saline (50mM Tris-HCl, pH 7.6; 150mM NaCl) with 0.05% Tween 20 and incubated with the secondary antibodies (anti-mouse-/anti-rabbit IgG peroxidase antibody, 1:5,000 (Sigma)). Detection was performed by chemiluminescence. Uncropped western blots are provided in <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 7 and 8</xref>.</p></sec><sec disp-level="2"><title>Real-time quantitative PCR</title><p>RNA of Flt3-L-BMDCs, sorted B and T cells was extracted with a &#x02018;High Pure RNA isolation kit&#x02019; (Roche) according to the manufacturer&#x02019;s manual. RNA samples were subjected to reverse transcription analysis using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo dT primers. Quantitative PCR analysis was performed using the DyNAmo Flash SYBR Green qPCR kit (Thermo Fisher Scientific). Following <italic>LAMTOR2</italic> primers were used: forward: 5&#x02032;-TCTGGGCCGCGTATGATAGG-3&#x02032; and reverse: 5&#x02032;-CACGCTGTTATGATGCTGCTACTT-3&#x02032;. Results were normalized to <italic>GAPDH</italic> cDNA. The amplification of cDNA was performed by PIKOREAL96 (Thermo Fisher Scientific). Resulting data were analysed via two-tailed type 2 Student&#x02019;s <italic>t</italic>-test.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>J.M.S., F.S, C.H.T., C.H., A.H. and J.B. did <italic>in vitro</italic> and <italic>in vivo</italic> experiments and analysed data. N.R and P.S. contributed with experimental design and edited the manuscript. L.A.H. conceived and supervised the study, and wrote and edited the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Scheffler, J. M. <italic>et al.</italic> LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling. <italic>Nat. Commun.</italic> 5:5138 doi: 10.1038/ncomms6138 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Figures</title><p>Supplementary Figures 1-8</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6138-s1.pdf"/></supplementary-material></sec>

Structural basis of IL-23 antagonism by an Alphabody protein scaffold

<p>Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein&#x02013;protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.</p>

<contrib contrib-type="author"><name><surname>Desmet</surname><given-names>Johan</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Verstraete</surname><given-names>Kenneth</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Bloch</surname><given-names>Yehudi</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lorent</surname><given-names>Eric</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wen</surname><given-names>Yurong</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Devreese</surname><given-names>Bart</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Vandenbroucke</surname><given-names>Karen</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Loverix</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hettmann</surname><given-names>Thore</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Deroo</surname><given-names>Sabrina</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Somers</surname><given-names>Klaartje</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Henderikx</surname><given-names>Paula</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lasters</surname><given-names>Ignace</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Savvides</surname><given-names>Savvas N.</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>COMPLIX N.V.</institution>, Technology Park 4, 9052 Ghent, <country>Belgium</country></aff><aff id="a2"><label>2</label><institution>Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University</institution>, K.L. Ledeganckstraat 35, 9000 Ghent, <country>Belgium</country></aff><aff id="a3"><label>3</label><institution>Unit for Biological Mass spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University</institution>, K.L. Ledeganckstraat 35, 9000 Ghent, <country>Belgium</country></aff><aff id="a4"><label>4</label>These authors contributed equally to this work</aff>

Nature Communications

<p>In the post-genomic era, the application and engineering of therapeutic antibodies to tackle cancer, as well as autoimmune and inflammatory disorders, has brought about a pronounced paradigm shift in the therapeutic targeting of protein&#x02013;protein interactions<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. At the same time, the elucidation of the molecular and structural basis of protein&#x02013;protein interactions has emerged as the cornerstone for understanding the extra- and intra-cellular context of signalling pathways and for the rational design of molecules with antagonistic or agonistic behaviour against molecular targets of biomedical importance<xref ref-type="bibr" rid="b3">3</xref>. The inherent challenges associated with targeting protein&#x02013;protein interfaces in a therapeutic setting<xref ref-type="bibr" rid="b4">4</xref> have stimulated considerable efforts towards designed protein interactions<xref ref-type="bibr" rid="b5">5</xref> and the development of engineered protein scaffolds that could serve as alternatives to antibodies in biomedical applications<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>. For instance, non-antibody molecular-binding platforms such as the DARPins<xref ref-type="bibr" rid="b8">8</xref> Monobodies<xref ref-type="bibr" rid="b9">9</xref>, Anticalins<xref ref-type="bibr" rid="b10">10</xref>, Affibodies<xref ref-type="bibr" rid="b11">11</xref>, Affitins<xref ref-type="bibr" rid="b12">12</xref> and the Adnectins<xref ref-type="bibr" rid="b13">13</xref> have led to a large expansion of the structural repertoire of engineered protein scaffolds and have contributed significant added value in terms of their diverse physicochemical properties, pharmacokinetics and delivery to and through tissues of interest<xref ref-type="bibr" rid="b6">6</xref>.</p><p>The Alphabody scaffold is a computationally designed protein scaffold of about 10&#x02009;kDa molecular weight, which was developed to serve as a therapeutic agent<xref ref-type="bibr" rid="b14">14</xref>. The scaffold does not have a counterpart in nature and is composed of a single contiguous polypeptide chain designed to adopt a triple-helix coiled-coil fold<xref ref-type="bibr" rid="b14">14</xref>. To explore the potential of the Alphabody platform in targeting biomedically relevant protein&#x02013;protein interactions, we opted to target the pro-inflammatory cytokine interleukin (IL)-23, a well-established therapeutic target for the treatment of inflammatory diseases<xref ref-type="bibr" rid="b15">15</xref>. IL-23 is produced by dendritic cells and macrophages and is required for the survival and expansion of pro-inflammatory Th17 cells, which by virtue of their production of IL-17 are associated with the pathogenesis of autoimmune inflammatory disorders, such as multiple sclerosis, rheumatoid arthritis, psoriasis and inflammatory bowel disease<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. In addition, IL-23 deficiency was recently shown to protect mice from tumour formation underscoring the general role of IL-23 in suppressing natural or cytokine-induced innate immunity and in promoting tumour development and metastasis<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>.</p><p>IL-23 adopts an atypical heterodimeric structure consisting of a p40 subunit encompassing three fibronectin-III-like domains, which is linked via a disulfide bond to an &#x003b1;-helical bundle subunit (p19) that topologically resembles long-chain helical cytokines<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref>. IL-12, also a heterodimeric cytokine secreted by the dendritic cell to promote development of Th1 cells, also features the p40 subunit but the latter is coupled to a p35 subunit instead<xref ref-type="bibr" rid="b15">15</xref>. While both cytokines employ their p40 subunits to bind to IL-12R&#x003b2;1 as a common receptor, IL-23 uses its p19 subunit to engage its cognate IL-23R, whereas IL-12 binds to IL-12R&#x003b2;2 via the p35 subunit. Interestingly, the monoclonal antibody Ustekinumab, originally developed to neutralize IL-12 for the treatment of autoimmune inflammatory disorders, was subsequently shown to also antagonize IL-23 due to its ability to bind to the common p40 subunit employed by the two cytokines<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. One of the reported side effects of the currently available anti-IL-12/IL-23 p40 therapeutic options is an increased susceptibility to infections, related to the important role IL-12 in mounting an appropriate immune system protection against pathogens<xref ref-type="bibr" rid="b21">21</xref>. In addition, several reports have described the protective role of and therapeutic potential of IL-12 in tumour development<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>.</p><p>We here report the design and development of Alphabodies as protein scaffolds not found in nature bearing unique physicochemical and structure&#x02013;function properties, and probe their potential to serve as antagonists against pro-inflammatory human IL-23. We show that affinity-matured Alphabodies can bind with ultra-high affinity to IL-23, but not IL-12, via an extensive interaction interface that engages the p19 subunit of IL-23 to abrogate IL-23-mediated signalling <italic>in vitro</italic> and <italic>in vivo</italic>. Together, these findings establish the Alphabody as a potent and versatile protein-based scaffold and set the stage for their application in disrupting diverse protein&#x02013;protein interactions of therapeutic relevance.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Design and development of a reference Alphabody scaffold</title><p>Our endeavours towards the design of the Alphabody scaffold originated from the wealth of information on diverse types of coiled-coils<xref ref-type="bibr" rid="b32">32</xref>, as recorded in the CC+ database<xref ref-type="bibr" rid="b33">33</xref>. Initially, the Alphabody scaffold was launched as an assembly of three non-covalently associated peptides designed to form a parallel coiled-coil trimer<xref ref-type="bibr" rid="b34">34</xref>. This design was then redefined as a three-helix coiled-coil scaffold, wherein all three helices are contained within a single contiguous polypeptide chain. The rationale behind this effort has been manifold. First, the folding of a single-chain polypeptide, as opposed to peptidic associations, is not dependent on the concentration. Second, the designed scaffold needed to be producible by cost-effective and scalable recombinant protein production methods. Third, a single-chain construct allows independent definition of each amino-acid residue in any &#x003b1;-helix of the sequence. Fourth, a stable and autonomously folded protein can, in principle, allow accommodation of both conformational and linear binding epitopes.</p><p>The reference scaffold, hereafter referred to as &#x02018;scRef_L16&#x02019;, was designed to feature three &#x003b1;-helices composed of four heptad repeats each, connected via glycine/serine-rich linkers (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The reference sequence of each heptad repeat was defined as &#x02018;I_aA_bA_cI_dQ_eK_fQ_g&#x02019;. Isoleucines were chosen at the heptad core positions (a and d) because of their specificity to induce three-stranded coiled-coil structures. Electrochemically neutral yet polar glutamines were placed at the interhelical &#x02018;groove&#x02019; positions (e and g). Alanines were chosen at the exposed b- and c-positions because of their high &#x003b1;-helical propensity. Positively charged lysine was chosen at the most exposed f-position. Minor deviations from this heptad motif were introduced near the helical termini to improve capping. The full amino-acid sequence of scRef_L16 can be written as N-HRS1-L1-HRS2-L2-HRS3-C, wherein heptad repeat sequences HRS<italic>x</italic> have the sequence IEEIQKQIAAIQKQIAAIQKQIYRM and linker sequences L<italic>x</italic> are TGGSGGGSGGGSGGGSGMS (the capping residues &#x02018;T&#x02019; and &#x02018;MS&#x02019; at the N- and C termini are formally included in the linker segments).</p><p>Isoleucine core residues are known to have a high tendency to induce parallel coiled-coil trimers<xref ref-type="bibr" rid="b35">35</xref>. However, molecular modelling using the parallel Ile-stabilized trimer GCN4-pII (PDB code 1GCM)<xref ref-type="bibr" rid="b35">35</xref> and the antiparallel Leu-stabilized coiled serine (PDB code 1COS)<xref ref-type="bibr" rid="b36">36</xref> suggested that core residue layers with Ile at the a- and d-positions in two parallel helices could also be complemented by Ile core residues in a third, antiparallel helix (<xref ref-type="fig" rid="f1">Fig. 1b,c</xref>). The linkers in scRef_L16 were therefore chosen to be long enough to allow antiparallel, as well as parallel folding (16 residues can theoretically bridge about 16 &#x000d7; 3&#x02009;&#x000c5;=48&#x02009;&#x000c5;, while 7 helical turns are 7 &#x000d7; 5.4&#x02009;&#x000c5;=38&#x02009;&#x000c5; in height). To explore the actual folding preferences in this regard, scRef_L16 and different variants were produced in soluble form and physicochemically characterized.</p><p>His-tagged scRef_L16 and other variants described below were purified from <italic>Escherichia coli</italic> using metal-affinity and size-exclusion chromatography (SEC), with yields reaching several milligrams of pure protein per liter of culture. Circular dichroism measurements showed that scRef_L16 adopted a predominantly &#x003b1;-helical structure that was remarkably stable (<italic>T</italic>_m value of ~120&#x02009;&#x000b0;C), as revealed by thermal denaturation studies in the presence of guanidine hydrochloride (<xref ref-type="fig" rid="f2">Fig. 2a&#x02013;c</xref>). Subsequently, a variant referred to as &#x02018;scRef_L8&#x02019; was produced in which both linker segments were shortened by 8 amino-acid residues. Such linkers would be physically too short to connect the helical termini in a fully parallel coiled-coil structure. Surprisingly, we found that scRef_L8 and scRef_L16 were equally thermostable (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). This finding suggested that both constructs adopt an antiparallel configuration. However, it remained possible that an unfavourably short linker could induce local unwinding near the termini without causing an energetic cost in stability.</p><p>To examine the local unwinding hypothesis, a panel of variants was produced based on a scaffold variant carrying one fewer heptad repeat (referred to as &#x02018;scShort&#x02019;), equipped with linkers composed of 6&#x02013;18 Gly/Ser residues (L6&#x02013;L18). Consistent with the loss of two heptad core layers per construct, all scShort variants generally showed T_m values that were about 40&#x02009;&#x000b0;C lower than the longer scRef constructs at comparable concentrations of GuHCl (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). The absence of any significant effect of the linker length in the context of a scaffold stabilized by only six core layers invalidated the local unwinding hypothesis and strengthened the notion of an antiparallel fold in an &#x02018;up-down-up&#x02019; configuration.</p></sec><sec disp-level="2"><title>Design of Alphabody libraries</title><p>To explore the potential of scRef_L16 for generating target-specific binders, three random libraries were designed (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). A first library, denoted &#x02018;scLib_AC11&#x02019;, comprised six randomized c- and g-positions in helix A and five randomized b- and e-positions in helix C. These positions form a contiguous surface contributed by 11/14 (79%) of all b-, c-, e- and g-positions of the groove surface in between helices A and C (<xref ref-type="fig" rid="f1">Fig. 1a&#x02013;c</xref>). On the basis of the same design principle applied to the scShort sequence, the &#x02018;scLib_AC7&#x02019; library was generated displaying four randomized positions in helix A and three in helix C. Both of these libraries are further referred to as &#x02018;groove libraries&#x02019;. A third library, &#x02018;scLib_C9&#x02019;, was derived from scRef_L16 and was intended to explore the surface functionality of helix C. Therefore, nine randomizations were introduced at b-, c- and f-positions in helix C. This library is further referred to as a &#x02018;helix surface library&#x02019;.</p></sec><sec disp-level="2"><title>Identification of Alphabody binders to human IL-23</title><p>The initial biopanning campaign employed the aforementioned generic libraries displayed as fusion constructs to the pIII protein of M13 phage. The library sizes ranged from 1.0 to 1.7 &#x000d7; 10⁸ unique clones. Enzyme-linked immunosorbent assay (ELISA) screening of output clones from consecutive biopanning rounds showed gradual enrichment of positive clones (optical densities (ODs) in screening ELISA at least three times higher than background), from about 33% in round 3 up to 95% in round 5 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). Next, 91 positive clones from selection rounds 3, 4 and 5 were sequenced. We found that all binding clones originated from the groove libraries (scLib_AC11: 80 clones, scLib_AC7: 11 clones and scLib_B9: 0 clones). The population comprised 36 unique sequences, with some convergence (duplicates) already seen in round 3 and gradually increasing in the next rounds. Strikingly, the majority of sequences (56%) showed severe deletions primarily in helix B, with 5 clones (3 unique) lacking 2 heptad repeats, 35 clones (12 unique) lacking the entire helix B and 5 clones (3 unique) comprising only helix C. In addition, a marked enrichment in proline and amber stop codons was observed in the helix A, especially among clones lacking deletions in the sequence. The observed deficiencies in the sequences are likely due to the usage of the PelB signal sequence, which mediates post-translational translocation of protein to the bacterial periplasm through the Sec pathway. However, this signal sequence was previously shown to be inefficient for the display of fast-folding proteins<xref ref-type="bibr" rid="b37">37</xref>, as we have now also observed in the case of Alphabodies. Evidently, while defective sequences may be true binders as polypeptides with or without tertiary structure, their inherent instability renders them less-well controllable in terms of structural and physicochemical properties, and thus not well suited for further development. We have now resolved this issue in current work by substituting the PelB signal sequence with the DsbA signal sequence, which directs protein translocation to the periplasm via the co-translational signal recognition particle (SRP) pathway<xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref>.</p><p><xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref> shows the top-10 unique clones without B-helix deletions ranked according to binding performance in phage ELISA, the degree of competition by the neutralizing monoclonal antibody B-Z23 directed against the p19 subunit of IL-23 and the amino-acid residues observed at the variable positions. Apart from the Pro and stop codon enrichments, clone 72 had three helix-destabilizing glycines in helix A. In contrast, four clones showing nearly complete inhibition of binding to IL-23 in the presence of the neutralizing antibody B-Z23 (clones 52, 44, 59 and 33) were less affected by destabilizing elements (only clone 44 had a Pro at position 1&#x02009;g). Further sequence comparisons elucidated the following apparent consensus binding motif: position A2g: aliphatic (M/I/V); A3c: small (A/G/S); A3g: aromatic (W/Y); C3b: aliphatic (V/I/L); C3e: aromatic (F/Y).</p></sec><sec disp-level="2"><title>Development of affinity-matured Alphabodies against IL-23</title><p>Clone 59 (hereafter referred to as &#x02018;Cl59&#x02019;) was purified as a soluble Alphabody following expression in <italic>E. coli</italic> and was subsequently tested in various binding and functional assays. We found that Cl59 bound with high affinity to human IL-23 (<italic>K</italic>_D=3.8&#x02009;nM in equilibrium ELISA and <italic>K</italic>_D=7.0&#x02009;nM in a kinetic ELISA assay). As described below, it also displayed moderate antagonistic activity in a functional assay on mouse splenocytes (<italic>K</italic>_I=69&#x02009;nM) (<xref ref-type="table" rid="t1">Table 1</xref>). In light of its promising initial characteristics, Cl59 was selected as the starting point for affinity maturation.</p><p>A maturation library termed &#x02018;matLib&#x02019; (<xref ref-type="fig" rid="f1">Fig. 1a</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>) was established based on the sequence of Cl59 and the apparent consensus binding motif (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>). This library was used to conduct two affinity-maturation campaigns in parallel. This resulted in a large panel of variants with improved apparent affinities in phage format, from which 14 unique clones were selected for further characterization in soluble format. To this set we added two consensus sequences from each campaign (&#x02018;MAcons&#x02019; and &#x02018;MBcons&#x02019;), three computationally designed variants (&#x02018;Cl59m&#x02019;, &#x02018;59m_C2eQ&#x02019; and &#x02018;59m_A3cA_A4cS_C2eQ&#x02019;) and the reference variant Cl59. <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref> lists the names and constitution of these 20 Alphabody variants in terms of selected residues at the variable positions, linker lengths and B-helix modification. All constructs were purified from <italic>E. coli</italic> with yields ranging from 0.37 to 10.1&#x02009;mg per liter culture (mean=2.7&#x02009;mg&#x02009;l^&#x02212;1).</p><p>We employed a broad set of binding assays to characterize this set of 20 Alphabodies, which revealed that the vast majority of maturation clones (# 1&#x02013;14) displayed subnanomolar affinities to IL-23 (<xref ref-type="table" rid="t1">Table 1</xref>). Both maturation campaigns yielded high-affinity binders (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The highest affinity in standard ELISA assays was measured for MA12 and MA5 (<italic>K</italic>_D=0.1&#x02009;nM), which is a factor 38 better than the non-matured variant Cl59. Kinetic ELISA assays (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>) and standard ELISA data (<xref ref-type="fig" rid="f3">Fig. 3a</xref>) were globally in good agreement, as inferred from the consistency between sub- and supra-nanomolar affinities (<xref ref-type="table" rid="t1">Table 1</xref>). The main improvement in affinities appears to have arisen from an increase in the <italic>k</italic>_on by about one order of magnitude, with some <italic>k</italic>_on values exceeding 10⁶&#x02009;M^&#x02212;1&#x02009;s^&#x02212;1 (<xref ref-type="table" rid="t1">Table 1</xref>). Furthermore, <italic>K</italic>_D values measured via phage-coated ELISA were generally in good agreement with <italic>K</italic>_D values obtained for the soluble constructs, indicating that affinities determined in the early-screening phase of phage display are good predictors of the affinities of the soluble constructs.</p></sec><sec disp-level="2"><title>Antagonistic potency of affinity-matured Alphabodies</title><p>To assess the antagonistic capacity of the selected Alphabodies <italic>in vitro</italic>, a mouse splenocyte assay was established<xref ref-type="bibr" rid="b40">40</xref>. Functional inhibition constants (<italic>K</italic>_I) were determined by measuring the inhibition of IL-23-induced IL-17 production in murine splenocytes. We observed that the majority of subnanomolar affinity binders also inhibited IL-23 with subnanomolar functional inhibition constants (<xref ref-type="table" rid="t1">Table 1</xref>). Over the whole range, the majority of <italic>K</italic>_I values deviated by less than a factor 3 from the binding <italic>K</italic>_D (median <italic>K</italic>_I/<italic>K</italic>_D=2.7) (<xref ref-type="fig" rid="f3">Fig. 3b</xref>). Alphabody Cl59, which had the lowest affinity, also showed the weakest inhibition. The second best binder, MA12, showed the strongest inhibition capacity (<italic>K</italic>_I=0.13&#x02009;nM). This suggests that the functional inhibition potencies are predominantly determined by the binding affinities. In addition, we sought to characterize the specificity of binding of matured Alphabodies against human IL-23 compared with IL-12 by ELISA, as this could have therapeutic implications in targeting the functional dichotomy between these two pro-inflammatory cytokines. We found that both representative Alphabodies tested, MA12 and MB23, exhibited exquisite specificity towards IL-23 (<xref ref-type="fig" rid="f3">Fig. 3c</xref>) suggesting that matured Alphabodies only target unique structural features in IL-23.</p><p>To confirm the binding and inhibition data in another cellular assay, the top-performing Alphabodies were tested on their capacity to neutralize IL-23-mediated signalling in a human DB lymphoma cell line<xref ref-type="bibr" rid="b41">41</xref>. For all Alphabodies tested, a clear dose-dependent inhibition of STAT3 phosphorylation was observed. Functional inhibition constants were determined using a calibration curve, in a similar manner as for the splenocyte assay. Here, Alphabody MB23 performed best, with a <italic>K</italic>_I of 0.16&#x02009;nM, while most other variants were also found to have subnanomolar inhibition constants (<xref ref-type="table" rid="t1">Table 1</xref>).</p><p>In addition, we attempted to investigate the <italic>in vivo</italic> efficacy of one of the best binders, MB23, in a mouse model for skin inflammation induced by intradermal injection of human IL-23 into the ear<xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref>. In this model, repeat dosing with human IL-23 resulted in psoriasis-like thickening of the ear skin epidermis and swelling of the ear, which could be monitored <italic>in vivo</italic> by calliper measurements (<xref ref-type="fig" rid="f4">Fig. 4</xref>). The negative-control group, which was injected with phosphate-buffered saline (PBS) showed no thickening, whereas the positive-control group injected with IL-23 only (group A) showed gradual thickening as of day 8. Mice in groups B and C were injected with both IL-23 and MB23, which was PEGylated (40&#x02009;kDa linear polyethylene glycol (PEG)) via a cysteine residue introduced in the B-helix of the Alphabody. Group B received intradermal MB23 injections and Group C was injected intraperitoneally. Indeed, both MB23-treated groups showed the same ear-thickness curves, only slightly above the negative-control curve, indicating that MB23 treatment effectively suppressed IL-23-induced ear swelling (<xref ref-type="fig" rid="f4">Fig. 4</xref>).</p></sec><sec disp-level="2"><title>Structure of Alphabody MA12 and its complex with IL-23</title><p>To obtain structural insights into the three-dimensional structure of the Alphabody scaffold and the structural and binding principles underlying its neutralizing interaction with IL-23, we carried out crystallographic studies of human IL-23 in complex with MA12 complemented by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Human IL-23 was transiently expressed by co-transfecting IL-12 subunit beta (p40) and IL-23 subunit alpha (p19) in HEK239T cells in the presence of kifunensine to limit the extent of N-linked glycosylation<xref ref-type="bibr" rid="b44">44</xref>. N-linked glycosylation on IL-23 was further trimmed during purification by treating IL-23 with EndoH. The free cysteine of the MA12 Alphabody was blocked by iodoacetamide and the N-terminal His-tag was removed by trypsin. Mixing of purified human IL-23 with an excess of purified MA12, followed by SEC, yielded a highly monodisperse fraction containing the IL-23:MA12 complex (<xref ref-type="fig" rid="f5">Fig. 5a</xref>). ITC experiments showed that MA12 binds to IL-23 with subnanomolar affinity (<italic>K</italic>_D=0.35&#x02009;nM) ), in what emerged as an enthalpically driven interaction with a 1:1 stoichiometry (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). Such binding affinity is consistent with the ELISA-based affinity determinations (<xref ref-type="table" rid="t1">Table 1</xref>). In addition, kinetic binding parameters obtained by SPR indicated a relatively fast on-rate (<italic>k</italic>_on=2.6 &#x000d7; 10⁵&#x02009;M^&#x02212;1&#x02009;s^&#x02212;1) and a slow off-rate (<italic>k</italic>_off=1.2 &#x000d7; 10^&#x02212;4&#x02009;s^&#x02212;1), yielding a <italic>K</italic>_D=0.46&#x02009;nM, in good agreement with values obtained from all other binding assays employed (<xref ref-type="fig" rid="f5">Fig. 5c</xref>; <xref ref-type="table" rid="t1">Table 1</xref>).</p><p>Crystallization trials of highly monodisperse preparations of the IL-23:MA12 complex yielded two distinct crystal forms that allowed us to determine crystal structures at 1.74 and 3.4&#x02009;&#x000c5; resolution (<xref ref-type="table" rid="t2">Table 2</xref>), providing the opportunity to visualize the binding epitope in detail and to cross-validate structural observations. MA12 folds into a left-handed triple-helix coiled-coil structure, with the binding surface located mainly at the C-terminal half of helices A and C (<xref ref-type="fig" rid="f6">Fig. 6a,b</xref>). The same structure and binding mode to IL-23 are maintained in both crystal forms (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). In agreement with the ITC-derived stoichiometry of the complex, one copy of MA12 binds to the p19 subunit of IL-23 and is oriented parallel to the longitudinal axis of the p40 subunit (<xref ref-type="fig" rid="f6">Fig. 6a,b</xref>). The respective binding interfaces feature 20 amino-acid residues contributed by MA12 and 28 amino-acid residues contributed by p19, burying a total surface area of 800 and 840&#x02009;&#x000c5;², respectively (<xref ref-type="fig" rid="f6">Fig. 6a</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>). This MA12 interface area constitutes 14.4% of the total surface area of the Alphabody. Five hydrogen bonds and two salt bridges are formed upon binding, while the rest of the interaction interface can be described in terms of van der Waals contacts (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>).</p><p>The binding interface is further hallmarked at its two ends by two prominent attachment points. At one end, Trp156 from IL-23 p19, a residue that was previously proposed to define a hotspot mediating the cognate interaction of IL-23 with IL-23R (ref. <xref ref-type="bibr" rid="b24">24</xref>), pokes the benzene ring part of its indole ring into the groove defined by helices A and C of the Alphabody, to interact with the Ile106 core residue (<xref ref-type="fig" rid="f6">Fig. 6d</xref>). At the same time, the N&#x02013;H group of the indole moiety engages in a water-mediated hydrogen bond with Gln13 projecting from helix A of MA12 (position A2c) (<xref ref-type="fig" rid="f6">Fig. 6d</xref>). At the other end, Tyr110 on MA12, a conserved residue in all matured binders (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>), inserts into a pocket on IL-23 p19 defined by the BC and AB loops and the N-terminal part of helix D (<xref ref-type="fig" rid="f6">Fig. 6e</xref>). Furthermore, we note that this segment of the AB loop in the p19 subunit of IL-23 is disordered in all currently available crystal structures of IL-23 (refs <xref ref-type="bibr" rid="b22">22</xref>, <xref ref-type="bibr" rid="b23">23</xref>, <xref ref-type="bibr" rid="b24">24</xref>, <xref ref-type="bibr" rid="b45">45</xref>), suggesting that the interaction with the Alphabody induces the observed conformation. Contrary to helices A and C of MA12, helix B does not participate at all in the binding epitope with p19 (<xref ref-type="fig" rid="f6">Fig. 6</xref>). The glycine-rich linkers connecting the three helices could not be visualized in the electron density maps and were not included in the model. Importantly, comparison of the variable positions in MA12 involved in the binding interface with IL-23 with those of MB23, another well-performing Alphabody, reveals strong sequence conservation levels (<xref ref-type="fig" rid="f6">Fig. 6f</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). Thus, such strong sequence conservation patterns in residues involved in contacts with IL-23 and the very similar binding profiles shared by the best binders (<xref ref-type="table" rid="t1">Table 1</xref>), essentially warrant that all matured high-affinity binders bind using similar structural principles.</p><p>We were intrigued by persisting residual difference electron density emanating from the C&#x003b4;1 position of Trp319 in IL-23 p40 up to the last rounds of refinement. Given previous reports that IL-12, a cytokine related to IL-23, can be C-mannosylated via a tryptophan residue<xref ref-type="bibr" rid="b46">46</xref>, we wondered whether this would also be the case for IL-23. Indeed, mass spectrometry on tryptic peptides of our recombinant human IL-23 confirmed that Trp319 in the p40 subunit of IL-23 is mannosylated (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a,b</xref>). However, modelling of a C-linked mannose to Trp319 proved challenging due to the likely structural heterogeneity of this adduct<xref ref-type="bibr" rid="b47">47</xref>, prompting us to omit the sugar moiety from the final models.</p><p>Finally, we note that the engineered Cys60, located in the middle of helix B of MA12 and all other affinity-matured Alphabodies (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>), remains freely accessible to solvent well away from the interaction epitope and is well positioned for introducing potential post-translational chemical modifications to improve bioavailability and modulate physicochemical properties. For example, the 40-kDa PEG-moiety attached to the MB23 Alphabody in the <italic>in vivo</italic> study on mice can be expected not to interfere with the binding, as also evidenced by <italic>in vivo</italic> data.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>The growing need for therapeutics that can efficiently antagonize biomedically relevant protein&#x02013;protein interactions has stimulated the expansion of the repertoire of novel and repurposed protein scaffolds. Despite good progress in the development of antibody-based<xref ref-type="bibr" rid="b48">48</xref> and non-antibody scaffolds<xref ref-type="bibr" rid="b6">6</xref> to serve as therapeutic biologicals in the areas of cancer, autoimmune diseases and inflammatory disorders<xref ref-type="bibr" rid="b1">1</xref>, the available approaches are yet to reach their full potential, as many drug discovery/development programmes are faced with high attrition rates<xref ref-type="bibr" rid="b49">49</xref>.</p><p>Here we reported on the development of a novel protein scaffold, termed the Alphabody, which emerged from a combination of <italic>de novo</italic> design and experimental validation. Alphabody sequences were found to fold as antiparallel triple-stranded &#x003b1;-helical coiled-coil structures, thus adopting a previously unknown fold. The core of the antiparallel coiled-coil fold features tightly packed isoleucines at conventional heptad a- and d-positions with a regular &#x02018;knobs-into-holes&#x02019; configuration, wherein the a- and d-residues of the antiparallel B-helix form core layers together with d- and a-residues, respectively, from the two parallel helices A and C. This type of packing is similar in configuration, yet distinct at the atomic level, from the leucine-stabilized antiparallel three-stranded &#x02018;coiled serine&#x02019; structure<xref ref-type="bibr" rid="b36">36</xref>, and possesses exceptional thermostability. Thus, such near-optimal core packing and the repetitive nature of the constituting heptad repeats allow for a scalable scaffold that is insensitive to multiple substitutions, thereby enabling the design of generic libraries for phage display that can exploit the entire scaffold surface, rather than the loop regions, as is the case for most other scaffold proteins. More specifically, Alphabodies can carry up to 25 variable positions that can be optimized for binding and other properties. This offers important advantages in the therapeutic targeting of protein&#x02013;protein interfaces, an area of increasing significance in drug development. In addition, the possibility to combine several tailor-made properties, including optimization of loop regions, on such a robust scaffold offers opportunities to develop Alphabodies that are minimally immunogenic, paving the way for (pre)clinical development of the scaffold. Current approaches to predict and assess the immunogenicity of protein therapeutics range from computational strategies to screening against human leukocyte antigen class II molecules, cellular models employing CD4+ T cells, as well as transgenic animal models including immunodeficient mice reconstituted with human hematopoietic stem cells<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b50">50</xref><xref ref-type="bibr" rid="b51">51</xref>.</p><p>Our Alphabody libraries were subdivided into structural &#x02018;themes&#x02019;, focusing on either the concave groove surface formed by the A- and C-helices, or the convex B-helix surface. The biopanning campaign against the present target, IL-23, using a mixture of the libraries converged towards groove binders only. Importantly, matured Alphabody variants were shown to potently suppress IL-23-mediated inflammatory phenotypes <italic>in vitro</italic>, with the best-performing Alphabodies reaching subnanomolar affinities in various binding assays (<xref ref-type="table" rid="t1">Table 1</xref>; <xref ref-type="fig" rid="f4">Fig. 4</xref>). The observed kinetic binding profiles of both the initial clones and the matured Alphabodies are within ranges observed for high-affinity protein&#x02013;protein interactions (<italic>k</italic>_on~10⁵&#x02013;10⁶&#x02009;M^&#x02212;1&#x02009;s^&#x02212;1 and <italic>k</italic>_off values ~10^&#x02212;4&#x02013;10^&#x02212;5&#x02009;s^&#x02212;1) (<xref ref-type="table" rid="t1">Table 1</xref>). In this regard, it was surprising to see that the gain in binding affinity in matured Alphabodies appeared to result primarily from improvements in the association rate constants by about an order of magnitude (<xref ref-type="table" rid="t1">Table 1</xref>). In combination with their favourable physicochemical properties, such binding potencies promise to render Alphabodies as multi-purpose anti-inflammatory agents for the treatment of inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis<xref ref-type="bibr" rid="b15">15</xref>. This was partly validated in this study by the successful <italic>in vivo</italic> results obtained via intradermal and intraperitoneal injection of MB23-PEG in mice. Furthermore, the fact that the developed Alphabodies exclusively target the p19 subunit of IL-23 and show no cross-reactivity with IL-12, offers the opportunity to decouple therapeutic modulation of signalling pathways mediated by IL-23 from those mediated by IL-12 (ref. <xref ref-type="bibr" rid="b15">15</xref>).</p><p>The design principles employed to develop the Alphabody scaffold and subsequent libraries were borne by our structural data. Arguably one hallmark of the Alphabody platform is the way it employs the surface defined by two of the scaffold helices to target a hitherto unexploited binding site on the p19 subunit of human IL-23 (<xref ref-type="fig" rid="f6">Figs 6</xref> and <xref ref-type="fig" rid="f7">7</xref>). The binding mode observed in the complex of MA12 with human IL-23 is strongly reminiscent of the principles employed in the two-sided design of a <italic>de novo</italic> binding pair<xref ref-type="bibr" rid="b52">52</xref>. In this binding mode, aromatic side chains with amphipathic character, such as tryptophan and tyrosine, are mutually contributed by the binding pair to mediate both aromatic and H-bonded interactions. Interestingly, Alphabody MA12 sequesters Trp156 on human IL-23 (<xref ref-type="fig" rid="f6">Fig. 6</xref>), a residue that has been proposed as a hotspot for the binding of cognate IL-23R (ref. <xref ref-type="bibr" rid="b24">24</xref>).</p><p>The potential to exploit the Alphabody scaffold to target diverse binding epitopes via classical biopanning and selection approaches and/or computational design is intrinsically large. Computational approaches are steadily emerging but are still lacking the robustness of experimental strategies based on screening and selection. The &#x003b1;-helical nature of the Alphabody scaffold, however, makes it particularly amenable to design techniques such as the grafting of binding motifs from natural &#x003b1;-helical ligands, at least in cases for which structural templates are available. The present study also illustrates how initial drug discovery can be accomplished by exploring different thematic libraries. Subsequently, once a preferred type of binding surface has been determined (for example, a groove, helix or a loop) and initial binding sequences are available, the rigid &#x003b1;-helical nature of the scaffold will essentially allow modelling of individual sequences onto a scaffold template structure with reasonable accuracy. In addition, the rigidity of the Alphabody helices may provide an entropic advantage when the helical surfaces are structurally compatible with the binding epitope. We envisage that such design options will considerably facilitate the rational design of focused libraries for further affinity maturation. Finally, having delineated a given critical binding region, the remainder of the scaffold can be independently optimized for other purposes such as solubility enhancement, biospecificity, labelling and PEGylation.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Recombinant production of Alphabodies</title><p>The complementary DNA (cDNA) of Alphabodies was cloned as N-terminally tagged decahistidine fusion proteins into the pET16b-vector (Novagen). Recombinant production was performed in transformed BL21(DE3)pLysS cells (Life Technologies), grown in Luria-Bertani (LB) medium at 37&#x02009;&#x000b0;C. Expression was induced by adding 1&#x02009;mM isopropylthio-&#x003b2;-galactoside at an OD_{600&#x02009;nm} of 0.6 and expression was continued for 4&#x02009;h at 37&#x02009;&#x000b0;C. Cells were harvested by centrifugation and resuspended in 50&#x02009;mM Tris, 500&#x02009;mM NaCl pH 7.8 and frozen at &#x02212;80&#x02009;&#x000b0;C. Cell lysis was performed by thawing and sonication. 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (0.1&#x02009;mM) was added to the lysed cells, together with 10&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 DNAseI and 5&#x02009;mM MgCl₂. The suspension was centrifuged for 20&#x02009;min at 40,000&#x02009;<italic>g</italic>. Typically, Alphabodies were purified from the soluble fraction, which was loaded on a 5-ml IMAC HP Ni sepharose column (GE Healthcare) and eluted with an imidazole gradient over 15 column volumes. Final polishing and desalting was performed on a Superdex 75 size-exclusion column (GEHealthcare) equilibrated in PBS.</p></sec><sec disp-level="2"><title>Development of Alphabody libraries</title><p>Genes with position-specific randomization (NNK) were designed wherein the Alphabody sequences were fused N-terminally to the pIII protein of bacteriophage M13. These libraries were ordered from GeneArt AG (Germany) and delivered as transformed <italic>E. coli</italic> cells (strain ER2738, supE strain). The library sizes ranged from 1.0 to 1.7 &#x000d7; 10⁸ unique clones. Alphabody displayed on pIII of the phagemid was confirmed by western blot analysis using anti-pIII (MoBiTec GmbH) and amounted to about 10% of total pIII protein.</p></sec><sec disp-level="2"><title>Biopanning procedure</title><p>A biopanning campaign was set up using a mixture of equal amounts of the three libraries scLib_AC11, scLib_AC7 and scLib C9. A soluble panning format was applied, that is, phages were pre-incubated with the target and subsequently captured on streptavidin beads. The preincubation was performed during 1&#x02009;h at room temperature (RT) on an end over end shaker, with hIL-23 (Biolegend) bound to biotinylated anti-IL-23p40 antibody (Biolegend) in a 2:1 molar ratio in PBS supplemented with 2% skimmed milk. The capturing step was performed using Dynabeads (50&#x02009;&#x003bc;l). Beads were washed 10 times with 1&#x02009;ml of 0.1% Tween 20 in PBS. Elution was performed with 200&#x02009;&#x003bc;l of buffer (100&#x02009;mM glycine&#x02013;HCl, pH 2), and the eluate was neutralized by adding 50&#x02009;&#x003bc;l of 1&#x02009;M Tris pH 8.0. Five consecutive panning rounds were conducted wherein the hIL-23 target concentration was decreased as of round 3 (rounds 1 and 2: 200&#x02009;nM, rounds 3&#x02013;5: 100, 10 and 1&#x02009;nM, respectively). Phagemid was propagated as follows. Neutralized phagemid solution was added to 1.75&#x02009;ml of LB. After taking out 50&#x02009;&#x003bc;l for further titration, the rest of the phagemid solution was added to 18&#x02009;ml of TG1 cells that had already grown to the exponential phase (OD_{600&#x02009;nm}=0.5). After 30&#x02009;min incubation at 37&#x02009;&#x000b0;C, the solution was centrifuged and the pellet was resuspended in 250&#x02009;ml of LB medium+ampicillin+glucose. Overnight (O/N) incubation was performed at 37&#x02009;&#x000b0;C (180&#x02009;r.p.m.).</p></sec><sec disp-level="2"><title>Screening and competition ELISA</title><p>Phage ELISA was used to screen for positive clones and to check which of them showed competition with the neutralizing antibody B-Z23, as follows. Nunc ELISA plates were coated with 100&#x02009;&#x003bc;l per well neutravidin (10&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1) in PBS for 1&#x02009;h at RT. In the following steps, the plate was washed 5 times with PBS, 0.05% Tween 20, between each incubation step. The plate was blocked O/N at 4&#x02009;&#x000b0;C with 200&#x02009;&#x003bc;l per well of PBS, 0.5% gelatin and 0.1% BSA. Biotinylated anti-human-p40 (100&#x02009;&#x003bc;l, 100&#x02009;nM, Biolegend) in 0.1% BSA&#x02013;PBS was added for 1&#x02009;h at RT. For the competition ELISA, 150&#x02009;nM hIL-23 (Biolegend) was pre-incubated with 150&#x02009;nM antibody B-Z23 (CellSciences) in 0.1% BSA&#x02013;PBS for 1&#x02009;h at RT. For the screening ELISA, the same sample without B-Z23 antibody was used. Then, 66.6&#x02009;&#x003bc;l of this sample (final concentration 100&#x02009;nM) and 33.3&#x02009;&#x003bc;l of rescued phagemid supernatant was added to the plate for 2&#x02009;h at RT. Bound phagemid was detected with anti-M13-HRP (GE Healthcare) for 0.5&#x02009;h at RT followed by 3,3&#x02032;,5,5&#x02032;-tetramethylbenzidine (TMB) (Sigma) staining. The colour reaction was stopped with 100&#x02009;&#x003bc;l 0.5&#x02009;M H₂SO₄.</p></sec><sec disp-level="2"><title>Phagemid rescue</title><p>TG1 cells, infected with selected phagemid, were titrated on an agar plate (2 &#x000d7; TY, agar, ampicillin). Individual clones were picked from the agar plate and cultured O/N in a microtiter plate containing 100&#x02009;&#x003bc;l 2 &#x000d7; TY AG per well<xref ref-type="bibr" rid="b53">53</xref>. One &#x003bc;l of each O/N culture was transferred to a microtiter plate containing 100&#x02009;&#x003bc;l of 2 &#x000d7; TY, 0.1% glucose, 100&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 ampicillin and 10⁸ helper phage per well and grown at 37&#x02009;&#x000b0;C for 2&#x02009;h while shaking. Kanamycin/ampicillin (30&#x02009;&#x003bc;l) was added to each well to a final concentration of 100 and 30&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1, respectively. After O/N incubation at 30&#x02009;&#x000b0;C while shaking, plates were spun (611<italic>g</italic> for 20&#x02009;min), and supernatant containing phagemid expressing Alphabody was used directly in ELISA.</p></sec><sec disp-level="2"><title>ELISA assays</title><p><italic>Phage-coated ELISA</italic>. To screen for IL-23-positive clones, we developed a phage-coated ELISA method. First, 100&#x02009;&#x003bc;l anti-M13 at 5&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 in PBS was coated O/N at 4&#x02009;&#x000b0;C (VWR, 27942001) onto a Nunc Maxisorp microtiter plate (Thermo Scientific, 42404). Then the plate was blocked with 130&#x02009;&#x003bc;l PBS, 0.1% BSA, 0.5% gelatin for 1&#x02009;h at RT. Between incubations, plates were washed with PBS containing 0.05% Tween 20. After addition of 70&#x02009;&#x003bc;l PBS with 0.1% BSA to the washed wells, 30&#x02009;&#x003bc;l phagemid from a 96-well rescue of clonal phagemid was added. Following an incubation of 1&#x02009;h at RT and a washing step, 1&#x02009;nM IL-23 (eBioscience, 34-8239-85) was added for 1&#x02009;h at RT. Then after washing, 100&#x02009;&#x003bc;l biotinylated anti-p40 in PBS 0.1% BSA (Imtec Diagnostics, BE 505302) was added. For each clone, the background (without IL-23) was measured as well. After washing, staining was performed with 100&#x02009;&#x003bc;l streptavidin-HRP in PBS 0.1% BSA (Biolegend, 405210) followed by a TMB reaction that was stopped with 0.5&#x02009;M H₂SO₄. The OD values were measured at 450&#x02009;nm (OD₄₅₀). A clone was scored as positive when OD_target&#x0003e;3 &#x000d7; OD_background.</p><p>To measure the affinities of the Alphabodies at the phagemid level, we optimized the phage-coated ELISA as described above and incubated different dilutions of target O/N at RT with the coated phagemid. Phagemid (30&#x02009;&#x003bc;l in 70&#x02009;&#x003bc;l PBS, 0.1% BSA) was coated indirectly via anti-M13 as described above, and IL-23 was diluted in a concentration range from 10 to 0.0098, nM (1/5 dilutions) in PBS, 0.1% BSA, and 100&#x02009;&#x003bc;l was added to the plate after washing away the non-bound phagemid. In the phage-coated ELISA format, the phagemid with IL-23 incubation was carried out O/N at RT. IL-23 binding was detected with biotinylated anti-p40 and streptavidin-HRP.</p><p><italic>Standard and kinetic ELISA assays</italic>. Neutravidin (10&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 in PBS) was coated onto a Nunc plate for 1&#x02009;h at RT. Plates were washed 5 &#x000d7; with PBS+0.05% Tween 20 and blocked O/N with 0.1% BSA+0.5% gelatin at 4&#x02009;&#x000b0;C. Biotinylated anti-p40 antibody (Imtec Diagnostics, 10&#x02009;nM in PBS+0.1% BSA) was incubated for 1&#x02009;h at RT, followed by washing. Next, human IL-23 (eBioscience, 10&#x02009;nM in PBS+0.1% BSA) was incubated for 1&#x02009;h at RT while shaking, followed by washing. For the standard ELISA, fivefold dilution series of Alphabodies in PBS+0.1% BSA were incubated for 24&#x02009;h while shaking (plates meanwhile covered with parafilm), followed by washing. For the kinetic ELISA, the same dilution series was added at different time points (24, 3, 1&#x02009;h, 15, 8 and 4&#x02009;min) before the washing step; wells not yet filled with Alphabody were temporarily filled with PBS+0.1% BSA. Both types of ELISA were developed with anti-penta-His-HRP (1/1,000 in PBS+0.1% BSA) for 1&#x02009;h at RT, while shaking. Further steps were as described for the phage-coated ELISA.</p><p>Kinetic binding data in the context of kinetic ELISA assays were derived as follows: ODs were divided by the maximum OD values to obtain the fraction of bound IL-23 (vertical axis in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). Each full data set was fitted with only two parameters, <italic>K</italic>_D and <italic>k</italic>_on, to yield <italic>k</italic>_off.</p></sec><sec disp-level="2"><title>Mouse splenocyte assay</title><p>To assess the inhibitory capacity of the selected Alphabodies in an <italic>in vitro</italic> assay, a mouse splenocyte assay was set up<xref ref-type="bibr" rid="b40">40</xref>. Human IL-23 in the presence of IL-2 stimulates the production of IL-17 in murine splenocytes at very low (picomolar) concentrations, which can be inhibited by co-incubation of inhibitors against either p40 or p19. The IL-17 response (measured using a Quantikine ELISA kit, R&#x00026;D Systems) was determined in parallel for a calibration experiment with increasing hIL-23 concentrations and for an inhibition experiment using a fixed (2.6&#x02009;pM) IL-23 concentration and increasing Alphabody concentrations (fivefold dilutions in the range 0&#x02013;1&#x02009;&#x003bc;M). The inhibition constants <italic>K</italic>_I were determined by fitting the data to a nonlinear equation describing the relationship between OD values and total Alphabody concentrations. The calibration and inhibition experiments were performed in fourfold and eightfold, respectively, on the same ELISA plate (data were averaged).</p></sec><sec disp-level="2"><title>DB cell assay</title><p>hIL-23 induces STAT3 phosphorylation upon engagement with IL-23R at the surface of DB (human lymphoma) cells (DSMZ, Germany). A calibration curve was established by determining the level of STAT3 phosphorylation in DB cells as a function of added human IL-23 within a concentration range of 0.01&#x02013;10&#x02009;nM. To measure the inhibitory activity of the hIL-23-binding Alphabodies, varying concentrations of Alphabodies (500&#x02013;0.01&#x02009;nM) were pre-incubated with a fixed concentration of hIL-23 (200&#x02009;pM) and pre-incubated samples were added to DB cells for 1&#x02009;h, in parallel to the calibration curve samples. Subsequently, DB cell lysates were prepared for STAT3 phosphorylation measurement by sandwich ELISA (PathScan ELISA). Inhibition curves were fitted to derive inhibition constants <italic>K</italic>_I of the tested Alphabodies.</p></sec><sec disp-level="2"><title>Psoriasis animal model</title><p>To determine the ability of affinity-matured Alphabodies to antagonize IL-23-driven inflammation <italic>in vivo</italic>, a 40-kDa linear PEGgylated form of variant MB23 (MB23-PEG) was tested in a mouse &#x02018;ear model&#x02019;<xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref>. Local ear skin inflammation was induced by repeated intradermal administrations of human IL-23 (1&#x02009;&#x003bc;g) every other day within a period of 15 days into the right ear pinna of 20 male C57Bl/6J mice (Harlan Laboratories, Horst, Netherlands) that were 10 weeks old at the start of the experiment. The control group received intradermal PBS injections under the same administration scheme. MB23-PEG was administered by intradermal (10&#x02009;&#x003bc;g; Group B) or intraperitoneal (40&#x02009;mg&#x02009;kg^&#x02212;1; Group C) administrations on days &#x02212;1, 0, 3, 6, 9, 12 and 15. Group A mice were not administered MB23-PEG, but PBS instead as a vehicle control. Skin swelling was measured by determining the ear skin thickness with a caliper on day 0 and subsequently every other day until day 16. The animal studies were approved by the Ethics Committee for Animal Protection under the jurisdiction of the regional administrative authority of Karlsruhe, Germany.</p></sec><sec disp-level="2"><title>Recombinant proteins for crystallographic studies</title><p>For large-scale production cDNA for the MA12_B2fC Alphabody was cloned into the pET16b-vector (Novagen) in frame with a N-terminal decahistidine tag. Recombinant production was performed in transformed BL21(DE3)pLysS cells, grown in LB medium at 37&#x02009;&#x000b0;C. Expression was induced by adding 1&#x02009;mM isopropylthio-&#x003b2;-galactoside at an OD_{600&#x02009;nm} of 0.7 and expression was continued for 4&#x02009;h at 37&#x02009;&#x000b0;C. Washed inclusion bodies were solubilized in 6&#x02009;M guanidine hydrochloride (GuHCl), loaded on a Ni Sepharose column, washed with buffer devoid of GuHCl, and eluted with imidazole. Next, the desalted protein sample was incubated for 2&#x02009;h at 37&#x02009;&#x000b0;C with magnetic trypsin beads to remove the N-terminal His-tag. The free cysteine of the MA12_B2fC Alphabody was blocked for 15&#x02009;min by incubating with 5&#x02009;mM iodoacetamide. Finally, as a polishing step the sample was injected on a Superdex 75 column equilibrated with 20&#x02009;mM HEPES, 150&#x02009;mM NaCl, pH 7.4.</p><p>Human IL-23 was produced by transient expression in mammalian cells<xref ref-type="bibr" rid="b23">23</xref>. cDNAs corresponding to the human IL-23 subunits, p19 (residues 1&#x02013;189) and p40 subunits (residues 1&#x02013;328) were purchased from GeneArt and cloned into the pHL expression vector<xref ref-type="bibr" rid="b54">54</xref> between the EcoRI and KpnI sites. The p19 subunit was cloned in frame with the C-terminal His-tag, while the p40 expression construct carried a stop codon before the KpnI site. Both p19 and p40 cDNA sequences corresponded to the reference sequences in the NCBI databank, NM_016584.2 and NM_002187.2, respectively. However, the AAT codon for Asn248 in the p40 sequence was replaced with AAC to remove an EcoRI site. Large-scale expression experiments in human embryonic kidney 293T (HEK-293T) cells were conducted in roller bottles. HEK-293T cells grown in roller bottles were co-transfected with the pHL-p19-His and pHL-p40 expression constructs in a 1:1 ratio, using polyethyleneimine as transfection reagent. Expression was performed in serum-free Dulbecco's modified Eagle's medium/F12 medium supplemented with 5&#x02009;&#x003bc;M kifunensine. Human IL-23 was purified from the conditioned medium by immobilized metal-ion affinity chromatography using Talon matrix (Clontech) followed by SEC using a Superdex 200 column (GE Healthcare).</p></sec><sec disp-level="2"><title>Crystal structure determination of the IL-23:MA12 complex</title><p>The IL-23:MA12 complex was formed by adding a molar excess of the MA12_B2fC Alphabody to recombinant human IL-23 that had been partially deglycosylated O/N with Endoglycosidase H (New England Biolabs). The complex was isolated from the excess of Alphabody by SEC using a Superdex 200 column with 20&#x02009;mM HEPES, 150&#x02009;mM NaCl, pH 7.4 as running buffer, and concentrated by ultracentrifugation to 6&#x02009;mg&#x02009;ml^&#x02212;1. Nanolitre crystallization trials were set up at RT using a Mosquito crystallization robot (TTP Labtech) against commercially available sparse matrix screens (Hampton Research and Molecular Dimensions). Numerous hits were obtained in the PEG/ION HT screen. Additional hits were found in various conditions of the ProPlex HT and Crystal Screen HT screens.</p><p>Single crystals from optimized conditions were transferred to a drop of reservoir solution with the use of a nylon loop mounted on a SPINE standard cryocap, and cryoprotected with 20% PEG 400 before being flash-frozen in liquid nitrogen. Diffraction experiments were conducted on the X06SA and X06DA beamlines at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland). Data were integrated and scaled using the XDS suite<xref ref-type="bibr" rid="b55">55</xref>. Crystals belonging to crystal form 1 were grown from 20.75% (w/v) PEG 3,350, 200&#x02009;mM Na₂SO₄. Crystals belonging to crystal form 2 were grown from 20.75% (w/v) PEG 3,350, 200&#x02009;mM potassium formate. The structure of the IL-23:MA12_B2fC complex was determined by maximum-likelihood molecular replacement as implemented in the program suite PHASER<xref ref-type="bibr" rid="b56">56</xref>, using the structure of human IL-23 (PDB entry 3DUH)<xref ref-type="bibr" rid="b24">24</xref> as a search model and structure-factors derived from X-ray diffraction data measured from crystal form 1 crystals. Model (re)building was carried out manually using the program COOT<xref ref-type="bibr" rid="b57">57</xref>. Crystallographic refinement and structure validation were carried out using PHENIX<xref ref-type="bibr" rid="b58">58</xref>.</p><p>Buried surface areas and analysis of interaction interfaces were calculated via the PISA server<xref ref-type="bibr" rid="b59">59</xref>. Rendering of the structures as illustrated in <xref ref-type="fig" rid="f3">Figs 3</xref>, <xref ref-type="fig" rid="f4">4</xref>, <xref ref-type="fig" rid="f5">5</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 4 and 5a</xref> was carried out in PyMOL<xref ref-type="bibr" rid="b60">60</xref>.</p></sec><sec disp-level="2"><title>ITC</title><p>For calorimetric measurements, recombinant human IL-23 and the MA12_B2fC Alphabody were prepared by SEC in the same running buffer (20&#x02009;mM Hepes, 150&#x02009;mM NaCl, pH 7.4). Protein concentrations were measured spectrophotometrically at 280&#x02009;nm using calculated theoretical extinction coefficients, and all solutions were extensively degassed before use. Experiments were carried out using a VP-ITC MicroCalorimeter (MicroCal, MA) at 30&#x02009;&#x000b0;C, and data were analysed using the Origin ITC analysis software package supplied by MicroCal. Human IL-23 (4.9&#x02009;&#x003bc;M in the microcalorimeter cell) was titrated with MA12 Alphabody (53.2&#x02009;&#x003bc;M in the titration syringe). Titrations were preceded by an initial injection of 3&#x02009;&#x003bc;l, and were carried out using 5-&#x003bc;l injections applied 300&#x02009;s apart, under constant stirring. The thermal titration data were fitted to the &#x02018;one binding site model&#x02019;, and apparent molar reaction enthalpy (&#x00394;<italic>H</italic>&#x000b0;), apparent entropy (&#x00394;<italic>S</italic>&#x000b0;), dissociation constant (<italic>K</italic>_D) and stoichiometry of binding (<italic>N</italic>) were determined.</p></sec><sec disp-level="2"><title>SPR</title><p>SPR experiments were carried out using a Biacore 3,000 instrument at 25&#x02009;&#x000b0;C with HBS-EP pH 7.4 (GE Healthcare) as running buffer. Forty-three RU of MA12_B2fC were immobilized on a CM5 chip by amine coupling. Recombinant human IL-23 (eBioscience) was used as analyte at concentrations between 0.5 and 40&#x02009;nM and kinetic data were collected at a flow rate of 30&#x02009;&#x003bc;l&#x02009;min^&#x02212;1. For each sample, a 3-min association phase was followed by a 20-min dissociation phase. The chip was regenerated between consecutive samples with 2 pulses of 30&#x02009;s with 10&#x02009;mM glycine&#x02013;HCl buffer pH 1.5. After double reference-subtraction, sensorgrams were analysed using the BiaEvaluation software (version 4.1). Kinetic parameters were fitted to a 1:1 Langmuir model.</p></sec><sec disp-level="2"><title>Mass spectrometry</title><p>Two &#x003bc;l of the same sample used in the crystallization experiments was diluted in 50&#x02009;&#x003bc;l of 50&#x02009;mM ammonium bicarbonate pH 7.5. The sample was subsequently digested with trypsin (Porcine, sequencing grade, Promega, Madison, WI) O/N at 37&#x02009;&#x000b0;C using an enzyme to protein ratio 1:20. Formic acid was added to a final v/v concentration of 0.1% before the liquid chromatography mass spectrometry analysis. The digest was separated using an ultra performance Nano Acquity system coupled to a Synapt G1 mass spectrometer (Waters, Milford, MA). The peptides were separated on an HSS T3 (75&#x02009;&#x003bc;m &#x000d7; 250&#x02009;&#x003bc;m, 1.8&#x02009;&#x003bc;m particles) column (Waters) with a gradient of 3&#x02013;40% of buffer A (0.1% formic acid) and buffer B (100% acetonitrile with 0.1% formic acid). The spectra were acquired in Liquid-chromatography mass spectrometry in elevated energy mode (LCMS^E) mode (alternating low and high collision energy) with mass range from 125 to 2,000&#x02009;<italic>m</italic>/<italic>z</italic> using a collision energy ramp (10&#x02013;40&#x02009;V)<xref ref-type="bibr" rid="b61">61</xref>. The data were analysed using the Proteinlynx Global Server V2.5 platform (Waters) against UniProt database using C-mannosylation as a variable modification.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>J.D., S.L., E.L. and I.L. designed and developed the Alphabody scaffold. K.V.B. expressed and purified recombinant proteins. K.V. and Y.B. carried out ITC and structural studies of the IL-23:MA12 complex with contributions from SNS in structural analysis. S.D. carried out SPR measurements. E.L. carried out CD and stability experiments. P.H. supervised and executed the biopanning campaigns, ELISAs and splenocyte assays. K.S. carried out the cellular assays and supervised the <italic>in vivo</italic> studies. Y.W. and B.D. carried out mass spectrometry. I.L. and S.N.S. designed and supervised the study. S.N.S. wrote the manuscript with contributions from all authors.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>Accession codes:</bold> Atomic coordinates and structure factors for the IL-23&#x02013;MA12 complex have been deposited in the Protein Data Bank with accession codes <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="PDB" xlink:href="4OE8">4OE8</ext-link> and <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="PDB" xlink:href="4OG9">4OG9</ext-link>.</p><p><bold>How to cite this article</bold>: Desmet, J. <italic>et al.</italic> Structural basis of IL-23 antagonism by an Alphabody protein scaffold. <italic>Nat. Commun.</italic> 5:5237 doi: 10.1038/ncomms6237 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-4, Supplementary Tables 1-4.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6237-s1.pdf"/></supplementary-material></sec>

Temporary hemodialysis catheters: recent advances

<p>The insertion of non-tunneled temporary hemodialysis catheters (NTHCs) is a core procedure of nephrology practice. While urgent dialysis may be life-saving, mechanical and infectious complications related to the insertion of NTHCs can be fatal. In recent years, various techniques that reduce mechanical and infectious complications related to NTHCs have been described. Evidence now suggests that ultrasound guidance should be used for internal jugular and femoral vein NTHC insertions. The implementation of evidence-based infection-control &#x02018;bundles' for central venous catheter insertions has significantly reduced the incidence of bloodstream infections in the intensive care unit setting with important implications for how nephrologists should insert NTHCs. In addition, the Cathedia Study has provided the first high-level evidence about the optimal site of NTHC insertion, as it relates to the risk of infection and catheter dysfunction. Incorporating these evidence-based techniques into a simulation-based program for training nephrologists in NTHC insertion has been shown to be an effective way to improve the procedural skills of nephrology trainees. Nonetheless, there are some data suggesting nephrologists have been slow to adopt evidence-based practices surrounding NTHC insertion. This mini review focuses on techniques that reduce the complications of NTHCs and are relevant to the practice and training of nephrologists.</p>

<contrib contrib-type="author"><name><surname>Clark</surname><given-names>Edward G</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>Barsuk</surname><given-names>Jeffrey H</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><aff id="aff1"><label>1</label><institution>Kidney Research Centre, Ottawa Hospital Research Institute</institution>, Ottawa, Ontario, <country>Canada</country></aff><aff id="aff2"><label>2</label><institution>Division of Nephrology, The Ottawa Hospital</institution>, Ottawa, Ontario, <country>Canada</country></aff><aff id="aff3"><label>3</label><institution>Department of Medicine, Northwestern University Feinberg School of Medicine</institution>, Chicago, Illinois, <country>USA</country></aff>

Kidney International

<p>Non-tunneled temporary hemodialysis catheter (NTHC) insertion is a required procedural skill for most nephrologists and nephrology trainees. For all central venous catheters (CVCs), including NTHCs, significant morbidity, mortality and expense can be attributed to their insertion and use.^{<xref ref-type="bibr" rid="bib1">1</xref>, <xref ref-type="bibr" rid="bib2">2</xref>} <xref ref-type="fig" rid="fig1">Figure 1</xref> details the more frequent and serious complications of NTHC insertion, according to insertion site. In the past decade, various techniques implemented at the time of NTHC insertion have reduced the risk of mechanical and infectious complications. However, these advances have largely been reported in the critical care, infection control and general internal medicine literature.</p><p>There is some evidence that the nephrology community has not fully adopted techniques shown to reduce complications related to NTHC insertion.^{<xref ref-type="bibr" rid="bib3">3</xref>} Previous work evaluating trainees' skills inserting NTHCs incorporated an assessment of techniques shown to reduce complications^{<xref ref-type="bibr" rid="bib4">4</xref>} and found that procedural competency at the end of nephrology training was poor. This is concerning because, while insertion of a NTHC may be essential for provision of life-saving renal replacement therapy, mechanical and infectious complications of catheter placement can themselves be fatal and are avoidable. This mini review of practices surrounding the use of NTHCs focuses on recent advances that are relevant to the practice and training of nephrologists.</p><sec><title>USE OF ULTRASOUND GUIDANCE TO PREVENT MECHANICAL COMPLICATIONS</title><p>Acute mechanical complications related to hemodialysis catheter insertion, such as vascular injury or hematoma, are common, occurring in up to 5&#x00025; of catheter insertions.^{<xref ref-type="bibr" rid="bib2">2</xref>} Other mechanical complications such as pneumothorax, pneumopericardium, air and guidewire embolism, and arrhythmia are less frequent but can be fatal.^{<xref ref-type="bibr" rid="bib2">2</xref>}</p><p>It has been well established that the use of real-time ultrasound guidance reduces the mechanical complications associated with CVC insertion at the internal jugular (IJ) and femoral sites. The benefits of ultrasound are likely largely due to anatomic variations in IJ (<xref ref-type="fig" rid="fig2">Figure 2</xref>) and femoral (<xref ref-type="fig" rid="fig3">Figure 3</xref>) vein locations relative to the artery.^{<xref ref-type="bibr" rid="bib5">5</xref>, <xref ref-type="bibr" rid="bib6">6</xref>} Although most data are from studies of non-dialysis catheters, a recent systematic review by Rabindranath <italic>et al.</italic>^{<xref ref-type="bibr" rid="bib7">7</xref>} specifically focused on the use of real-time ultrasound guidance for insertion of dialysis catheters. Seven randomized controlled trials (RCTs) involving 767 patients and 830 IJ catheter insertions (including both tunneled and non-tunneled catheters inserted at the femoral or IJ site) were included. The authors concluded that the use of real-time ultrasound at the time of catheter insertion resulted in a significant reduction in arterial punctures and hematomas, improved the rate of successful insertion on the first attempt and reduced the time taken for successful venous puncture compared with the anatomic landmark-guided technique with no ultrasound.^{<xref ref-type="bibr" rid="bib7">7</xref>} This accords with current recommendations regarding the use of ultrasound for NTHC insertion by renal organizations internationally.^{<xref ref-type="bibr" rid="bib8">8</xref>, <xref ref-type="bibr" rid="bib9">9</xref>} However, it is unclear the extent to which these guidelines have been adopted. Ultrasound is used less commonly for NTHC insertions at the femoral site^{<xref ref-type="bibr" rid="bib3">3</xref>} which might be expected given that there is no risk of pneumothorax. Nonetheless, serious and even fatal complications related to femoral hemodialysis catheter insertion have been reported with the incidence of severe hemorrhage (usually retroperitoneal) estimated at 0.5&#x00025;.^{<xref ref-type="bibr" rid="bib10">10</xref>} One study by Prabhu <italic>et al.</italic>^{<xref ref-type="bibr" rid="bib11">11</xref>} randomized 110 patients to real-time ultrasound-guided anatomic landmark-guided insertion of a femoral NTHC. The use of real-time ultrasound resulted in significantly better &#x02018;first attempt' and overall success rates compared with the use of anatomic landmarks alone: 85.5&#x00025; vs. 54.5&#x00025; (<italic>P</italic>&#x0003c;0.001) and 98.2&#x00025; vs. 80&#x00025; (<italic>P</italic>=0.002), respectively. In addition, the complication rate was 18.2&#x00025; for the landmark-based technique vs. 5.5&#x00025; with ultrasound guidance (<italic>P</italic>=0.039). Subgroup analyses conducted according to operator experience of more or less than 6 years did not show any significant differences from what was observed overall. The current level of evidence suggests that all operators use real-time ultrasound guidance for insertion of NTHCs at the femoral site.</p></sec><sec><title>EVIDENCE-BASED TECHNIQUES TO REDUCE INFECTIOUS COMPLICATIONS</title><sec><title>Infection-control techniques at the time of catheter insertion</title><p>Central line-associated bloodstream infections (CLABSIs) and exit-site infections cause significant morbidity and mortality.^{<xref ref-type="bibr" rid="bib1">1</xref>} Fortunately, the overall incidence of CLABSIs in the United States has markedly declined in recent years likely as a result of infection-control interventions targeted to the intensive care unit (ICU) setting.^{<xref ref-type="bibr" rid="bib1">1</xref>} With respect to NTHCs in particular, a systematic review reported that the rate of CLABSIs for NTHCs is higher than for other types of CVCs (4.8 vs. 2.7 per 1000 catheter days).^{<xref ref-type="bibr" rid="bib12">12</xref>}</p><p>In the ICU setting, multiple interventions at the time of CVC insertion have reduced the rate of associated infections^{<xref ref-type="bibr" rid="bib13">13</xref>} and include: <list list-type="bullet"><list-item><p>adequate hand hygiene.</p></list-item><list-item><p>maximal barrier precautions at the time of CVC insertion (sterile gown, mask, gloves and cap) plus full head-to-toe sterile covering of the patient using a specialized sterile drape as opposed to a &#x02018;standard-sized' sterile field.</p></list-item><list-item><p>2&#x00025; chlorhexidine skin antisepsis applied using a &#x02018;back-and-forth' scrubbing motion for times indicated by the manufacturer on dry areas (IJ site) and moist areas (femoral site).</p></list-item><list-item><p>avoidance of the femoral site (to be discussed in more detail below).</p></list-item><list-item><p>systematic, daily review of the need for a catheter and removal if no longer required.</p></list-item></list></p><p>An infection-control &#x02018;bundle' consists of interventions such as these, applied together, in conjunction with staff education, facilitation of access to the required equipment in the form of a standardized kit and a program of nurse-led monitoring and real-time feedback that uses a detailed checklist to ensure compliance. Infection-control &#x02018;bundles' have been shown to dramatically reduce the rate of CLABSIs^{<xref ref-type="bibr" rid="bib14">14</xref>} and subsequently maintain this reduced rate^{<xref ref-type="bibr" rid="bib15">15</xref>} in the ICU setting. While &#x02018;bundled-interventions' have primarily been studied in ICUs, it is likely that this approach could be similarly effective in the various settings that nephrologists and trainees may insert hemodialysis catheters. This is particularly relevant given that the reduction in CLABSIs observed in recent years in ICUs has not been observed in other settings^{<xref ref-type="bibr" rid="bib1">1</xref>} and may reflect an opportunity for more widespread application of these types of interventions. As such, the various aspects of an infection-control &#x02018;bundle', as described, should be integrated into the practice and training of nephrologists.</p></sec><sec><title>Catheter-locking solutions</title><p>The properties of different catheter-locking solutions may influence both thrombus and biofilm formation and associated complications of catheter malfunction and infection. Multiple smaller-scale RCTs have shown that the use of various antibacterial catheter locks (ACLs) for tunneled hemodialysis catheters (and some studies including NTHCs in addition to tunneled hemodialysis (HD) catheters) can reduce the likelihood of infectious outcomes compared with conventional locking solutions such as heparin alone.^{<xref ref-type="bibr" rid="bib16">16</xref>} An RCT by Maki <italic>et al.</italic>^{<xref ref-type="bibr" rid="bib17">17</xref>} included 407 hemodialysis outpatients with tunneled HD catheters and compared heparin locks with ones containing a mixture of sodium citrate, methylene blue, methylparaben and propylparaben (C&#x02013;MB&#x02013;P). The authors demonstrated that C&#x02013;MB&#x02013;P locks were associated with significantly fewer CLABSIs and were significantly less likely to be discontinued due to poor flows.^{<xref ref-type="bibr" rid="bib17">17</xref>} While this study presents an exciting avenue for further research, it also highlights one of the difficulties in assessing the evidence with respect to ACLs for NTHCs: studies involving the use of ACLs for tunneled HD catheters^{<xref ref-type="bibr" rid="bib18">18</xref>, <xref ref-type="bibr" rid="bib19">19</xref>} may not be directly applicable to NTHCs given the different settings and clinical circumstances in which they are typically used. One study to focus exclusively on the use of ACLs for NTHCs was an RCT by Kim <italic>et al.</italic>^{<xref ref-type="bibr" rid="bib20">20</xref>}, which included 120 new hemodialysis patients using NTHCs while awaiting placement or maturation of a fistula or graft. This study compared ACLs containing gentamicin (5&#x02009;mg/ml), cefazolin (10&#x02009;mg/ml) and heparin (1000&#x02009;IU/ml) with locks containing heparin alone.^{<xref ref-type="bibr" rid="bib20">20</xref>} The ACL group had significantly fewer CLABSIs (0.44 per 1000 catheter days vs. 3.12 per 1000 catheter days, <italic>P</italic>=0.031) and no adverse events were reported.^{<xref ref-type="bibr" rid="bib20">20</xref>} Although this study did not detect any methicillin-resistant <italic>Staphyloccocus aureus</italic> resulting from the ACLs, it was underpowered to do so.</p><p>Overall, in addition to the lack of large scale, RCT-based evidence favoring the use of any particular ACL, there are additional concerns that have limited their broad usage: higher costs, practical issues related to the compounding of ACL solutions at individual dialysis centers and, most importantly, the possibility of promoting antibiotic resistance.^{<xref ref-type="bibr" rid="bib16">16</xref>}</p><p>Given the possibility of antibiotic resistance, it is an appealing concept to utilize antimicrobial locking solutions containing different antibiotics than those routinely used to treat CLABSIs. A recent RCT that utilized such an approach compared EDTA (30&#x02009;mg/ml)&#x0002b;minocycline (3&#x02009;mg/ml) to heparin (5000&#x02009;U/ml) as the catheter lock solution in 187 catheters (144 were NTHCs).^{<xref ref-type="bibr" rid="bib21">21</xref>} This study concluded that there were no significant differences in the rate of catheter removal for dysfunction. However, there was a significant improvement in catheter-related bacteremia-free survival (hazard ratio 0.32; 95&#x00025; CI 0.14&#x02013;0.71) and 90 day catheter-related bacteremia-free survival (91.3&#x00025; vs. 69.3&#x00025;) with EDTA&#x0002b;minocycline.^{<xref ref-type="bibr" rid="bib21">21</xref>}</p><p>Another consideration is whether catheter locks should contain trisodium citrate (hereafter referred to as citrate) or heparin as the primary anticoagulant to maintain catheter patency. A recent systematic review and meta-analysis compared the use of citrate (with or without antimicrobials) to heparin locks in hemodialysis catheters. This review included 13 studies, two of which considered only NTHCs and two that included both NTHCs and tunneled HD catheters.^{<xref ref-type="bibr" rid="bib22">22</xref>} The authors concluded that antimicrobial-containing citrate solutions with a low to moderate concentration of citrate (i.e., &#x02a7d;4&#x00025;) reduced the incidence of CLABSIs compared with heparin-containing locks. There was no significant difference in exit-site infections or catheter patency.^{<xref ref-type="bibr" rid="bib22">22</xref>} Locks using higher concentrations of citrate (&#x02a7e;30&#x00025;) have been associated with additional safety concerns such as hypocalcemia and arrhythmia related to accidental systemic administration^{<xref ref-type="bibr" rid="bib22">22</xref>} and were subjected to a manufacturer's recall in the United States for this reason.^{<xref ref-type="bibr" rid="bib18">18</xref>} While variable outcome measures used across the included studies prevented a subgroup analysis on the basis of catheter type (NTHCs or tunneled HD catheters, specifically), the overall conclusions accord generally with what was observed for studies that involved NTHCs.</p><p>A 2011 study of exclusively NTHCs (<italic>n</italic>=177) compared three types of locks: 4&#x00025; citrate&#x0002b;1.35&#x00025; taurolidine, 5000&#x02009;U/ml heparin&#x0002b;gentamicin and 5000&#x02009;U/ml heparin alone.^{<xref ref-type="bibr" rid="bib23">23</xref>} In this study, citrate&#x0002b;taurolidine significantly reduced CLABSI rates more than heparin alone (RR: 0.37; 95&#x00025; CI, 0.16&#x02013;0.84).^{<xref ref-type="bibr" rid="bib23">23</xref>}</p><p>Overall, the evidence supports a recommendation that citrate (&#x02a7d;4&#x00025;) locks be favored over heparin locks for NTHCs. Currently, 4&#x00025; citrate is used in most HD units in Canada in the form of prefilled 5&#x02009;ml syringes (Citralok, MED-XL, Montreal, QC, Canada)^{<xref ref-type="bibr" rid="bib24">24</xref>} for patients with tunneled HD catheters. The extent to which it is used for NTHCs is unknown particularly given that NTHCs are often used in ICUs or other critical care areas. It should be noted that, in the United States, none of the most commonly used catheter-locking solutions, including heparin at the 1000&#x02009;U/ml concentration, are approved by the Food and Drug Administration for use in HD catheters.^{<xref ref-type="bibr" rid="bib25">25</xref>} This also includes 4&#x00025; citrate which is only available in 250- or 500-ml bags and requires further preparation before being used as a catheter lock.^{<xref ref-type="bibr" rid="bib24">24</xref>}</p><p>While citrate catheter locks should be favored over heparin ones, the efficacy and safety of specific ACLs that contain antibiotics or other antimicrobials, including those that also utilize &#x02a7d;4&#x00025; citrate, have yet to be established by large scale RCTs. Nonetheless, some experts have advocated for the more widespread use of ACLs with hemodialysis catheters (tunneled and NTHCs) in view of the significant burden of morbidity and mortality associated with CLABSIs amongst HD patients.^{<xref ref-type="bibr" rid="bib21">21</xref>} While we agree that the findings of recent studies are encouraging, in view of the potential risks and costs, we feel that large scale, multicenter RCTs of specific ACLs are required before recommending their routine use.</p></sec><sec><title>Specialized antimicrobial catheters</title><p>Reduction of thrombus and biofilm formation is another potential method to reduce CLABSIs because they are often sources for infection. As such, specialized CVCs with antithrombotic and/or antimicrobial properties (e.g., surface coatings, antimicrobial- or antithrombotic-impregnated materials) have been developed. For CVCs in general, there is insufficient evidence for routine use, although they may be indicated in circumstances where high rates of CLABSIs persist despite successful implementation of a &#x02018;bundled' program to reduce them.^{<xref ref-type="bibr" rid="bib13">13</xref>} While there is experimental evidence for silver having antimicrobial properties,^{<xref ref-type="bibr" rid="bib26">26</xref>} studies that have assessed the use of silver-coated tunneled HD catheters have not demonstrated a benefit^{<xref ref-type="bibr" rid="bib27">27</xref>, <xref ref-type="bibr" rid="bib28">28</xref>} and silver-coated NTHCs have not been studied prospectively. A recent RCT that included 77 patients requiring acute dialysis showed significantly less bacterial colonization with a bismuth-coated NTHC, but no significant difference in the primary endpoint of time-to-catheter-removal compared with a conventional NTHC.^{<xref ref-type="bibr" rid="bib29">29</xref>} At the present time, the efficacy and safety of specialized NTHCs for reducing CLABSIs have not been evaluated in large scale RCTs and there are potential barriers to their routine use in the future such as the likelihood of higher costs and, for specialized NTHCs that might be impregnated with antibiotics, the possibility of promoting antibiotic resistance.</p></sec><sec><title>Catheter dressings</title><p>The Centers for Disease Control and Prevention guidelines^{<xref ref-type="bibr" rid="bib13">13</xref>} provide some guidance about the types of dressings to be used on dialysis catheters, but often do not distinguish between other CVCs and tunneled HD catheters or NTHCs. Sterile transparent semipermeable dressing or sterile gauze can be used as line dressings. Gauze dressing are recommended for patients with bleeding or diaphoresis, but must be changed every 48&#x02009;h.^{<xref ref-type="bibr" rid="bib13">13</xref>} Transparent dressings afford the benefit of visualizing the line-entry site for evidence of infection and don't need to be changed for 7 days.^{<xref ref-type="bibr" rid="bib13">13</xref>} During dressing changes, 2&#x00025; chlorhexidine should be used to clean the skin.^{<xref ref-type="bibr" rid="bib13">13</xref>} If there is a contraindication to chlorhexidine, povidodine&#x02013;iodine or 70&#x00025; alcohol can be used as alternatives.^{<xref ref-type="bibr" rid="bib13">13</xref>} Topical antibacterial ointment or creams (povidone&#x02013;iodine or bacitracin/gramicidin/polymixin B) on insertion sites are not recommended except for consideration on tunneled dialysis catheters at the time of insertion and at the end of each dialysis session.^{<xref ref-type="bibr" rid="bib13">13</xref>} Chlorhexidine-impregnated sponge dressing for NTHC cannot be recommended, because studies showing its efficacy in reducing CLABSIs excluded all types of HD catheter.</p></sec><sec><title><italic>S. aureus</italic> decolonization strategies</title><p>Bacterial decolonization with intranasal mupirocin has been shown to significantly reduce the incidence of <italic>S. aureus</italic> bacteremia for chronic hemodialysis patients with tunneled HD catheters.^{<xref ref-type="bibr" rid="bib18">18</xref>} We are unaware of any studies that have assessed decolonization strategies specifically for patients with NTHCs.</p><p>Given that NTHCs are often used for AKI in the context of critical illness, studies of decolonization strategies for unselected ICU patients are relevant to patients who have NTHCs in that setting. A recent, large, cluster-randomized trial (<italic>n</italic>=74,256 patients in 73 ICUs) utilized a 5-day decolonization protocol consisting of twice daily intranasal mupirocin and daily bathing with chlorhexidine-impregnated cloths.^{<xref ref-type="bibr" rid="bib30">30</xref>} This protocol, when applied to all ICU patients, significantly reduced the rate of bloodstream infections (from any pathogen) as compared with a strategy of applying the same decolonization protocol only to methicillin-resistant <italic>S. aureus</italic> carriers or using a strategy that only involved isolation of methicillin-resistant <italic>S. aureus</italic> carriers (i.e., no decolonization).^{<xref ref-type="bibr" rid="bib30">30</xref>} It should be noted that this study did not report any analysis on the basis of whether or not patients had a NTHC in place and its applicability to non-ICU patients with NTHCs is tenuous.</p></sec></sec><sec><title>INITIATION OF DIALYSIS WITH TEMPORARY CATHETERS AND OPTIMAL TIMING OF PERMANENT HEMODIALYSIS ACCESS FOR PATIENTS WITH ACUTE KIDNEY INJURY</title><p>Due to the increased risk of infection associated with NTHCs as compared with tunneled HD catheters, Centers for Disease Control and Prevention guidelines^{<xref ref-type="bibr" rid="bib13">13</xref>} suggest that tunneled catheters be considered if dialysis access is expected to be required for more than 3 weeks. For the same reason, Kidney Disease Outcomes Quality Initiative guidelines (2006 update) for vascular access suggest that NTHCs should not be used for more than 1 week at the IJ or SC sites and a maximum of 5 days at the femoral site.^{<xref ref-type="bibr" rid="bib8">8</xref>} This recommendation was based on a study that showed significantly higher infection rates for patients initiating hemodialysis with NTHCs compared with tunneled HD catheters and an exponential increase in the risk of infection after 1 week for NTHCs.^{<xref ref-type="bibr" rid="bib31">31</xref>} These recommendations are applicable to patients who are not anticipated to recover renal function and when dialysis is not urgently indicated. In such circumstances, initial placement of a tunneled HD catheter or other access is clearly preferable. However, NTHCs are often required to initiate urgent dialysis treatment in the setting of AKI and critical illness when the duration of dialysis that will be required cannot be readily predicted.^{<xref ref-type="bibr" rid="bib32">32</xref>} In addition, the requirement for specialized procedural expertise and equipment to place a tunneled HD catheter may present a barrier to timely initiation of dialysis treatment and may not be safe or feasible if it requires transferring a critically ill patient out of the ICU setting. Finally, while tunneled HD catheters can often be removed as easily as NTHCs within the first weeks after being inserted, their removal typically becomes more invasive and time consuming over time.</p><p>As highlighted by the commentary accompanying the Kidney Disease&#x02013;Improving Global Outcomes (KDIGO) Clinical Practice Guidelines for AKI (2012),^{<xref ref-type="bibr" rid="bib9">9</xref>} recent large RCTs that assessed the optimal dose of RRT for AKI (the ATN and RENAL studies) indicated that RRT was required for a mean duration of 12&#x02013;13 days.^{<xref ref-type="bibr" rid="bib33">33</xref>, <xref ref-type="bibr" rid="bib34">34</xref>} It is unclear at this time if the increased infectious risks associated with a NTHC outweigh the relative burdens of placing tunneled catheters in unselected patients with dialysis-requiring AKI. An RCT that included 34 patients with AKI who required incident dialysis randomized them to receive femoral NTHCs versus tunneled femoral HD catheters.^{<xref ref-type="bibr" rid="bib35">35</xref>} This study found fewer infections and better catheter function but more hematomas and longer insertion times for those assigned to receive a tunneled catheter.^{<xref ref-type="bibr" rid="bib35">35</xref>} While this study suggests an interesting avenue for future research, its small sample size and other methodologic limitations prevent it from providing any guidance with respect to the question of whether the optimal first choice of dialysis access for AKI is a tunneled HD catheter or a NTHC. This is especially the case given that the femoral site is not an ideal first choice of site of catheter insertion in many situations (for either tunneled or NTHCs) and that tunneled femoral HD catheters are rarely used in clinical practice. In the absence of other prospective evidence, the KDIGO guidelines suggest that NTHCs be used to initiate RRT for AKI while acknowledging that this suggestion is made without high-level evidence.^{<xref ref-type="bibr" rid="bib9">9</xref>}</p><p>A related issue is that of the optimal timing for switching from a NTHC to a tunneled HD catheter when ongoing dialysis is required following AKI. To our knowledge, and echoing the commentary accompanying the KDIGO guidelines,^{<xref ref-type="bibr" rid="bib9">9</xref>} at this time there is no prospective data to guide this decision and it seems reasonable that a NTHC be replaced with a tunneled HD catheter once it becomes clear that renal recovery is unlikely in the near-term.</p></sec><sec><title>CHOOSING THE OPTIMAL SITE FOR TEMPORARY HEMODIALYSIS CATHETER INSERTION</title><p>A variety of factors should be taken into consideration to assess the optimal site for NTHC insertion for a particular patient. One aspect of this assessment that is unique to NTHC site selection compared with site selection for other types of CVCs relates to preservation of the vasculature should longer term hemodialysis access be required in the future. As summarized in the KDIGO Guidelines for AKI (2012),^{<xref ref-type="bibr" rid="bib9">9</xref>} whenever possible, the subclavian site should be avoided for NTHC insertion to reduce the likelihood of central venous stenosis.^{<xref ref-type="bibr" rid="bib9">9</xref>} Use of left-sided IJ and subclavian veins may also be associated with a greater risk of central venous stenosis than right-sided veins, possibly as a result of more contact between the catheter and the vessel wall throughout the more tortuous anatomic course.^{<xref ref-type="bibr" rid="bib9">9</xref>} Taking this into account, as well as past recommendations and observational data regarding the likelihood of increased risk of infection at the femoral site, the KDIGO guidelines provided an ungraded recommendation regarding site selection: &#x02018;first choice: right jugular vein; second choice: femoral vein; third choice: left jugular vein; last choice: subclavian vein with preference for the dominant side'.^{<xref ref-type="bibr" rid="bib9">9</xref>} This recommendation was made in accordance with prospective, observational studies that indicated a higher rate of infection at the femoral site.^{<xref ref-type="bibr" rid="bib36">36</xref>, <xref ref-type="bibr" rid="bib37">37</xref>} However, there is more recent evidence that a patient's body mass index (BMI) should be an additional factor to be considered in selected circumstances. The risk of CLABSI may be lower in patients with femoral NTHCs if BMI is &#x0003c;24.2 and for IJ NTHCs if the BMI &#x0003e;28.4^{<xref ref-type="bibr" rid="bib38">38</xref>} according to a pre-specified analysis from the Cathedia Study based on the lowest and highest BMI tertiles of included patients.^{<xref ref-type="bibr" rid="bib38">38</xref>} This RCT of femoral versus IJ NTHC insertion for patients requiring acute dialysis in the ICU included 750 bed-bound patients who required acute renal replacement therapy.^{<xref ref-type="bibr" rid="bib38">38</xref>} Overall, based on catheter colonization rates, this study demonstrated that the use of the femoral site was not associated with a higher infectious risk.^{<xref ref-type="bibr" rid="bib38">38</xref>} An extension of the Cathedia Study which analyzed data from those patients who subsequently required a second NTHC insertion at the alternative site included 134 patients and demonstrated consistent results: time to catheter-tip colonization at removal did not differ significantly between femoral and IJ sites.^{<xref ref-type="bibr" rid="bib39">39</xref>} Despite some limitations, such as the infrequent use of ultrasound for catheter insertions, the Cathedia Study provided the first high-quality RCT-based evidence regarding selection of the optimal site for NTHC insertion. Given the findings of the Cathedia Study, we suggest that for critically ill, bed-bound patients, in the absence of overriding considerations, the first choice of site for NTHC insertion should consider a patient's ambulatory status and BMI if below 24 (femoral) or above 28 (IJ). <xref rid="tbl1" ref-type="table">Table 1</xref> details some of the considerations that might favor a particular site for NTHC insertion.</p><p>Another consideration regarding the optimal choice of insertion site for NTHCs pertains to the risk of catheter dysfunction and consequent &#x02018;under-dialysis'. Notably, the Cathedia Study showed no significant differences between femoral and IJ groups in terms of mean urea reduction ratio or time to catheter dysfunction.^{<xref ref-type="bibr" rid="bib39">39</xref>, <xref ref-type="bibr" rid="bib40">40</xref>} In addition, no significant difference in the risk of deep vein thrombosis (detected using ultrasound screening) was observed.^{<xref ref-type="bibr" rid="bib38">38</xref>}</p></sec><sec><title>SIMULATION-BASED MASTERY TRAINING FOR TEMPORARY HEMODIALYSIS CATHETER INSERTION</title><p>Many medical licensing authorities require candidates seeking certification in nephrology to be competent in NTHC insertion. However, traditional nephrology fellowships may not provide the necessary skills to perform this procedure safely. Two recent surveys showed that up to 1/3 of nephrology fellows received little-to-no training and did not feel confident in inserting NTHCs.^{<xref ref-type="bibr" rid="bib3">3</xref>, <xref ref-type="bibr" rid="bib41">41</xref>} In addition, during a simulated NTHC insertion assessment of graduating nephrology fellows from three fellowship programs in the United States, the mean score on a skills checklist was 53&#x00025; items correct (only one fellow met the minimum passing score on the assessment).^{<xref ref-type="bibr" rid="bib2">2</xref>} A 2008 publication called for evidence-based guidelines to establish standards in procedural competency for nephrology fellows.^{<xref ref-type="bibr" rid="bib42">42</xref>}</p><p>To ensure today's medical trainees are proficient and competent in the procedures they are expected to perform, simulation-based mastery learning (SBML) has been promoted as an ideal training method.^{<xref ref-type="bibr" rid="bib43">43</xref>} Simulation-based training has been used in medical education to increase knowledge, provide opportunities for deliberate and safe practice and shape the development of clinical skills. Mastery learning is a rigorous form of competency-based learning where knowledge and skills are measured against a precise achievement standard.^{<xref ref-type="bibr" rid="bib43">43</xref>} All learners must reach this preset standard so that educational results are equivalent. This is accomplished by allowing varying practice times for individual learners, as necessary, to achieve these results.</p><p>First-year nephrology fellows who were trained with SBML for NTHC insertion skills demonstrated 67&#x00025; higher scores on a simulated skills exam than graduating fellows (<italic>P</italic>&#x0003c;0.001).^{<xref ref-type="bibr" rid="bib2">2</xref>} Other medical trainees who participated in an SBML curriculum for CVC insertion had lower mechanical complications (including arterial punctures, number of needle passes, need for line readjustment and failed insertions) during actual line insertions on patients.^{<xref ref-type="bibr" rid="bib44">44</xref>} In addition, SBML training in central line insertion reduced CLABSI rates by 85&#x00025;^{<xref ref-type="bibr" rid="bib45">45</xref>} and lowered healthcare costs with a 7:1 return on investment.^{<xref ref-type="bibr" rid="bib46">46</xref>} Based on this evidence, we believe that using SBML for NTHC insertion should be considered at all nephrology fellowship programs.</p></sec><sec sec-type="conclusions"><title>CONCLUSIONS</title><p>Given that the complications of NTHC insertion are frequent and can be fatal,^{<xref ref-type="bibr" rid="bib2">2</xref>} it is important that nephrologists and trainees practice techniques that limit these risks and are evidence based. Recent data suggest, in addition to using real-time US guidance for all NTHC insertions at the IJ site, US guidance should also be used for NTHC insertions at the femoral site.^{<xref ref-type="bibr" rid="bib11">11</xref>} Infection-control &#x02018;bundles' of specific evidence-based practices to reduce the risk of CLABSIs and exit-site infections should be implemented in all settings in which NTHCs are inserted and used. This should include the use of detailed checklists for the insertion of catheters, as well as for a daily assessment of whether or not a NTHC is still required or should be removed. Citrate (&#x02a7d;4&#x00025;) catheter locks should be used for NTHCs rather than heparin.^{<xref ref-type="bibr" rid="bib22">22</xref>} There is currently insufficient evidence to support the routine use of ACLs or specialized catheters with antimicrobial properties.^{<xref ref-type="bibr" rid="bib13">13</xref>} A variety of factors must be taken into consideration when determining the optimal site for NTHC insertion. If possible, the subclavian site should be avoided due to the long-term risk of central venous stenosis.^{<xref ref-type="bibr" rid="bib9">9</xref>} RCT evidence now suggests that the femoral site may not be associated with a higher risk of infection and is possibly even preferable in patients who are critically ill and bed-bound with a BMI less than 24.^{<xref ref-type="bibr" rid="bib38">38</xref>} Last, the use of SBML to teach NTHC insertion to nephrology fellows has been shown to significantly improve their procedural competency.^{<xref ref-type="bibr" rid="bib4">4</xref>} This type of educational program can reduce the risk of CLABSIs,^{<xref ref-type="bibr" rid="bib45">45</xref>} result in significant cost savings^{<xref ref-type="bibr" rid="bib46">46</xref>} and should be considered for implementation by all nephrology training programs.</p></sec>

Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress

<p>Plants capture solar energy and atmospheric carbon dioxide (CO₂) through photosynthesis, which is the primary component of crop yield, and needs to be increased considerably to meet the growing global demand for food. Environmental stresses, which are increasing with climate change, adversely affect photosynthetic carbon metabolism (PCM) and limit yield of cereals such as rice (<italic>Oryza sativa</italic>) that feeds half the world. To study the regulation of photosynthesis, we developed a rice gene regulatory network and identified a transcription factor HYR (HIGHER YIELD RICE) associated with PCM, which on expression in rice enhances photosynthesis under multiple environmental conditions, determining a morpho-physiological programme leading to higher grain yield under normal, drought and high-temperature stress conditions. We show HYR is a master regulator, directly activating photosynthesis genes, cascades of transcription factors and other downstream genes involved in PCM and yield stability under drought and high-temperature environmental stress conditions.</p>

<contrib contrib-type="author"><name><surname>Ambavaram</surname><given-names>Madana M. R.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Basu</surname><given-names>Supratim</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Krishnan</surname><given-names>Arjun</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n2">&#x02020;</xref></contrib><contrib contrib-type="author"><name><surname>Ramegowda</surname><given-names>Venkategowda</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Batlang</surname><given-names>Utlwang</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n3">&#x02021;</xref></contrib><contrib contrib-type="author"><name><surname>Rahman</surname><given-names>Lutfor</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Baisakh</surname><given-names>Niranjan</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Pereira</surname><given-names>Andy</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>Virginia Bioinformatics Institute, Virginia Tech</institution>, Blacksburg, Virginia 24061, <country>USA</country></aff><aff id="a2"><label>2</label><institution>Department of Crop, Soil, and Environmental Sciences, University of Arkansas</institution>, Fayetteville, Arkansas 72701, <country>USA</country></aff><aff id="a3"><label>3</label><institution>School of Plant, Environmental, and Soil Sciences, Louisiana State University Agricultural Center</institution>, Baton Rouge, Louisiana 70803, <country>USA</country></aff>

Nature Communications

<p>High crop yield under optimal as well as environmental stress conditions is a valuable crop-stability trait that is targeted for improvement using classical breeding as well as genetic engineering<xref ref-type="bibr" rid="b1">1</xref>. Many approaches have been proposed to boost intrinsic yield, such as enhancement of growth or increase in photosynthetic rate and capacity<xref ref-type="bibr" rid="b2">2</xref>. Photosynthesis, the basis of life on earth that converts light energy to chemical energy in integrated photosynthetic carbon metabolism (PCM) processes, is complex and requires a systems-wide approach to coordinately improve plant productivity and yield<xref ref-type="bibr" rid="b3">3</xref> that is stable under environmental stresses. Transcription factors (TFs) have shown promise in coordinately improving specific traits in rice, such as photosynthetic assimilation and plant biomass<xref ref-type="bibr" rid="b4">4</xref> or grain yield (GY) components under drought<xref ref-type="bibr" rid="b5">5</xref>, and have the potential to coordinately regulate photosynthesis and PCM for crop yield.</p><p>Although photosynthesis is accepted as the basis of absolute yield, yield improvement via direct improvement of photosynthetic efficiency has not yet been successful<xref ref-type="bibr" rid="b6">6</xref>. Nonetheless, evidence that elevated CO₂ can increase leaf photosynthesis in crops by as much as 22.6% over the growing season suggests that increasing photosynthesis can increase productivity and yield<xref ref-type="bibr" rid="b7">7</xref>. One of the primary strategies has been on engineering RuBisCO to improve photosynthetic efficiency<xref ref-type="bibr" rid="b8">8</xref>, although many more metabolic reactions in PCM and associated processes in sucrose synthesis and photorespiration have been shown to play an equivalent role. Metabolic analysis using a dynamic model of PCM<xref ref-type="bibr" rid="b9">9</xref> suggests that the partitioning of resources among enzymes of PCM in C₃ crop leaves is not optimal for maximizing the light-saturated rate of photosynthesis, and under elevated CO₂ predicted for the future, this problem is amplified. The selection of changes to the photosynthetic process intended to improve biomass production and crop yield must take into account a complex matrix of interacting genes and mechanisms. It is recognized that combining systems modelling with modern breeding and transgenic technologies holds promise to design new pathways, such as improved CO₂ fixation and photorespiratory pathways<xref ref-type="bibr" rid="b10">10</xref>, or new genetic-regulatory networks<xref ref-type="bibr" rid="b11">11</xref> to improve photosynthetic efficiency.</p><p>GY in cereals such as rice is limited by environmental stresses such as drought and high temperature, which are also increasing due to climate-change effects. Photosynthesis and related carbon metabolism is primarily affected by stress, thereby reducing GY<xref ref-type="bibr" rid="b12">12</xref>. Understanding of this complex interaction in a systems biology approach will provide the genetic tools to maintain yield under stress. Amongst cereals, rice as a paddy field crop is particularly susceptible to water stress and it is estimated that 50% of the world rice production is affected by drought. Major research efforts are directed at understanding the mechanism of plant responses to drought stress to identify gene products that confer adaptation to water deficit. Molecular mechanisms of water stress response have been investigated primarily in the model plant species Arabidopsis. Upon exposure to drought-stress conditions, many stress-related genes are induced, and their products are thought to function as cellular protectors from stress-induced damage<xref ref-type="bibr" rid="b13">13</xref>.</p><p>The expression of stress-related genes is largely regulated by TFs. The rice and Arabidopsis genomes code for &#x0003e;1,500 TFs, and about 45% of them are reported from plant-specific families. Various drought-stress studies have identified TF families with putative functions in drought including MYB, bZIP, Zinc finger, NAM and APETALA2 (AP2)<xref ref-type="bibr" rid="b13">13</xref>. The AP2 family is one of the plant-specific TFs whose members share a highly conserved DNA-binding domain known as AP2, and members of this family have been associated with various developmental processes and stress tolerance<xref ref-type="bibr" rid="b14">14</xref>. The AP2 TF CBF4, also known as DREB1, was shown by overexpression analysis to lead to drought adaptation in Arabidopsis<xref ref-type="bibr" rid="b14">14</xref>; the Arabidopsis AP2 TF called HARDY was reported to provide enhanced drought tolerance and water-use efficiency (WUE) in Arabidopsis and rice<xref ref-type="bibr" rid="b4">4</xref>. Ectopic expression of these genes confer drought tolerance and/or adaptation by modifying cellular structures of leaves and roots, CO₂ exchange and parameters such as WUE, which correlate with the transformed plants&#x02019; ability to withstand drought. Taken together, these and other findings indicate that AP2 TFs offer the potential to engineer plants in a way that makes them more productive under stress conditions.</p><p>Although drought stress can alter the growth and development of a plant at any time during its life cycle, water limitations during reproductive growth stages can be especially conducive to yield losses in crops such as rice and maize (<italic>Zea mays</italic>)<xref ref-type="bibr" rid="b15">15</xref>. Accordingly, the reproductive phases in these plants should be an important stage to study for identifying stress-responsive genes that might have a protective, or yield-altering, function in drought. Advances in plant genomics, including the availability of the complete genome sequence of rice, have provided an opportunity to identify stress-related TFs that control yield under drought. To this end, a genome-wide analysis of drought-stress responses was conducted and led to the identification of a candidate drought-induced AP2/ERF TF in reproductive tissues.</p><p>To determine whether the TF could play a role in enhancing the tolerance of rice and possibly other crops to drought stress, transgenic plants were generated that contain the candidate gene driven by the CaMV 35S promoter. The <italic>HYR</italic> (<italic>HIGHER YIELD RICE</italic>) gene-expressing transgenic plants here are referred to as HYR lines, as they showed higher GY under well-watered and drought-stress conditions. In addition the HYR lines expressed multiple component traits involved in photosynthesis, sugar levels, root and shoot biomass and WUE under well-watered and drought-stress conditions. The enhanced productivity and the drought-resistant phenotype of the transgenic plants compared with the wild type (WT) are discussed. These studies provide an insight into improvement of plant productivity through enhancement of photosynthesis and multiple downstream biological processes (BPs) in combination with stress tolerance in plants.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Rice regulatory association network analysis</title><p>Since environmental stresses such as drought perturb many essential BPs important for growth and development<xref ref-type="bibr" rid="b12">12</xref>, we reasoned that a global analysis of the regulation of stress responses would reveal the underlying transcription network regulating photosynthesis, PCM and growth. For PCM-related BPs&#x02014;for example, photosynthesis and carbohydrate biosynthesis&#x02014;we sought to identify transcription factors (TFs), whose functions were also perturbed/implicated under environmental stress. To characterize the network of genes and BPs involved in stress response and tolerance, we developed separate &#x02018;regulatory association networks&#x02019; in rice using genome-wide expression profiles of rice genes under &#x02018;control&#x02019; and &#x02018;stress&#x02019; conditions (see Methods, <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). Briefly, we used numerous publicly available whole-genome expression profiles of rice to calculate &#x02018;specific&#x02019; correlation scores (<italic>S</italic>_{<italic>ij</italic>}) of all genes in the genome to each TF, first under control conditions, and then under a variety of stress conditions. We then used these correlations to determine the association of each TF to functionally related sets of genes (diverse gene ontology (GO)<xref ref-type="bibr" rid="b16">16</xref> BPs). The process generated two conditional&#x02014;control and stress&#x02014;TF-function association networks in rice. The networks individually represent the association of each of 328 &#x02018;specific&#x02019; BP/pathway to every TF (among 3,082) in the rice genome for control and stress conditions.</p><p>Using this framework, we focused on choosing candidate rice TFs that potentially play a role in growth, yield and tolerance under drought. The japonica rice genome contains 182 AP2/ERF-domain-containing TFs, many of which have been shown to exhibit functions under growth and stress<xref ref-type="bibr" rid="b17">17</xref>. We first analysed publicly available expression profiles of rice genes under drought<xref ref-type="bibr" rid="b18">18</xref> and identified 59 drought-regulated AP2/ERF TFs (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). We then selected two broad (GO) functional categories, &#x02018;carbohydrate metabolism&#x02019; and &#x02018;photosynthesis&#x02019; (and all their descendants), related to growth and energy allocation to query the &#x02018;control&#x02019; TF-function association network. Examination of the associations of these biological functions with the 59 AP2/ERF TFs showed that some of the AP2 TFs have large positive associations with genes annotated to these functions, and many were negatively associated with some key functional subsets. The exception was Os03g02650 (named HYR, for reasons described above); this was the only drought-inducible rice AP2/ERF TF having positive associations with all of the carbohydrate or photosynthetic functional categories. We used a similar procedure to identify HYR&#x02019;s association with PCM processes in the &#x02018;stress&#x02019; TF-functional network. Contrasting these &#x02018;stress&#x02019; associations with the positive function associations of HYR in the &#x02018;control&#x02019; network, we noted that most of these functions have drastically different associations with HYR (<xref ref-type="fig" rid="f1">Fig. 1d</xref>). Under &#x02018;stress&#x02019;, many of the positive associations are reduced to no (zero) associations (for example, &#x02018;photosynthesis&#x02019; and &#x02018;chlorophyll biosynthesis&#x02019;), while several others have flipped into negative associations (for example, &#x02018;carbohydrate biosynthesis&#x02019;). This analysis, thus, revealed that HYR&#x02019;s positive association with PCM processes under control conditions is significantly perturbed under strong environmental stimuli (<xref ref-type="fig" rid="f1">Fig. 1d</xref>), which together suggested a role for HYR in regulating PCM for basal level protection against environmental stresses.</p><p>Quantitative reverse transcriptase-PCR (qRT-PCR) experiments carried out on Nipponbare plants following 4&#x02013;8 days of progressive drought, confirmed that drought causes upregulation of <italic>HYR</italic> in rice at different developmental stages including two critical reproductive phases&#x02014;pre-anthesis (end of booting stage, panicle elongation) and post-anthesis (2 weeks after flowering) (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). <italic>HYR</italic> is predominantly induced in panicles, at about threefold at pre-anthesis and 1.5-fold at post-anthesis under severe drought relative to well-watered conditions, which include the critical reproductive phases at which drought stress reduces cereal yield<xref ref-type="bibr" rid="b19">19</xref>.</p></sec><sec disp-level="2"><title>Expression of the <italic>HYR</italic> gene enhances photosynthesis in rice</title><p>To assess the role of increased HYR expression in rice, an overexpression construct of the <italic>HYR</italic> gene under control of the CaMV 35S promoter was transformed into rice cultivar Nipponbare (see Methods for details), and five hygromycin-resistant lines (HYR-2, HYR-4, HYR-12, HYR-16 and HYR-45) were identified that showed segregation for a single hygromycin resistance locus and presence of the T-DNA locus by PCR. The five lines expressed <italic>HYR</italic> at approximately 2&#x02013;3 fold higher than the expression level of <italic>HYR</italic> under drought (<xref ref-type="fig" rid="f1">Fig. 1b,c</xref>).</p><p>Morphological analysis showed that the HYR lines had brilliant dark-green leaves compared with the WT (<xref ref-type="fig" rid="f2">Fig. 2a</xref>), with ~15% increased chlorophyll levels (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>) and chloroplast number (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). HYR lines also displayed higher accumulation of starch granules in flag-leaf parenchyma (<xref ref-type="fig" rid="f2">Fig. 2c</xref>), signifying a carbohydrate reserve proximal to the panicle during grain development. In response to increased CO₂ concentration and irradiance levels HYR lines revealed increased photosynthetic capacity, as well as higher CO₂ and light-saturation points than WT (<xref ref-type="fig" rid="f2">Fig. 2e,f</xref>).</p><p>Under low irradiance (&#x02264;400&#x02009;&#x003bc;mol photons m^&#x02212;2&#x02009;s^&#x02212;1), the net CO₂ assimilation was not much different compared with that of WT (<xref ref-type="fig" rid="f2">Fig. 2f</xref>). However, at higher irradiance (400&#x02013;1,500&#x02009;&#x003bc;mol photons m^&#x02212;2&#x02009;s^&#x02212;1) the HYR lines exhibited significantly higher CO₂ assimilation compared with the WT. The increased photosynthesis of HYR lines at present CO₂ levels of 400&#x02009;p.p.m. and above (<xref ref-type="fig" rid="f2">Fig. 2e</xref>) indicates a high photosynthetic capacity of the system to synthesize more carbohydrates and thereby a major component of GY.</p></sec><sec disp-level="2"><title>Rice HYR lines are drought tolerant</title><p>The five <italic>HYR</italic> overexpression lines were evaluated for multiple drought-tolerance physiological parameters in greenhouse tests. The HYR lines were compared with WT plants in a progressive drought experiment in which seedlings were allowed to dry down, and the HYR lines showed better growth than WT (<xref ref-type="fig" rid="f3">Fig. 3a</xref>) and survived 8 days without watering. More importantly, the HYR lines maintained higher relative water content (&#x02265;65% RWC) compared with WT plants (<xref ref-type="fig" rid="f3">Fig. 3b</xref>), throughout the drought treatment and indicating a physiological tolerance drought mechanism.</p><p>To make detailed drought physiological measurements at the adult plant level, the HYR and WT plants were grown side by side to the late-vegetative stage under well-watered/semi-flooded conditions for 8 weeks, and half of the plants from each genotype along with WT were allowed to dry down for 4&#x02013;8 days until plants showed drought-stress symptoms but not leaf rolling. A day before gas-exchange measurements, the soil moisture in the pots with drought stress was adjusted to 75% of field capacity to maintain drought stress. The CO₂ gas-exchange parameters showed that <italic>HYR</italic> lines maintained a significantly higher rate of photosynthetic carbon assimilation compared with WT under both well-watered (32% higher) and drought-stress (60% higher) conditions (<xref ref-type="fig" rid="f3">Fig. 3d</xref>).</p><p>Chlorophyll fluorescence measurements of Fv/Fm (Fv is variable fluorescence and Fm maximum fluorescence) values that represent the maximum photochemical efficiency of PSII in a dark-adapted state, were 5% and 35% higher in the HYR lines, than in the WT plants under well-watered and drought-stress conditions, respectively (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). These results indicate that the effect of drought stress in the fluorescence parameter Fv/Fm, which is a measure of accumulated photooxidative damage to PSII, were considerably smaller in the HYR lines than in the WT plants. Transmission electron microscopy analysis of the rice genotypes under drought revealed that the structure of the chloroplasts in HYR lines under drought was not affected (<xref ref-type="fig" rid="f2">Fig. 2d</xref>), whereas in WT plants their shapes changed from oblong to spherical with severely damaged thylakoid membranes under the same level of drought stress, inferring that maintenance of the HYR chloroplast membrane structure contributes to the sustained photosynthetic capacity under drought<xref ref-type="bibr" rid="b20">20</xref>.</p><p>Drought-response analysis of HYR lines was also carried out under controlled water-deficit stress in soil maintained at 75% of field capacity (see Methods). The HYR lines showed significantly higher shoot biomass under both well-watered and drought-stress conditions (<xref ref-type="fig" rid="f3">Fig. 3c</xref>), contributed by the higher photosynthetic capacity (<xref ref-type="fig" rid="f3">Fig. 3d</xref>), and also showed higher WUE (<xref ref-type="fig" rid="f3">Fig. 3e</xref>), with no significant changes in cumulative water use and stomatal conductance (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). Since abscisic acid (ABA) is known to play a key role in drought-stress response of plants, HYR lines were tested for ABA response and showed less sensitivity to ABA, monitored by seed germination as well as seedling growth parameters (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 5 and 6</xref>). Under drought stress, ABA mainly regulates stomatal behaviour to reduce transpirational water loss, which can reduce photosynthesis<xref ref-type="bibr" rid="b21">21</xref>.</p><p>The HYR lines and WT grown under controlled water-deficit stress were also analysed to study the effect of drought on carbohydrate metabolism. Leaf material of HYR lines and WT was harvested, and dry matter analysed for glucose, fructose and sucrose content (<xref ref-type="fig" rid="f3">Fig. 3f</xref>). Drought stress increased glucose, fructose and sucrose; the HYR lines produced more free glucose, fructose, sucrose and total sugars than the WT under well-watered and drought-stress conditions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>). This suggests a similar hypothesis as in wheat, one of the mechanisms utilized by the plants to overcome water-stress effects via accumulation of compatible soluble sugars<xref ref-type="bibr" rid="b22">22</xref>.</p></sec><sec disp-level="2"><title>HYR promotes a vigorous root system</title><p>Analysis of the root system of HYR lines revealed a robust root system with increased number of adventitious roots (<xref ref-type="fig" rid="f4">Fig. 4a,b</xref>), which grew longer and thicker (<xref ref-type="fig" rid="f4">Fig. 4b&#x02013;d</xref>) than that of WT plants. Microscopic studies of the roots of HYR lines show an enlarged stele and larger size of cortical cells with expansive aerenchyma (<xref ref-type="fig" rid="f4">Fig. 4f,g</xref>). Further, gravimetric analysis of root biomass in HYR lines showed a significant increase of 42% and 72% under well-watered and controlled drought, respectively, compared with the WT (<xref ref-type="fig" rid="f4">Fig. 4e</xref>).</p><p>Root thickness in rice was found to confer drought resistance, as roots are capable of increasing root length density and water uptake by producing more and larger root branches<xref ref-type="bibr" rid="b5">5</xref>. The contribution of these traits clearly indicates efficient translocation and increased surface area available for the uptake of water from the soil, thus imparting drought tolerance to HYR lines. An increase in root dry weight (DW) under stress indicates remobilization of assimilates from shoot to root, and higher root biomass increases the plant&#x02019;s ability to find less-available water and thus increased drought resistance. These phenotypes of increased root surface area available for water and nutrient uptake from the soil are related to field drought resistance and GY of rice<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b23">23</xref>.</p></sec><sec disp-level="2"><title>HYR regulates GY under normal and stress conditions</title><p>The GY potential of rice is determined by three major components: number of panicles (NPs) per plant (associated to tiller number), number of spikelets per panicle and grain weight<xref ref-type="bibr" rid="b24">24</xref>. Under well-watered conditions, the HYR lines exhibited as high as 29% increase in GY with half the genotypes ranging between 27 and 29% increase (<xref ref-type="table" rid="t1">Table 1</xref>). The increase in GY was represented in the yield components plant biomass (<xref ref-type="fig" rid="f3">Fig. 3c</xref>), NP and panicle length, spikelet number per panicle (NSP) and grain number per panicle (NGP) (<xref ref-type="fig" rid="f5">Fig. 5b</xref>; <xref ref-type="table" rid="t1">Table 1</xref>), corroborating other GY component studies<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b24">24</xref>.</p><p>Drought-stress treatment at the sensitive reproductive stage showed that HYR lines produced 14&#x02013;39% higher GY through improvement in multiple yield components (<xref ref-type="fig" rid="f3">Figs 3c</xref> and <xref ref-type="fig" rid="f5">5a,b</xref>; <xref ref-type="table" rid="t2">Table 2</xref>), exhibiting higher biomass with larger panicles and more grains than WT under drought conditions (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). This means that GY increased due to higher single-grain weight and grain number and NSP in HYR. In summary, the results indicate that the HYR lines produced larger panicles with more and larger grains, as well as more total biomass compared with WT under well-watered and even drought-stress conditions. An increase in the number of filled grains might be due to the contribution of carbohydrates from photosynthesis, with more and efficient translocation into the grain and thus increase in the GY<xref ref-type="bibr" rid="b24">24</xref>. The above results clearly indicate that HYR plays a significant role in conferring drought tolerance and improving GY under drought in rice.</p><p>Heat stress is another problem affecting rice yield worldwide due to increases in nighttime temperature caused by climate change<xref ref-type="bibr" rid="b25">25</xref>. The response of HYR lines to high day and night temperatures was tested at flowering and grain maturation. The HYR lines showed increased GY under high temperatures (<xref ref-type="fig" rid="f5">Fig. 5c</xref>), independent of spikelet sterility and grain weight (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8</xref>), and more fertilized spikelets developed to maturity. Because high nighttime temperatures decrease grain quality<xref ref-type="bibr" rid="b26">26</xref>, the harvested threshed grain were examined for chalkiness, which showed significant reduction in HYR lines (<xref ref-type="fig" rid="f5">Fig. 5d,e</xref>), supporting a mechanism of HYR maintaining photosynthate supply and starch deposition in the developing grain under high nighttime temperature.</p></sec><sec disp-level="2"><title>HYR regulates expression of genes in PCM and stress response</title><p>To identify genes and BPs regulated by HYR, gene expression profiles of WT and a HYR line were analysed by microarrays and a set of genes confirmed by qRT-PCR (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 9</xref>). GO analysis revealed that photosynthesis and PCM processes were upregulated by HYR (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Data1</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 10</xref>), and corroborates the positive association of HYR with PCM in the TF-function association network (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). These results indicate that HYR is a key regulator of genes involved in PCM processes essential for plant growth and yield (<xref ref-type="fig" rid="f1">Fig. 1d</xref>), and its upregulation confers drought and heat tolerance.</p><p>To obtain evidence of the HYR regulatory network, rice transformants expressing affinity-tagged (tandem affinity protein (TAP)-tagged) HYR protein were used to isolate HYR-bound chromatin<xref ref-type="bibr" rid="b27">27</xref>, and assayed by chromatin immunoprecipitation (ChIP)-qPCR for binding to promoters of predicted HYR-regulated genes. Promoter sequences of photosynthesis genes, PCM TFs and drought- and temperature-regulated genes were found enriched in the chromatin complex by qPCR analysis (<xref ref-type="fig" rid="f6">Fig. 6a,c</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 11&#x02013;13</xref>), demonstrating binding of the HYR protein to promoters of these genes <italic>in vivo</italic>. Transcriptional regulation of HYR target photosynthesis genes (<xref ref-type="fig" rid="f6">Fig. 6a</xref>) and PCM TFs (<italic>GASR2</italic>, <italic>ARF1</italic> and <italic>WRKY72</italic>) (<xref ref-type="fig" rid="f6">Fig. 6c</xref>) was confirmed by transactivation assays in rice protoplast transformation experiments expressing <italic>HYR</italic> co-transformed with target promoter-luciferase constructs. The PCM genes predicted to be downstream targets of HYR-regulated TFs (GASR2/ARF1) revealed transcriptional regulation by GASR2/ARF1 in co-transformation experiments (<xref ref-type="fig" rid="f6">Fig. 6c</xref>). Similarly, drought-regulated and heat-regulated genes predicted to be targets of HYR were also shown to be regulated by HYR in promoter-luciferase transactivation assays (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 12,13</xref>).</p><p>The rice <italic>WRKY72</italic> gene, orthologue of the Arabidopsis <italic>WRKY75</italic> gene that induces root growth when repressed and involved in phosphate acquisition<xref ref-type="bibr" rid="b28">28</xref>, is interestingly also repressed by <italic>HYR</italic> expression (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 11</xref>). This was further tested for regulation of the expansin gene EXPA8 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14</xref>), identified from gene expression data of HYR lines, which had been shown to improve root growth and architecture<xref ref-type="bibr" rid="b29">29</xref>. Overexpression of <italic>HYR</italic> in rice protoplasts represses WRKY72 and induces EXPA8, while overexpression of WRKY72 represses EXPA8. This supports the hypothesis that WRKY72 regulates the expression of EXPA8, which is known to affect rice root growth and architecture<xref ref-type="bibr" rid="b29">29</xref>, and provides an explanation for the enhanced root growth in HYR lines.</p><p>To confirm direct regulation of target genes, human oestrogen receptor (HER) protein fusions<xref ref-type="bibr" rid="b30">30</xref> of HYR and downstream TFs (<italic>GASR2</italic> and <italic>ARF1</italic>) were tested in estradiol induction assays with cycloheximide that inhibits new protein synthesis (<xref ref-type="fig" rid="f6">Fig. 6</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 11&#x02013;13</xref>). These assays demonstrated that HYR is a direct transcriptional activator of the photosynthesis and heat-responsive genes identified, and of other co-regulated TFs that in turn transcriptionally activate or repress genes involved in carbon metabolism and drought response (<xref ref-type="fig" rid="f6">Fig. 6</xref>).</p><p>The broader significance of HYR regulation of plant processes was explored further. Since HYR increases GY through a regulatory network of genes, we asked the question whether genes known to be involved in different yield components were regulated by HYR. A set of genes (<italic>RCN</italic>, <italic>MOC1</italic>, <italic>TB1</italic>, <italic>LAX</italic> and <italic>GIF</italic>) involved in different components of yield<xref ref-type="bibr" rid="b31">31</xref> were tested for their expression in HYR lines and showed differential expression. There was enhanced expression of tillering genes <italic>MOC1</italic> and <italic>TB1</italic>, and panicle differentiation gene <italic>RCN</italic>, and repression of panicle development gene <italic>LAX</italic> and grain-filling gene <italic>GIF</italic> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 15</xref>). To assess the potential role of HYR in diverse rice germplasm, the expression of <italic>HYR</italic> in rice plants was tested by qPCR and showed significant differences in expression between genotypes under normal growth, with higher expression of <italic>HYR</italic> in genotypes N22, Vandana and Pokkali (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 16</xref>) that are known to express traits for GY under stress<xref ref-type="bibr" rid="b32">32</xref>.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Improvement of GY is the primary objective in breeding and improvement of cereal crops. Rice lines overexpressing the <italic>HYR</italic> gene produce up to 29% increase in GY grown under well-watered ambient conditions (<xref ref-type="fig" rid="f5">Fig. 5b</xref>; <xref ref-type="table" rid="t1">Table 1</xref>), showing a general improvement in all yield components in most HYR lines. The HYR lines showed increased levels of net photosynthesis measured under ambient conditions, as well as with increasing levels of CO₂ and light intensity. This enhanced photosynthetic capacity is supported by an increase in chloroplast number and chlorophyll content, and also represented by increase in the photosynthetic products of soluble sugars, starch and plant biomass. These results concur with previous studies that have shown increased biomass production is concomitant with improved photosynthesis in Arabidopsis<xref ref-type="bibr" rid="b10">10</xref> and rice<xref ref-type="bibr" rid="b4">4</xref>.</p><p>As a result of increased photosynthesis, total soluble sugars including sucrose, the primary phloem-mobile carbohydrate, are increased in HYR lines (<xref ref-type="fig" rid="f3">Fig. 3f</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>), but do not seem to cause the expected feedback inhibition of photosynthesis<xref ref-type="bibr" rid="b33">33</xref>. This could be due to the putative feedback loop (whereby &#x02018;excess&#x02019; sucrose inhibits photosynthesis) that is less sensitive in HYR lines, or the sucrose levels at critical sites (presumably the chloroplasts) are less because of stronger sink activity in the larger, faster-growing plants. The evidence suggests that the higher sink capacity of growing roots, flag leaf, shoots and developing grain of HYR plants can be a reservoir for the increased photosynthate, reducing the sugar accumulation in leaves and the downregulation of photosynthesis<xref ref-type="bibr" rid="b34">34</xref>.</p><p>Under progressive drought stress with the soil allowed to dry down, HYR lines survived longer than WT controls (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The higher RWC observed in the HYR lines under drought could be due to the accumulation or presence of sugars, especially sucrose, leading to osmotic adjustment. In osmotic adjustment, leaves develop a more negative osmotic potential by accumulating solutes. They can then maintain a higher RWC during a period of leaf water-potential reduction. Solute accumulation and osmotic adjustment have been associated with drought tolerance in many crop plants<xref ref-type="bibr" rid="b12">12</xref>. With controlled drought-stress treatment (see Methods), the rice HYR lines maintained higher photosynthesis and WUE, resulting in higher shoot and root biomass compared with WT plants. Since the WUE of HYR lines increased, although the cumulative water use and transpiration remained the same as WT, the enhanced photosynthetic CO₂ assimilation determines the improved productivity of HYR lines. In C₃ plants, WUE is generally determined by, among other factors, stomatal control of the ratio of the instantaneous rates of photosynthesis and transpiration. Stomatal closure typically leads to decrease in photosynthetic CO₂ assimilation due to restricted diffusion of CO₂ into the leaf and altered CO₂ metabolism that is mainly responsible for the decline in photosynthesis in C₃ plants under drought<xref ref-type="bibr" rid="b35">35</xref>. However, the HYR lines have reduced sensitivity to ABA (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 5 and 6</xref>) and therefore have lower stomatal response to drought stress with less effect on stomatal conductance. The results for HYR plants imply that the WUE is not determined by stomatal control of photosynthesis, but by effective CO₂ fixation as reported previously for another C₃ plant<xref ref-type="bibr" rid="b36">36</xref>, suggesting that CO₂ metabolism of HYR lines is more resistant to dehydration.</p><p>Soluble carbohydrates (glucose, sucrose, fructose, sorbitol and mannitol) have been reported to accumulate in plants under drought stress<xref ref-type="bibr" rid="b37">37</xref>. This is due to a shift in C-partitioning from non-soluble carbohydrates (starch) to soluble carbohydrates, which can help maintain turgor for a longer period during drought and also participate in stress-protective functions<xref ref-type="bibr" rid="b37">37</xref>. Under drought stress the accumulation of soluble carbohydrates can maintain plant turgor and contribute to stress-protective functions, such as maintenance of RWC in HYR lines due to osmotic adjustment, a mechanism of drought tolerance<xref ref-type="bibr" rid="b38">38</xref>.</p><p>The increase in number of filled grains, evident in HYR lines under drought or high-temperature stress, can be due to efficient translocation of carbohydrates from photosynthesis into the grain and increase in GY as suggested previously<xref ref-type="bibr" rid="b39">39</xref>. Silencing studies of the rice <italic>OsBP-73</italic> gene that reduced photosynthetic rate, NGP and filled spikelets<xref ref-type="bibr" rid="b20">20</xref>, indicate an association between these processes. Likewise, even small increases in the rate of net photosynthesis were shown to cause large increases in biomass and yield in wheat<xref ref-type="bibr" rid="b40">40</xref>. The results described here characterize the role of the TF HYR in coordinating the expression of genes involved in enhancement of the energy source photosynthesis and PCM, along with an increase of biomass and GY sink, which are sustained under normal and environmental stress conditions.</p><p>GY from rice plants is severely affected by reduction in the NPs and/or spikelets, when they are exposed to drought stress at the reproductive stage<xref ref-type="bibr" rid="b13">13</xref>. If the drought is severe during the grain-filling period, grain filling can be impaired and mean grain weight reduced. In rice, grain-filling process depends on two main carbon resources: photosynthetic assimilates and carbohydrates stored during pre-anthesis and transported to the grain from vegetative tissues. Under drought stress at pre-anthesis stage, the number of spikelets and total GY declined markedly. Moreover tiller (panicle) number per plant was also reduced in WT and some extent in HYR lines. Competition for assimilates during stem extension is believed to be an important factor influencing tiller and spikelet mortality under drought<xref ref-type="bibr" rid="b13">13</xref>. However, the lower reduction in the filling rate and number of spikelets of HYR plants under drought conditions implies that the developmental processes for panicles and spikelets had been protected from drought stress, indicating drought tolerance at the reproductive stage. HYR lines showed about 14&#x02013;40% more yield under drought stress compared with the WT, probably due to the contribution of carbohydrates because of higher photosynthetic rate, which are efficiently translocated into the panicle and thus increase GY. Even small increases in the rate of net photosynthesis can translate into large increases in biomass and hence yield<xref ref-type="bibr" rid="b40">40</xref>. These studies along with our observations in HYR, deduce a positive correlation between leaf photosynthesis and crop biomass and/or yield, evidence of the essential relationship between photosynthesis and crop yield.</p><p>The rice genome is predicted to contain 139 AP2/ERF-domain-containing TF genes<xref ref-type="bibr" rid="b17">17</xref>. Other AP2 TFs have been previously found to provide enhanced root strength and increased number of secondary and tertiary roots in transgenic Arabidopsis and rice plants<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. HYR is one of the novel AP2/ERF TFs that impacts multiple processes by regulation of biochemical pathways, growth, development and response to drought and heat. This work provides a functional characterization of the gene for its effect in plant productivity under well-watered and water-limiting conditions.</p><p>Functional analysis of the HYR protein activity as a TF show that HYR is primarily involved in direct transcriptional activation of multiple photosynthesis-related processes (<xref ref-type="fig" rid="f6">Fig. 6d</xref>), HYR is also involved in a regulatory cascade activating the auxin-responsive TF <italic>ARF1</italic> involved in vegetative growth and seed development in rice<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b41">41</xref>, capable of binding to the <italic>DRO1</italic> drought avoidance gene promoter<xref ref-type="bibr" rid="b23">23</xref>. HYR was shown to repress <italic>OsWRKY72</italic>, which is the orthologue of Arabidopsis <italic>AtWRKY75</italic> that induces root growth when repressed<xref ref-type="bibr" rid="b28">28</xref>, supporting the HYR-enhanced root growth phenotype. This was experimentally verified by expressing HYR and <italic>OsWRKY72</italic> in rice protoplasts, HYR expression causing WRKY72 repression and EXP8 induction, and WRKY72 overexpression causing EXP8 repression (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14</xref>)</p><p>HYR induces transcription of the glucose-6-phosphate isomerase gene. This glycolytic enzyme PGI is localized to plastids and is required for starch accumulation in Arabidopsis<xref ref-type="bibr" rid="b42">42</xref>. Through transcriptional activation of the gibberellin-mediated regulation of <italic>GASR2</italic> TF, HYR also causes repression of starch phosphorylase (<italic>OsPHO1</italic>) responsible for reversible phosphorylation of starch precursors and starch accumulation<xref ref-type="bibr" rid="b43">43</xref>. These and other carbon metabolism genes support the role of HYR in coordinate regulation of PCM genes for biomass accumulation. These genes are coordinated in expression with yield-related genes involved in tillering, <italic>MOC1</italic> and <italic>TB1</italic>, and the panicle differentiation gene <italic>RCN</italic><xref ref-type="bibr" rid="b31">31</xref>. The HYR-mediated regulation of genes that are induced by heat and drought implies a role for HYR in the stress-response regulatory pathway leading to tolerance and robust plant growth. One of genes regulated by heat and HYR, <italic>CRY2</italic>, is a blue-light photoreceptor<xref ref-type="bibr" rid="b44">44</xref> involved in regulation of leaf sheath elongation, and thereby growth and biomass.</p><p>The above results provide strong evidence that HYR is a master regulator of multiple BPs, directly acting as an activator/repressor of TFs and other genes in a network involved in PCM and stress-protective pathways (<xref ref-type="fig" rid="f6">Fig. 6</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 11&#x02013;13</xref>). The morpho-physiological programme regulated by HYR expression conditioning superior photosynthetic capacity under elevated CO₂, light and temperature offers the potential use of HYR-expressing plants to maintain crop growth and yield under environmental stresses associated with climate change.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Rice regulatory association network analysis</title><p>To identify transcriptional regulators (TFs), of PCM-related BPs (photosynthesis and carbohydrate biosynthesis), whose functions were perturbed/implicated under environmental stress, we developed a &#x02018;regulatory association network&#x02019; in rice as follows (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). Genome-wide expression profiles of 35,161 rice genes (denoted here without the locus loc_ prefix identifier) from various tissue/developmental stages under control conditions (243 samples; 6 data sets) were aggregated in the form of a normalized gene expression matrix (<italic>E</italic>_{<italic>ij</italic>}) (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). Raw data were downloaded from Gene Expression Omnibus and background corrected, quantile normalized and summarized using robust multi-array average (RMA)<xref ref-type="bibr" rid="b45">45</xref> based on a custom computable document format (CDF)<xref ref-type="bibr" rid="b46">46</xref> and replicate values were averaged. The distribution of gene expression values within a developmental stage were then scaled to have mean 0 and s.d. 1, resulting in the expression matrix <italic>E</italic>. Next, 3,100 rice TFs were curated from multiple public databases<xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b47">47</xref>, out of which 3,082 were present in the rice Affymetrix microarray. Using the expression matrix <italic>E</italic>, Pearson correlations were calculated between all gene pairs<xref ref-type="bibr" rid="b48">48</xref>, which were then Fisher Z-transformed<xref ref-type="bibr" rid="b49">49</xref> and standardized to get normalized correlations with a <italic>N</italic> (0,1) distribution. From these values, correlations between gene-TF pairs (35,161times;3,082) were extracted to populate a gene-TF matrix <italic>C</italic> with each entry <italic>C</italic>_{<italic>ij</italic>} corresponding to the normalized correlation of gene <italic>i</italic> with TF <italic>j</italic>. Following the CLR algorithm<xref ref-type="bibr" rid="b50">50</xref>, for each correlation value <italic>C</italic>_{<italic>ij</italic>} between TF <italic>j</italic> and a potential target gene <italic>i</italic>, its likelihood given the background distribution of correlations for all TF-gene pairs that involve either TF <italic>j</italic> or gene <italic>i</italic> were computed. This was done by combining the two <italic>z</italic>-scores of <italic>C</italic>_{<italic>ij</italic>} compared with the distribution of <italic>C</italic>_{<italic>i</italic>} and <italic>C</italic>_{<italic>j</italic>} values using Stouffer&#x02019;s method<xref ref-type="bibr" rid="b51">51</xref> to acquire &#x02018;specific&#x02019; correlation scores (<italic>S</italic>_{<italic>ij</italic>}) of all genes in the genome to each TF. Using functional annotations from GO (see below), these gene-level correlation scores were summarized into more robust process-level association scores to the TF<xref ref-type="bibr" rid="b52">52</xref>, to generate a &#x02018;control&#x02019; TF-process association network in rice. We repeated the same procedure with a set of rice expression profiles under a variety of stress conditions (~130 samples; 10 data sets) aggregated in the form of a normalized gene expression matrix (<italic>E</italic>_{<italic>ij</italic>}) (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). Calculating process/function-level association scores to TFs using this expression data resulted in a &#x02018;stress&#x02019; TF-process association network in rice.</p></sec><sec disp-level="2"><title>Functional annotations of rice genes</title><p>Functional information&#x02014;GO BP annotations&#x02014;for rice genes was obtained from AgBase<xref ref-type="bibr" rid="b53">53</xref>. A gene annotated to a term was also annotated to its ancestors in the GO hierarchy following the &#x02018;is_a&#x02019; and &#x02018;part_of&#x02019; relationships. Then, to retain only &#x02018;specific&#x02019; annotations, terms that annotate &#x0003c;1,500 genes were selected. Furthermore, to remove high redundancy in these annotations&#x02014;large overlaps between genes annotated to several GO terms&#x02014;going down the list of terms sorted based on the number of annotated genes, for each term, any term lower in the list that satisfied the following two conditions were removed: (i) differed in the number of genes by &#x0003c;5, and (ii) shared a number of genes with a Jaccard coefficient of 0.9. This provided a set of non-redundant specific GO BPs for enrichment analysis and in building the TF-process association network.</p></sec><sec disp-level="2"><title>Vector construction and rice transformation</title><p>The rice <italic>HYR</italic> gene (Loc_Os03g02650) as annotated in the database of rice TFs<xref ref-type="bibr" rid="b47">47</xref>, was used to make an overexpression construct by assembling the individual fragments (CaMV 35S promoter, <italic>HYR</italic> gene Os03g02650 and CaMV 35S terminator) with appropriate compatible cohesive ends and ligated into the binary vector pCAMBIA1301 as described below. The CaMV 35S promoter fragment from &#x02212;526 to the transcription start site was obtained as a 0.55-kb HindIII&#x02013;SalI fragment from a pBS-SK+ derivative of pDH51 (ref. <xref ref-type="bibr" rid="b54">54</xref>). The full-length coding region of rice <italic>HYR</italic> was amplified using <italic>pfu</italic> DNA polymerase from genomic DNA of rice cv. Nipponbare, and a CaMV 35S terminator fragment was obtained as a 0.21-kb PstI&#x02013;EcoRI fragment from a pBS-SK+ derivative of pDH51 (ref. <xref ref-type="bibr" rid="b54">54</xref>). The construct was made in the binary vector pCAMBIA1301 containing a CaMV 35S-hygromycin phosphotransferase-tNos for selection during transformation.</p><p>A TAP-tagged <italic>HYR</italic> construct was made in pUC19 assembling the fragments of the CaMV 35S promoter<xref ref-type="bibr" rid="b54">54</xref> and the NOS terminator, coding sequence for six His repeat (6 &#x000d7; His), a 9 &#x000d7; myc peptide, a 3C protease cleavage site and two copies of the IgG-binding domain (2 &#x000d7; IgG-BD) together known as the TAP tag<xref ref-type="bibr" rid="b55">55</xref>. The entire cassette was cloned between the Xba1&#x02013;EcoR1 sites of pMOG22 (Zeneca-Mogen), which contains a chimeric CaMV 35S-hygromycin phosphotransferase-tNos for selection during transformation.</p><p>Agrobacterium-mediated transformation of <italic>Oryza sativa</italic> ssp. <italic>japonica</italic> cv. Nipponbare, plant regeneration and selection conditions were performed<xref ref-type="bibr" rid="b56">56</xref>, using the Agrobacterium strain LBA4404. Regenerated transgenic plantlets were transferred to environmentally controlled growth chambers maintained at 28&#x02009;&#x000b0;C&#x000b1;1 day and 25&#x02009;&#x000b0;C&#x000b1;1 night temperature, 65% relative humidity (RH) with a daily photoperiodic cycle of 14&#x02009;h light and 10&#x02009;h dark, and the plants were grown in soil till maturity under greenhouse conditions.</p></sec><sec disp-level="2"><title>Chlorophyll fluorescence and gas-exchange parameters</title><p>Chlorophyll fluorescence was measured using the modulated chlorophyll fluorometer OS1-FL (Opti-Sciences Inc, USA). Flag leaves of stressed and unstressed genotypes was placed in close contact with the photosynthetically active radiation clip, which provides basic data to the OS1-FL system on ambient conditions. The maximal quantum yield of PSII was calculated as Fv/Fm=(Fm&#x02212;Fo)/Fm, where the minimum fluorescence (Fo) was recorded after dark adaptation for 10&#x02009;min and the maximum fluorescence (Fm) was monitored by application of a 0.8-s saturating light pulse (6,000&#x02009;&#x003bc;mol photons m^&#x02212;2&#x02009;s^&#x02212;1) from white LED light. Gas-exchange measurements were done using a LI-6400XT (LI-COR Inc., NE, USA) in attached leaves of WT and <italic>HYR</italic> transgenic plants under well-watered and drought-stress conditions. CO₂ gas-exchange measurements were performed after 4&#x02009;h of illumination with a daily photoperiodic cycle of 14&#x02009;h light and 10&#x02009;h dark at leaf temperature of 25&#x02009;&#x000b0;C, CO₂ at 400&#x02009;&#x003bc;mol&#x02009;s^&#x02212;1 and RH of 55&#x02013;60%. Instantaneous WUE (WUEi) was calculated as described<xref ref-type="bibr" rid="b4">4</xref> using the formula: WUEi=(Pn/<italic>E</italic>).</p></sec><sec disp-level="2"><title>Measurements of chlorophyll and RWC</title><p>Chlorophyll was extracted from 2-week-grown seedlings with 80% acetone, and determined as described<xref ref-type="bibr" rid="b57">57</xref>. Plant water status was determined by measurement of RWC (%)<xref ref-type="bibr" rid="b58">58</xref>, in which the leaf used for photosynthesis measurement was excised, and an ~6-cm section had its fresh weight (FW) determined immediately. The leaf sections were floated in deionized water at room temperature for 6&#x02009;h, their rehydrated weight (RW) determined, dried in an oven at 70&#x02009;&#x000b0;C overnight and weighed to obtain the DW. The RWC% was calculated as: RWC%=(FW&#x02212;DW)/(RW&#x02212;DW) &#x000d7; 100.</p></sec><sec disp-level="2"><title>Drought-stress treatment and WUE analysis</title><p>Controlled drought treatment was done on rice lines at late-vegetative developmental stages grown in 250-ml pots filled with a 1:1 mix of top-soil and compost, and placed in water-filled trays to simulate flooded/paddy conditions, supplemented with a general-purpose 20-20-20 fertilizer dissolved in water to provide 50&#x02009;kg N, P₂O₅ and K₂O&#x02009;ha^&#x02212;1 applied weekly during the growing period. Twenty-eight days after planting (DAP), the pots were adjusted to equal weights (soil+water) by adding water as needed, mulched with a layer of perlite of fixed weight to minimize evaporative water loss and placed on tared bases. Multiple pots of each genotype were divided in two sets, for drought-stress treatment and well-watered controls. In the drought-stress treatments pots were dried down to ~70% field capacity. At this time (31 DAP), shoots on half of the plants (four for drought and four for control treatment) for each line were harvested and dried at 72&#x02009;&#x000b0;C for 96&#x02009;h and this biomass was designated as BIO-31. For the remaining plants, gravimetric soil moisture was maintained at 70% (drought stress) or at field capacity (well-watered) by replacing water lost through transpiration. The amount of water added daily for each pot was noted for calculation of cumulative water used (WUc) for a watering schedule of 14 days. At that point (45 DAP), shoots were harvested and dried at 72&#x02009;&#x000b0;C for 96&#x02009;h, and this biomass was designated BIO-45. WUc was calculated using the formulae: daily WU=(Total weight)&#x02212;(tared pot+tared base+tared mulch), and WUc=&#x02211;(daily WU) over 14 days. Gravimetric WUE (WUEg) was calculated as: WUEg=[(BIO-45)&#x02212;(BIO-31)]/(WUc).</p></sec><sec disp-level="2"><title>Measurement of sugar composition and content</title><p>For sugar analysis<xref ref-type="bibr" rid="b59">59</xref>, plants were harvested (above-ground biomass) and dried at 40&#x02009;&#x000b0;C for 72&#x02009;h. Sugars were extracted from 20-mg ground samples in 2&#x02009;ml of 80% ethanol in an 80 &#x000b0;C water bath for 15&#x02009;min. The cooled crude extract was centrifuged at 3,000<italic>g</italic> for 10&#x02009;min, 20&#x02009;mg charcoal added to the supernatant, the extract centrifuged at 2,200<italic>g</italic> for 15&#x02009;min, 20&#x02009;&#x003bc;l transferred to a microtitre plate and dried at 50&#x02009;&#x000b0;C for 1.5&#x02009;h. A series of standard solutions of glucose, fructose and sucrose were co-analysed with the extracts. After drying, 20&#x02009;&#x003bc;l per well deionized-distilled water was added, and after 1&#x02009;h, 100&#x02009;&#x003bc;l of glucose reagent (Sigma, St Louis, MO); the plate was kept at room temperature for 30&#x02009;min, and glucose measured on a microplate reader (SpectroMax plus 386, Molecular Devices Corp., Sunnyvale, CA) at 340&#x02009;nm. Ten &#x003bc;l of 0.25 enzyme unit (EU) phosphoglucose isomerase was next added to each well, incubated at room temperature for 30&#x02009;min and fructose measured at 340&#x02009;nm. Ten &#x003bc;l of 83&#x02009;EU invertase solution was next added and incubated for 30&#x02009;min before measuring at 340&#x02009;nm for sucrose.</p></sec><sec disp-level="2"><title>Reproductive stage drought stress and GY analysis</title><p>For drought treatment at the reproductive stage, plants were grown in 500-ml pots in environmentally controlled growth chambers (14&#x02009;h light/10&#x02009;h dark cycles with light intensity 600&#x02009;&#x003bc;mol&#x02009;m^&#x02212;2&#x02009;s^&#x02212;1 and around 65% RH), fertilized regularly with a general-purpose fertilizer (N:P:K 20-20-20) and maintained well-watered conditions until the panicle-emergence stage<xref ref-type="bibr" rid="b60">60</xref>. Drought treatments were started at the pre-anthesis stage by withholding water for 4&#x02013;8 days followed by re-watering, and physiological state monitored by chlorophyll fluorescence and gas-exchange measurements. The plants that survived after re-watering and growth till maturity (&#x0003e;95%), and well-watered controls were maintained under well-watered condition, with minimal six plants per genotype per treatment for GY component analysis.</p><p>At maturity/stage R9 (ref. <xref ref-type="bibr" rid="b60">60</xref>), the panicle on the main culm was harvested, and spikelets with grains and unfilled spikelets were counted. The grains (caryopses) with hulls (palea and lemma attached) were threshed by hand and dried at 37&#x02009;&#x000b0;C for 7 days and subsequently weighed. The main culm was also harvested and dried at 70&#x02009;&#x000b0;C for 72&#x02009;h and weighed. The yield components assessed were NPs per plant, panicle length, NSP, number of filled grains per plant, spikelet fertility (number of spikelets with filled grains divided by the total number of spikelets), NGP, GY (weight of grain per plant) and average single-grain weight (GY divided by grain number). The harvest index was calculated as the ratio of total grain weight to total above-ground DW.</p></sec><sec disp-level="2"><title>High-temperature stress treatment</title><p>For high-temperature stress during the vegetative stage, 50-day-old plants were transferred into a controlled environmental growth chamber (Conviron Model PGW36, Winnipeg, Manitoba, Canada) set at day/night temperature of 34/24&#x02009;&#x000b0;C for 10 days, with light intensity 60&#x02009;cm above the canopy 600&#x02009;&#x003bc;mol&#x02009;m^&#x02212;2&#x02009;s^&#x02212;1 and RH of 65%. For reproductive stage temperature stress, plants at the early-booting stage were exposed to high day/night temperature of 36/26&#x02009;&#x000b0;C for 20 days. The panicles affected by high temperature were marked and used for analysis of yield components. At both vegetative and reproductive stage, a set of control plants were maintained in the greenhouse conditions with day/night temperature of 26/22&#x02009;&#x000b0;C, light intensity of 800&#x02009;&#x003bc;mol&#x02009;m^&#x02212;2&#x02009;s^&#x02212;1 and 65% RH. The day/night cycles for temperature and photoperiod were 10&#x02009;h day and 14&#x02009;h night in both growth chamber and greenhouse.</p></sec><sec disp-level="2"><title>Histochemical staining and microscopy</title><p>One-week-old hydroponically grown roots of HYR and WT plants were excised, fixed and stained with 0.5% (w/v) uranyl acetate at 4&#x02009;&#x000b0;C overnight as described<xref ref-type="bibr" rid="b5">5</xref>. Samples were dehydrated through an ethanol gradient, embedded in Spurr&#x02019;s medium and 1-&#x003bc;m ultrathin sections made with a diamond knife by an ultra-microtome (RMC MTXL). The stained sections were examined and photographed with a light microscope (Nikon Eclipse E600). For transmission electron microscopy, samples were embedded in LR White resin, thin sections (50&#x02009;nm) made with an ultra-microtome (RMC MTXL) and collected on nickel grids. The sections were stained with uranyl acetate and lead citrate and viewed with a JEM-1010 electron microscope (JEOL) operating at 60&#x02009;kV.</p></sec><sec disp-level="2"><title>Gene expression analysis</title><p>For Affymetrix GeneChip analysis, total RNA was isolated from leaf tissue of 1-month-old HYR lines (HYR-4, HYR-12 and HYR-16) along with WT plants plant tissue using the RNeasy plant kit (Qiagen, USA). The RNA quantity/quality was measured using the Agilent 2100 Bioanalyzer (Agilent Technolgies, USA), and 4&#x02009;&#x003bc;g of total RNA was used to generate first-strand complementary DNA with a T7-Oligo(dT) primer. Following second-strand synthesis, <italic>in vitro</italic> transcription was performed using the GeneChip IVT Labelling Kit. The preparation and processing of labelled and fragmented complementary RNA targets, as well as hybridization to rice Affymetrix GeneChips, washing, staining and scanning were carried out according to the manufacturer&#x02019;s instructions ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.affymetrix.com">http://www.affymetrix.com</ext-link>).</p><p>For qRT-PCR analysis three independent biological replicates were used with protocol following the comparative threshold cycle (Ct) method of quantitation with the actin or ubiquitin gene as reference<xref ref-type="bibr" rid="b61">61</xref>. For each sample, 2&#x02009;&#x003bc;g total DNAse-treated RNA was used with GoScript Reverse Transcription System (Promega), qRT-PCR experiments were carried out using GoTaq qPCR Master Mix (Promega) with ubiquitin as standard in a CFX-96 Bio-Rad thermocycler (Bio-Rad). Melting-curve analysis was done by applying increasing temperature from 55 to 95&#x02009;&#x000b0;C (0.5&#x02009;&#x000b0;C/10&#x02009;s), and gel electrophoresis of the final product confirmed single amplicons. Untranscribed RNA was also run as a negative control to check DNA contamination. The relative difference in expression for each sample in individual experiments was determined by normalizing the Ct value for each gene against the Ct value of ubiquitin or actin and was calculated relative to a calibrator using the equation 2^{&#x02212;&#x00394;&#x00394;Ct} (ref. <xref ref-type="bibr" rid="b62">62</xref>).</p></sec><sec disp-level="2"><title>Analysis of differential gene expression</title><p>Raw data from the HYR expression microarray experiment were background corrected, normalized and summarized according to the custom CDF<xref ref-type="bibr" rid="b46">46</xref> using RMA<xref ref-type="bibr" rid="b45">45</xref>, followed by nonspecific filtering of genes that do not have enough variation (interquartile range across samples&#x0003c;median interquartile range) to allow reliable detection of differential expression. A linear model was then used to detect differential expression of the remaining genes<xref ref-type="bibr" rid="b63">63</xref>. The <italic>P</italic> values from the moderated <italic>t</italic>-tests were converted to <italic>q</italic>-values to correct for multiple hypothesis testing<xref ref-type="bibr" rid="b64">64</xref>, and genes with <italic>q</italic>-value &#x0003c;0.01 were declared as differentially expressed in response to HYR expression (compared with WT).</p><p>Genes differentially expressed in response to HYR were tested for enrichment of specific GO BPs using the hypergeometric test. To adjust for multiple comparisons, a Benjamini&#x02013;Hochberg false discovery rate<xref ref-type="bibr" rid="b65">65</xref> (<italic>q</italic>-value) was calculated from the <italic>P</italic> values, and a <italic>q</italic>-value threshold of 0.01 was used for significance. The results from the enrichment analysis were visualized in the form of a gene set graph, with significantly overlapping gene sets connected by an edge and the network visualized using Cytoscape<xref ref-type="bibr" rid="b66">66</xref>.</p></sec><sec disp-level="2"><title>HYR ChIP and regulation of genes</title><p>HYR:TAP-tagged chromatin was used for ChIP assays<xref ref-type="bibr" rid="b67">67</xref>. Rice leaf samples (5&#x02009;g) were crosslinked in buffer containing formaldehyde (2%), nuclei isolated and the chromatin sheared by sonication into 200&#x02013;600-bp-sized fragments<xref ref-type="bibr" rid="b68">68</xref>. Sonicated chromatin was precleared with 100&#x02009;&#x003bc;l protein A agarose for 1.5&#x02009;h, and immunoprecipitated into three fractions: with anti-His (R932-25, Invitrogen, 1.5&#x02013;2&#x02009;&#x003bc;g) and anti-MYC (AHO0062, Invitrogen, 1.5&#x02013;2&#x02009;&#x003bc;g) for HYR-bound chromatin, and anti-Histone H3 (AHO1432, Invitrogen, 1.5&#x02013;2&#x02009;&#x003bc;g) conjugate as nonspecific chromatin. Chromatin-bound DNA fragments were eluted after reverse crosslinking by incubation overnight with proteinase K at 37&#x02009;&#x000b0;C, repeated again at 65&#x02009;&#x000b0;C overnight and DNA purified following the manufacturer&#x02019;s instructions. Real-time PCR was carried out in a CFX-96 thermal cycler (Bio-Rad) using the qPCR master mix (Promega) with the cycle 95&#x02009;&#x000b0;C for 3&#x02009;min, 40 cycles of 95&#x02009;&#x000b0;C for 15&#x02009;s and 59&#x02009;&#x000b0;C for 1&#x02009;min. Fold enrichment for DNA bound to chromatin isolated with the specific anti-His antibody to the nonspecific anti-Histone H3 was calculated using the formula: Ct (target)&#x02212;Ct (nonspecific Ab)=dCt (ref. <xref ref-type="bibr" rid="b27">27</xref>), normalized against the calibrator using the equation 2^{&#x02212;&#x00394;&#x00394;Ct} (ref. <xref ref-type="bibr" rid="b62">62</xref>). The ChIP experiments were performed with three biological replicates, qPCR assay done in triplicate with primers from putative HYR-regulated gene promoters for each ChIP assay (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>), of differentially expressed genes based on HYR microarray data and tested for significance using the <italic>t</italic>-test (<italic>P</italic>&#x02264;0.01).</p><p>To obtain purified HYR protein for electrophoretic mobility shift assay (EMSA) experiments, a full-length HYR fragment was amplified using primers containing attB1 and attB2 sites, respectively, and cloned into pDEST42 (C-6 &#x000d7; -His tag Gateway expression vector) (12276-010, Invitrogen) vector using the Gateway cloning strategy (BP and LR reaction system, Invitrogen) and transformed into <italic>Escherichia coli</italic> strain BL-21. The bacterial protein (6 &#x000d7; -His-tagged HYR) was induced with 1&#x02009;mM isopropyl-&#x003b2;-<sc>D</sc>-thiogalactoside and purified using Ni-NTA agarose (Invitrogen) following the manufacturer&#x02019;s instructions. Specific sets of primers (250&#x02009;bp) spanning the binding sites (GCC core) were used to amplify the promoter region of target genes of HYR genomic DNA of Nipponbare rice as template (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>). Gel-purified promoter fragments were labelled using the Biotin 3&#x02032; End DNA Labelling Kit (Pierce). The protein&#x02013;DNA-binding assay, according to the manufacturer&#x02019;s instructions (Light Shift Chemiluminescent EMSA kit), was carried out in binding buffer (10&#x02009;mM Tris&#x02013;HCl, pH 7.5, 50&#x02009;mM KCl, 1&#x02009;mM dithiothreitol), by incubation on ice for 10&#x02009;min after adding 100-fold excess of unlabelled competitor DNA (gel-purified promoter DNA fragments) followed by the addition of labelled DNA and further incubation on ice for 20&#x02009;min before loading on to a 5% native polyacrylamide gel. The resolved DNA&#x02013;protein complexes were electroblotted onto Nylon membrane (Biodyne) and gel shift was detected using Chemidoc (Bio-Rad) as described<xref ref-type="bibr" rid="b68">68</xref>.</p><p>For transactivation dual luciferase assays of HYR and other TFs, constructs with: (a) putative promoter targets amplified using primers (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>) and cloned upstream of the firefly luciferase, (b) a CaMV 35S Renilla luciferase construct control for transformation and (c) the coding regions of HYR or other TF genes (ARF1, WRKY72 and GASR2) cloned in pUC19 between the CaMV 35S promoter and NOS terminator, were co-transformed into rice protoplasts. Luminescence was measured by the Glomax Luminometer (Promega) and the relative luciferase activity calculated<xref ref-type="bibr" rid="b68">68</xref>. The data presented are means of three biological replicates and tested for significance using the <italic>t</italic>-test (<italic>P</italic>&#x02264;0.01).</p><p>To prove direct activation of promoters by a TF, an oestrogen-inducible expression system (HER)<xref ref-type="bibr" rid="b30">30</xref> was developed in rice, &#x02018;HER&#x02019; constructs of HYR and its target downstream genes (ARF1, GASR2 and WRKY72) were generated by ligating the PCR-amplified complementary DNAs (primers shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Table 5</xref>) at the <italic>Kpn</italic>1 sites fused with the regulatory regions of HER at the C terminus<xref ref-type="bibr" rid="b30">30</xref> between the CaMV 35S promoter and NOS terminator in pUC19. These constructs were used for analysis of direct transactivation of promoters in rice.</p></sec><sec disp-level="2"><title>Statistical analysis</title><p>The Student&#x02019;s <italic>t</italic>-test was used for statistical analysis of the data in the experiments of gas-exchange measurements, chlorophyll fluorescence, gravimetric WUE, biomass measurements, root length, soluble sugars and qRT-PCR. GY components were subjected to analysis of variance and the means were tested using Fisher&#x02019;s least significant difference test using statistical analysis software ( <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.sas.com">www.sas.com</ext-link>). In all the experiments <italic>t-</italic>tests performed were two sided, and quantitative differences between the two groups (for example, WT and HYR lines) of data for comparison were deemed statistically significant at <italic>P</italic>&#x02264;0.01 or <italic>P</italic>&#x02264;0.05 as indicated for each comparison in the figures and tables.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>M.M.R.A. performed the plant phenotypic, physiological, molecular and biochemical experiments. S.B. performed ChIP, protein-interaction and transactivation experiments. A.K. conducted the bioinformatics and statistical analysis on gene expression and network analysis. V.R. performed temperature stress and photosynthesis experiments. U.B. generated the rice transgenic plants and conducted plant phenotypic and physiological experiments. L.R. generated and analysed transgenic rice lines. N.B. supported the generation of transgenic lines and stress tests. A.P. designed and supervised the research, and together with M.M.R.A., A.K., V.R. and S.B. analysed data and wrote the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>Accession codes</bold>: Microarray data are deposited in the NCBI Gene Expression Omnibus (GEO) under accession number <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="NCBI:geo" xlink:href="GSE60936">GSE60936</ext-link>.</p><p><bold>How to cite this article:</bold> Ambavaram, M. M. R. <italic>et al.</italic> Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress. <italic>Nat. Commun.</italic> 5:5302 doi: 10.1038/ncomms6302 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-16, Supplementary Tables 1-5.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6302-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Data Set</title><p>GO Biological processes enrichment analysis of rice HYR lines</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6302-s2.xls"/></supplementary-material></sec>

Inductively guided circuits for ultracold dressed atoms

<p>Recent progress in optics, atomic physics and material science has paved the way to study quantum effects in ultracold atomic alkali gases confined to non-trivial geometries. Multiply connected traps for cold atoms can be prepared by combining inhomogeneous distributions of DC and radio-frequency electromagnetic fields with optical fields that require complex systems for frequency control and stabilization. Here we propose a flexible and robust scheme that creates closed quasi-one-dimensional guides for ultracold atoms through the &#x02018;dressing&#x02019; of hyperfine sublevels of the atomic ground state, where the dressing field is spatially modulated by inductive effects over a micro-engineered conducting loop. Remarkably, for commonly used atomic species (for example, ⁷Li and ⁸⁷Rb), the guide operation relies entirely on controlling static and low-frequency fields in the regimes of radio-frequency and microwave frequencies. This novel trapping scheme can be implemented with current technology for micro-fabrication and electronic control.</p>

<contrib contrib-type="author"><name><surname>Sinuco-Le&#x000f3;n</surname><given-names>German A.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Burrows</surname><given-names>Kathryn A.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Arnold</surname><given-names>Aidan S.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Garraway</surname><given-names>Barry M.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Physics and Astronomy, University of Sussex, Falmer</institution>, Brighton BN1 9QH, <country>UK</country></aff><aff id="a2"><label>2</label><institution>Department of Physics, SUPA, University of Strathclyde</institution>, Glasgow G4 0NG, <country>UK</country></aff>

Nature Communications

<p>Ultracold atomic gases of alkali atoms are suitable for exploring fundamental questions in physics and developing quantum technologies. Such a double utility stems from the possibility of varying the interatomic interaction and potential landscape, through electromagnetic fields that can be precisely produced. Advances in this area have lead to impressive experimental demonstrations of macroscopic quantum phenomena such as matter-wave interferometry<xref ref-type="bibr" rid="b1">1</xref> and persistent matter flux<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref>. This is possible because appropriately tuned radiation addresses the atomic level structure and affects the dynamics of both internal as well as motional degrees of freedom<xref ref-type="bibr" rid="b4">4</xref>. This approach has been exploited to create complex miniaturized potentials for the atomic motion, utilizing laser radiation that addresses optical dipole transitions. Thus, the features of the resulting potential landscape vary with a length-scale limited by optical diffractive effects, being of the same order of magnitude as the laser wavelength and corresponding to a few hundred nanometres for alkali atoms.</p><p>In contrast, atom chips can create microscopic trapping structures utilizing long wavelength radiation<xref ref-type="bibr" rid="b5">5</xref>. In this case, the atomic potential landscape is tailored on a micron scale using electromagnetic fields radiating from micron-sized conductors. Developments in this area have made it possible to address atomic energy levels separated by low-frequency photons (radio-frequency and microwave) where decoherence effects are substantially reduced in comparison with optical transitions. These ideas were central for achieving radio-frequency-assisted coherent splitting of Bose-Einstein condensates for the first time in ref. <xref ref-type="bibr" rid="b1">1</xref>, and, more recently, for beating the standard quantum limit with a scanning probe atom interferometer<xref ref-type="bibr" rid="b6">6</xref>. Micron and sub-micron control over atomic gases is at the heart of promising technological applications in metrology<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>, quantum information technology<xref ref-type="bibr" rid="b8">8</xref> and quantum simulation<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, and atom chips are platforms with great potential for experimental realization of many of such proposals.</p><p>Microscopic ring traps (and toroidal traps) are of particular interest because of the possibility they offer to study physical phenomena in a non-trivial geometry with true periodic boundary conditions and to create atomic analogues of solid state electronic devices (for example refs <xref ref-type="bibr" rid="b7">7</xref>, <xref ref-type="bibr" rid="b11">11</xref>). Trapping of cold gases in such geometries has been demonstrated with a variety of experimental techniques, requiring control over optical fields<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref> or distributions of magnetic fields<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. There are several proposals for creating ring traps that rely solely on the field produced by DC current carrying conductors, suitable to be implemented with atom-chip technology (for example, refs <xref ref-type="bibr" rid="b17">17</xref>, <xref ref-type="bibr" rid="b19">19</xref>), but having the downside that feed wires can break desirable symmetries. Such an effect can be mitigated by employing low-frequency inductive coupling<xref ref-type="bibr" rid="b20">20</xref>, where the atoms are confined by time-averaged magnetic potentials. This idea has been experimentally demonstrated in millimetre-sized ring traps<xref ref-type="bibr" rid="b18">18</xref> and proposed to produce microscopic ring traps based on generalizing the radio-frequency dressing approach<xref ref-type="bibr" rid="b21">21</xref> to an inductive system<xref ref-type="bibr" rid="b22">22</xref>.</p><p>In this work, we show that highly configurable quasi-one-dimensional microscopic guides for ultra-cold alkali atoms result from the response of an inductive loop to AC magnetic fields tuned near the atomic ground state <italic>hyperfine</italic> splitting. We find various advantages of this approach over optical schemes and dressing of Zeeman split levels (that is, coupling states within the same hyperfine manifold)<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b22">22</xref>. For example, the present proposal does not require sophisticated optical control and it is free from potential symmetry breaking current carrying wires in the vicinity of the trapping volume<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>. In addition, our system can be designed to create multiply connected atomic circuits, for example, arrays of connected ring traps, having in mind applications that benefit from matter-wave interferometry as in ref. <xref ref-type="bibr" rid="b7">7</xref>. Importantly, in this work we also demonstrate the experimental feasibility of our scheme using current atom-chip technology<xref ref-type="bibr" rid="b5">5</xref>.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Device concept and trapping mechanism</title><p>A sketch of the physical set-up is shown in <xref ref-type="fig" rid="f1">Fig. 1</xref>. It comprises a micro-engineered conducting loop (metallic or superconducting), a static magnetic field <inline-formula id="d33e194"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e195" xlink:href="ncomms6289-m1.jpg"/></inline-formula> (single-headed arrow), and a homogeneous AC magnetic field <inline-formula id="d33e197"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e198" xlink:href="ncomms6289-m2.jpg"/></inline-formula> (double-headed arrows). Both fields are transverse to the plane defined by the loop (<italic>x</italic>&#x02212;<italic>y</italic> in <xref ref-type="fig" rid="f1">Fig. 1</xref>). In response to the electro-motive force induced by the time variation of the magnetic flux across the area enclosed by the loop, an electric current circulates within it. The induced current produces, in its turn, an inhomogeneous magnetic field of the form <bold>B</bold>_ind(<bold>r</bold>) cos(<italic>&#x003c9;t</italic>+<italic>&#x003b4;</italic>), that modifies the total AC magnetic field. For sufficiently large frequencies that the inductive reactance of the loop dominates its Ohmic resistance, the external and induced fields are almost in anti-phase. Thus, the resulting field has an approximately quadrupole distribution, schematically shown in <xref ref-type="fig" rid="f2">Fig. 2a</xref>, whose centre is located close to the conducting loop at the position where the amplitude of induced and external fields satisfy <italic>B</italic>_ind=<italic>B</italic>_AC cos(<italic>&#x003b4;</italic>) (ref. <xref ref-type="bibr" rid="b20">20</xref>), where <italic>&#x003b4;</italic>+<italic>&#x003c0;</italic> is the relative phase between external and induced fields.</p><p>By tuning the driving angular frequency <italic>&#x003c9;</italic> near the atomic ground state hyperfine transition, the AC magnetic field couples hyperfine Zeeman split sub-levels as depicted in <xref ref-type="fig" rid="f2">Fig. 2b,c</xref>. The induced energy shifts lead to state-dependent potential energy landscapes for the atomic centre-of-mass motion, which have been used for weakly trapping neutral alkali atoms<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref> and coherent manipulation of Bose-Einstein condensates<xref ref-type="bibr" rid="b29">29</xref>. The electric field associated with the oscillating magnetic field can be ignored, as the time-averaged quadratic Stark shift is proportional to the atomic DC polarizability of the ground state and thus independent of the quantum numbers <italic>F</italic> and <italic>m</italic>_F (ref. <xref ref-type="bibr" rid="b30">30</xref>). The energy shifts are conveniently described in terms of the field components in spherical unit vectors <inline-formula id="d33e275"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e276" xlink:href="ncomms6289-m3.jpg"/></inline-formula>, and corresponding Rabi frequencies <inline-formula id="d33e278"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e279" xlink:href="ncomms6289-m4.jpg"/></inline-formula> with <italic>&#x02113;</italic>=&#x02212;1,0,1 and <italic>g</italic>_{<italic>J</italic>} the Land&#x000e9; factor of the electronic angular momentum <italic>J</italic>. After the rotating-wave approximation and utilizing second order perturbation theory, near the quadrupole centre the energy shifts are given by<xref ref-type="bibr" rid="b30">30</xref></p><p><disp-formula id="eq5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e297" xlink:href="ncomms6289-m5.jpg"/></disp-formula></p><p>with <italic>&#x0210f;</italic>&#x00394;_Zeeman=<italic>&#x003bc;</italic>_B<italic>g</italic>_F<italic>B</italic>_DC and the detuning</p><p><disp-formula id="eq6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e321" xlink:href="ncomms6289-m6.jpg"/></disp-formula></p><p>where the zero field hyperfine splitting of the ground state is 2<italic>A</italic>, and <italic>g</italic>_F the hyperfine Land&#x000e9; factor. The &#x000b1; sign in <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> corresponds to the states of the hyperfine manifolds <italic>F</italic>=<italic>I</italic>&#x000b1;1/2. Dynamical control over the potential landscape can be exerted via the amplitude of the applied fields and the detuning. With respect to these parameters, trapping characteristics scale in a similar manner to those of optical dipole traps<xref ref-type="bibr" rid="b14">14</xref>. First, the trap depth, defined as the difference between the energy at the trap centre (<bold>r</bold>₀:=(<italic>x</italic>₀,0,0)) and at the geometrical centre of the conducting ring (<bold>r</bold>:=(0,0,0)), is proportional to the power of the applied AC field and the inverse of the detuning. Second, the trap frequency scales in proportion to the amplitude of the field and the inverse of the square root of the detuning. Notice also that as a consequence of the linear relation of induced current with both the amplitude <italic>B</italic>_AC and angular frequency <italic>&#x003c9;</italic> of the applied field, the location of the trap centre (<italic>x</italic>₀) is extremely robust to noise on both parameters. In addition, as shown below, for typical working parameters the ring trap is sufficiently far from the edge of the conducting ring that proximity effects can be neglected.</p><p>For illustrative purposes, we present calculations for the hyperfine level structure of ⁸⁷Rb, denoted by |<italic>F</italic>, <italic>m</italic>_F&#x0203a;, and shown in <xref ref-type="fig" rid="f2">Fig. 2b,c</xref>. Nevertheless, our conclusions are straightforwardly extended to other atomic species with similar energy level structure. To give an explicit example of the potential landscape emerging from <xref ref-type="disp-formula" rid="eq5">equation (1)</xref>, we consider a circular loop of gold with radius <italic>a</italic>=100&#x02009;&#x003bc;m and diameter <italic>s</italic>=10&#x02009;&#x003bc;m, corresponding to approximate resistance <italic>R</italic> &#x02248;0.26&#x02009;&#x003a9; and inductance <italic>L</italic> &#x02248;0.33&#x02009;nH (ref. <xref ref-type="bibr" rid="b31">31</xref>). In this case, the total field distribution produces a circular trapping region with typical landscapes as shown in <xref ref-type="fig" rid="f3">Fig. 3a&#x02013;d</xref>, for states |<italic>F</italic>=2, <italic>m</italic>_F=1&#x0203a; and |<italic>F</italic>=1, <italic>m</italic>_F=&#x02212;1&#x0203a; of ⁸⁷Rb, and applied fields of <italic>B</italic>_DC=1&#x02009;G and <italic>B</italic>_AC=2&#x02009;G. Notice that in this example the centre of the quadrupole distribution is located at <italic>x</italic>₀ &#x02248;70&#x02009;&#x003bc;m, corresponding to a distance of &#x02248;20&#x02009;&#x003bc;m from the edge of the conducting ring.</p><p>The quadrupole AC field distribution produces harmonic confinement, as the linear dependence of the field amplitude with the distance to the quadrupole centre translates into a quadratic variation of the energy shift in <xref ref-type="disp-formula" rid="eq5">equation (1)</xref>. The tightness of the trap, quantified by the spatial curvature of <inline-formula id="d33e453"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e454" xlink:href="ncomms6289-m7.jpg"/></inline-formula> along the <inline-formula id="d33e456"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e457" xlink:href="ncomms6289-m8.jpg"/></inline-formula> and <inline-formula id="d33e459"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e460" xlink:href="ncomms6289-m9.jpg"/></inline-formula> directions, is shown in <xref ref-type="fig" rid="f3">Fig. 3e,f</xref> as a function of the detuning of the driving field (see <xref ref-type="disp-formula" rid="eq6">equation (2)</xref>). According to <xref ref-type="disp-formula" rid="eq5">equation (1)</xref>, the trapping tightness increases arbitrarily by reducing the detuning with respect to pairs of transitions, resulting in the divergent behaviour in <xref ref-type="fig" rid="f3">Fig. 3e,f</xref> (vertical dashed lines) at integer multiples of <inline-formula id="d33e475"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e476" xlink:href="ncomms6289-m10.jpg"/></inline-formula> for <italic>B</italic>_DC=1&#x02009;G.</p><p>This trapping scheme provides confinement of two hyperfine states in overlapping regions, which can be useful for applications in quantum information processing and metrology<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b8">8</xref>. In our example of <xref ref-type="fig" rid="f3">Fig. 3e,f</xref>, detuning in the range &#x00394;₀/2<italic>&#x003c0;</italic>&#x003b5;[&#x02212;0.5, 0.5]MHz produces energy-shift landscapes for states |<italic>F</italic>=2, <italic>m</italic>_F=1&#x0203a; and |<italic>F</italic>=1, <italic>m</italic>_F=&#x02212;1&#x0203a; with approximately equal curvatures for both states. These states experience exactly the same potential landscape for a driving field resonant to the hyperfine splitting, that is, with &#x00394;₀=0. Note that the static magnetic field makes this resonant driving blue (red) detuned with respect to coupling of states with <italic>m</italic>_F=&#x02212;1 (<italic>m</italic>_F=1), as schematically shown by the solid arrows in <xref ref-type="fig" rid="f2">Fig. 2b</xref> (<xref ref-type="fig" rid="f2">Fig. 2c</xref>).</p><p>The detuning of the driving field also provides control over the shape of the trapping cross section, as seen in the potential landscapes in <xref ref-type="fig" rid="f3">Fig. 3a&#x02013;d</xref>. This is because the relative weights of the terms in <xref ref-type="disp-formula" rid="eq5">equation (1)</xref> can be adjusted by changing the offset field and the driving frequency that determine <inline-formula id="d33e541"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e542" xlink:href="ncomms6289-m11.jpg"/></inline-formula>.</p></sec><sec disp-level="2"><title>Connected double loop trap</title><p>So far we have focused on the trapping produced by a circular conductor. However, our scheme offers the possibility of creating complex atomic guides shaped by the conducting loop. We illustrate this by considering a demanding case where we impose a severe &#x02018;pinch&#x02019; in the shape of the conducting loop, as depicted in <xref ref-type="fig" rid="f4">Fig. 4</xref>, creating a double loop with a variety of junction geometries. The field distribution corresponding to this case can be understood as follows: away from the pinch centre, the field distribution is similar to the quadrupole field in <xref ref-type="fig" rid="f1">Fig. 1c</xref>, while in its neighbourhood the total field results from combining two quadrupole-like distributions associated with conducting segments at each side of the constriction. In particular, when the induced field balances the applied one at the centre of the pinch, the field distribution acquires a hexapolar character. The geometry of the resulting potential landscape is sensitive to the shape of the conductor, while its energy scale is determined by the amplitude and detuning of the applied fields. This is illustrated in <xref ref-type="fig" rid="f4">Fig. 4b,g</xref>, where field distributions and energy landscapes have been obtained for three different constrictions with sizes differing by &#x02248;1&#x02009;&#x003bc;m, producing significantly different junction geometries. Consideration of this case can be straightforwardly applied to more complex geometries of the conductor, which can be used to create more involved atomic guides.</p></sec><sec disp-level="2"><title>Finite size effects</title><p>Modelling the loop as a single current filament is insufficient to describe the potential landscape associated with conductors whose cross sectional radius is comparable to the loop length<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b32">32</xref>. In such a case, the induced current distributes itself unevenly across the conductor and produces a magnetic field that differs significantly from the one produced by a single filament, having direct impact on the quality of the trapping potential (see <xref ref-type="supplementary-material" rid="S1">Supplementary Methods</xref> for details about calculation of the current distribution within metallic and superconducting loops<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>). An illustration of these effects is shown in <xref ref-type="fig" rid="f5">Fig. 5</xref>, where we consider circular loops with square and circular cross sections made of two different conducting materials commonly used in atom-chip experiments: gold (Au) and superconducting niobium (Nb) (ref. <xref ref-type="bibr" rid="b32">32</xref>).</p><p>In the case of a normal conductor, the combination of small skin depth at high frequency with a radially dependent magnetic flux pushes the induced current towards the outer edge of the conductor, spreading the current along its surface (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). For superconducting loops, the Meissner effect, here described by the London equations<xref ref-type="bibr" rid="b33">33</xref>, contributes to a more marked confinement of the current close to the conductor surface (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). In both cases, as a consequence of distributing the current over a wide area, the gradient of the magnetic field is reduced in comparison with the single-filament case. In terms of the atomic potential landscape, this translates to modifying the trapping position (that is, the centre of the quadrupole field distribution) and reducing its tightness (here quantified through the trap frequency along the <italic>x</italic> direction, <italic>&#x003bd;</italic>_{<italic>x</italic>}). Our numerical results indicate that both position and trap frequency, although dependent on the conducting material and cross sectional shape, do not vary strongly with these parameters. In both cases, the most relevant parameter is the thickness of the conductor, favouring the use of thin conductors to produce strong trapping potentials.</p></sec><sec disp-level="2"><title>Experimental considerations</title><p>The design of atom-chips including current carrying elements is limited by several technical issues that restrict the range of experimentally accessible parameters<xref ref-type="bibr" rid="b5">5</xref>. In the present case, for example, the goal of obtaining the tightest possible trap, via small detuning or large driving fields, should be balanced against an increase in heating of the conductors and atom-loss rates. In what follows, we briefly consider these two problems.</p><p>Ohmic loses due to the induced current must be restricted to avoid thermal destruction of the conductive loop, or undesirable alteration of the trapping track due to thermal deformations of the conductor. For typical experimental parameters, such as those in <xref ref-type="fig" rid="f3">Fig. 3</xref>, the average current densities (see <xref ref-type="fig" rid="f5">Fig. 5b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1,2</xref>) are significantly lower than the maximal tolerable values demonstrated in experiments with normal and superconducting materials operating under DC and high-frequency conditions (&#x02248;10⁶ A&#x02009;cm^&#x02212;2)<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>, suggesting that the heat generated in our proposed trapping set-up can be efficiently transferred to the supportive structures of the device. Also, although our numerical results for heating power favours using thick conductors, this should be balanced against the higher trapping frequency and better thermal coupling achievable with thin wires, which can support large current densities and are also convenient for fabrication<xref ref-type="bibr" rid="b5">5</xref>.</p><p>We estimate non-adiabatic atom losses in our trapping set-up by considering an atom moving at speed <italic>u</italic> in the plane defined by the conducting loop. After the rotating-wave approximation, the atom-field interaction is described by the two-level Hamiltonian<xref ref-type="bibr" rid="b8">8</xref>:</p><p><disp-formula id="eq12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e646" xlink:href="ncomms6289-m12.jpg"/></disp-formula></p><p>where <italic>&#x003c3;</italic>_{<italic>i</italic>} with <italic>i</italic>=<italic>x</italic>, <italic>y</italic>, <italic>z</italic> are Pauli matrices, and the spatially dependent phase <italic>&#x003d5;</italic> and Rabi frequency &#x003a9;₀, are defined by the combination of the applied and induced fields. Atom-loss processes are modelled as transitions between the position-dependent eigenvectors of Hamiltonian <xref ref-type="disp-formula" rid="eq12">equation (3)</xref>, denoted by {|1&#x0203a;,|2&#x0203a;} in the present treatment<xref ref-type="bibr" rid="b4">4</xref>. Such dressed states consist of linear combinations of hyperfine states with the same projection of angular momentum <italic>m</italic>_F that depends on the amplitude of the magnetic field. For example, at the centre of the quadrupole field distribution, where the field is null, the dressed states |1&#x0203a;,|2&#x0203a; coincide with the hyperfine states |<italic>F</italic>, <italic>m</italic>_F&#x0203a;,|<italic>F</italic>&#x02212;1, <italic>m</italic>_F&#x0203a;, while very far from the zero they are equal superposition of these two states. In the trapping geometry produced by a circular loop of inductance <italic>L</italic> and radius <italic>a</italic>, the rate of transitions between pairs of dressed states is approximately<xref ref-type="bibr" rid="b4">4</xref>:</p><p><disp-formula id="eq13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e712" xlink:href="ncomms6289-m13.jpg"/></disp-formula></p><p>Under typical experimental conditions, for example, an atom moving with speed <italic>u</italic>&#x02248;10&#x02009;mm&#x02009;s^&#x02212;1 (corresponding to a temperature of 1&#x02009;&#x003bc;K), and for the trap configuration presented in <xref ref-type="fig" rid="f3">Fig. 3</xref>, <xref ref-type="disp-formula" rid="eq13">equation (4)</xref> predicts non-adiabatic transitions with a rate of ~10^&#x02212;5 Hz, allowing enough time for manipulation of the trapped atoms.</p><p>Feeding the external field into the conducting loop presents another potential challenge. However, in the case of ⁶Li and light atoms, the driving frequency falls in the MHz range, where several well-known techniques can easily be employed<xref ref-type="bibr" rid="b5">5</xref>. For the case of Rb and Cs isotopes, the driving field should have a frequency in the GHz range, for which near-surface fields of a microwave source could be suitable<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>. For example, a sufficiently uniform field of 2&#x02009;G at a frequency of 6.8&#x02009;GHz can be generated by a feeding structure consisting of a single-turn circular conductor of radius 1.7&#x02009;mm, positioned concentrically on the same chip as the inductive loop. The dimensions of this feeding conductor are an order of magnitude larger than the inductive loop, and it is sufficiently far away that a small break for connecting a coaxial signal will not cause significant end effects near the smaller loop. Moreover, the amplitude of the produced field is uniform over the smaller loop at the &#x0003c;1% level (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). The inductance of the feeding loop is ~6&#x02009;nH, with a resistance ~0.2&#x02009;&#x003a9;, due to the 1&#x02009;&#x003bc;m skin depth at this frequency. The inductive impedance of the feeding loop (~250&#x02009;&#x003a9;) can be cancelled at resonant microwave frequencies by a small capacitor of ~0.1&#x02009;pF. In addition, a resistor in series with the feeding loop provides control of the resonance full-width-half-maximum, which naturally is ~7&#x02009;MHz. The feeding loop needs to carry a current of ~0.5 A to generate a 2&#x02009;G field at its geometrical centre, which is achievable using a transformer to drive the low impedance load via a (high impedance) commercial microwave source and amplifier<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>.</p></sec><sec disp-level="2"><title>Possibilities for loading the induced trapping loop</title><p>Atoms can be transferred to the induced trapping loop using an auxiliary near-surface magnetic trap, as commonly done in other micro-trapping geometries<xref ref-type="bibr" rid="b5">5</xref>. Following a slow variation of the trapping parameters, the potential landscape is deformed and atoms can be transported between regions separated by several hundreds of microns. For example, a typical magnetic microtrap, created by a set of conductors carrying DC currents and a uniform static field, can initially hold the atoms above the centre of the trapping loop. Subsequent modification of the current and applied fields deforms the trap and places the atoms in a region of significant influence of the induced ring trap. This procedure is schematically shown in <xref ref-type="fig" rid="f6">Fig. 6</xref>, where we plot contours of the potential energy produced by the combination of a two-wire magnetic trap (see figure caption) with the energy shift caused by the AC field distribution. The figure shows four snapshots of a sequence that transport atoms from a position above the centre of the ring to the plane of the centre of the induced trapping region. The sequence starts with a magnetic trap centred above the ring, (<bold>r</bold>₀=(0,0,<italic>z</italic>₀)) (panel a), then, by adjusting the currents, the amplitude of applied fields and the driving frequency, the trap centre moves towards the plane of the ring, (<bold>r</bold>₁=(0,0,0)) (panel b). The loading scheme then proceeds to slowly increase the field producing the induced ring trap and simultaneously reduce the strength of the magnetic trap (panels c and d). To reduce atom losses during the loading process, all parameters defining the trapping potential should be selected in such a way that the trap depth and frequencies are approximately constant through the procedure. A similar sequence can be planned by replacing the initial magnetic trap with a mirror-MOT (as in ref. <xref ref-type="bibr" rid="b37">37</xref>), having the advantage of providing a uniform distribution of atoms along the trapping loop.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In summary, we show that complex one-dimensional guides for ultracold matter can be defined by inductive effects over metallic and superconducting loops. A very flexible wave-guide shape is possible as the guide simply follows the curves of a metal track laid down as a loop on the surface of an atom-chip or carved into it. For operation, the loop should receive a magnetic field that oscillates near to resonance with the hyperfine splitting of the atomic ground state of the atoms. The field induces an electric current on the conducting track without the need of leading wires that might introduce undesired asymmetries in the potential landscape. The combined applied and induced fields form a trapping structure for the atoms.</p><p>Our numerical investigations indicate that the experimental realization of this type of trap is realistic with current technology, predicting trapping frequencies varying from a few hundred Hz to a few kHz. This allows applications such as exploring low-dimensional looped traps for Bose-Einstein condensates, novel quantum interference devices and multiply looped structures for cold atom gyroscopes. Interestingly, our scheme can produce overlapping trapping regions for two different hyperfine states, which might be of practical interest for experiments with atomic species where a low magnetic field Feshbach resonance is available, such as in ⁶Li.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Current distribution in circular conductors</title><p>We evaluate the current distribution in conducting loops with circular and square cross sections. The trapping configuration works in the regime of long-wavelength compared with the size of the loop, which allows us to use quasi-static Maxwell equations coupled to constitutive relations between the fields and the current (for example, Ohm&#x02019;s law and London equations)<xref ref-type="bibr" rid="b31">31</xref>. We use the open-source package FEMM<xref ref-type="bibr" rid="b38">38</xref>, which implements a Finite-Element algorithm for magnetic problems, to calculate the current distribution in metallic conductors. The corresponding total field distribution is then employed to evaluate the trapping frequency as a function of the conductor width in <xref ref-type="fig" rid="f5">Fig. 5</xref>.</p><p>We also consider superconducting loops of Niobium and calculate its current distribution, <bold>J</bold>(<bold>r</bold>). To do so, we adapted the procedure detailed in ref. <xref ref-type="bibr" rid="b39">39</xref> as follows: First, as the frequency of the applied magnetic field is much smaller than the superconducting gap, the current can be described by the London equation<xref ref-type="bibr" rid="b33">33</xref>:</p><p><disp-formula id="eq14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e816" xlink:href="ncomms6289-m14.jpg"/></disp-formula></p><p>where <italic>m</italic> and <italic>e</italic> are the electron mass and charge, respectively, <italic>n</italic>_s is the density of superconducting electrons and the vector potential <bold>A</bold> satisfies the Coulomb gauge &#x02207;&#x000b7;<bold>A</bold>=0. Second, the solution to quasi-static Maxwell equations for the magnetic field provides us a second relation between the vector potential and the current distribution:</p><p><disp-formula id="eq15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e838" xlink:href="ncomms6289-m15.jpg"/></disp-formula></p><p>where <bold>A</bold>_AC is the vector potential corresponding to the applied field and the integral is limited to the volume where the current is defined<xref ref-type="bibr" rid="b39">39</xref>.</p><p>Then, combining these two equations and considering circular loops with homogeneous cross section, we obtain a relation between the applied flux of magnetic field across sections of loops with radius <italic>&#x003c1;</italic> and the current distribution:</p><p><disp-formula id="eq16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e855" xlink:href="ncomms6289-m16.jpg"/></disp-formula></p><p>in which we have used cylindrical coordinates. In <xref ref-type="disp-formula" rid="eq16">equation (7)</xref> <italic>B</italic>_AC is the amplitude of the oscillating applied field, <italic>J</italic>(<italic>&#x003c1;</italic>&#x02032;, <italic>z</italic>&#x02032;) is the current density at points (<italic>&#x003c1;</italic>&#x02032;, <italic>z</italic>&#x02032;) within the superconductor, <inline-formula id="d33e882"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e883" xlink:href="ncomms6289-m17.jpg"/></inline-formula> denotes a unitary vector along the azimuthal direction and the integral is restricted to the volume of the superconducting loop. After discretizing the superconductor cross section, we obtain an algebraic problem relating the currents passing through finite cross sections of the conductor and the applied flux. Finally, this set of equations is solved with a standard computing package for linear algebra (LAPACK). A fully detailed description of this procedure is shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Methods</xref>.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>B.M.G. and A.S.A. conceived the trapping scheme discussed. G.A.S.-L. and K.A.B. performed theoretical and numerical analysis. G.A.S.-L. and B.M.G. wrote the manuscript. All authors discussed the results and implications, and commented on the manuscript at all stages.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Sinuco-Leon, G. A. <italic>et al</italic>. Inductively guided circuits for ultracold dressed atoms. <italic>Nat. Commun.</italic> 5:5289 doi: 10.1038/ncomms6289 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-4, Supplementary Methods and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6289-s1.pdf"/></supplementary-material></sec>

Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients

<p>Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-&#x003b3;-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans.</p>

<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Tae Jin</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Jin</surname><given-names>Hyun-Tak</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Hur</surname><given-names>Soo-Young</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Hyun Gul</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Seo</surname><given-names>Yong Bok</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Hong</surname><given-names>Sung Ran</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Chang-Woo</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Suhyeon</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Woo</surname><given-names>Jung-Won</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Ki Seok</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Hwang</surname><given-names>Youn-Young</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Jaehan</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>In-Ho</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lim</surname><given-names>Kyung-Taek</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Ki-Heon</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jeong</surname><given-names>Mi Seon</given-names></name><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Surh</surname><given-names>Charles D.</given-names></name><xref ref-type="aff" rid="a4">4</xref><xref ref-type="aff" rid="a8">8</xref></contrib><contrib contrib-type="author"><name><surname>Suh</surname><given-names>You Suk</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Jong Sup</given-names></name><xref ref-type="corresp" rid="c3">c</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Sung</surname><given-names>Young Chul</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a4">4</xref></contrib><aff id="a1"><label>1</label><institution>Department of Obstetrics and Gynecology, Cheil General Hospital and Women&#x02019;s Healthcare Center, College of Medicine, Kwandong University</institution>, Seoul 100-380, <country>Korea</country></aff><aff id="a2"><label>2</label><institution>Research Institute, Genexine Inc., Korea Bio Park</institution>, Seongnam, Gyeonggi-do 463-400, <country>Korea</country></aff><aff id="a3"><label>3</label><institution>Department of Obstetrics and Gynecology, Seoul St Mary&#x02019;s Hospital, College of Medicine, The Catholic University of Korea</institution>, Seoul 137-701, <country>Korea</country></aff><aff id="a4"><label>4</label><institution>Division of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology</institution>, Pohang 790-784, <country>Korea</country></aff><aff id="a5"><label>5</label><institution>Department of Pathology, Cheil General Hospital and Women&#x02019;s Healthcare Center, College of Medicine, Kwandong University</institution>, Seoul 100-380, <country>Korea</country></aff><aff id="a6"><label>6</label><institution>Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine</institution>, Suwon, Gyeonggi-do 440-746, <country>Korea</country></aff><aff id="a7"><label>7</label><institution>Laboratory of R&#x00026;D for Genomics, Cheil General Hospital and Women&#x02019;s Healthcare Center, College of Medicine, Kwandong University</institution>, Seoul 100-380, <country>Korea</country></aff><aff id="a8"><label>8</label><institution>Academy of Immunology and Microbiology, Institute of Basic Science</institution>, Pohang 790-784, <country>Korea</country></aff>

Nature Communications

<p>Cervical cancer is one of the leading causes of cancer death in women worldwide<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>, and about 75% of its cases are caused by persistent infection with the most common high-risk human papilloma virus (HPV) types, namely HPV16 and HPV18 (refs <xref ref-type="bibr" rid="b3">3</xref>, <xref ref-type="bibr" rid="b4">4</xref>). HPV persistence is usually associated with the lack of demonstrable HPV-specific T-cell immunity, and the virus-specific T cells found in pre-malignant and malignant patients are reported to be generally dysfunctional and sometimes even suppressive<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>. These findings suggest that the functional impairment of virus-specific T cells is a contributing factor for the emergence of HPV-induced cervical cancer.</p><p>The pre-malignant cervical intraepithelial neoplasia 2 and 3 (CIN2/3), in particular those positive for HPV16, are considered as high-grade lesions that have approximately a 30% chance of developing into invasive cancer<xref ref-type="bibr" rid="b7">7</xref>. Current treatment for CIN2/3 is limited to surgical excision, which is associated with about 10% recurrence rate and pregnancy-related complications, such as preterm delivery, low birth weight and premature rupture of membrane<xref ref-type="bibr" rid="b8">8</xref>. Recently introduced prophylactic HPV vaccines (Gardasil and Cervarix) have been shown to be effective in preventing HPV infection<xref ref-type="bibr" rid="b9">9</xref>, but without any therapeutic efficacy against pre-existing HPV infection or pre-malignant lesions. Therefore, there is an urgent need for an effective therapeutic vaccine that can eradicate HPV-related neoplasia without surgical manipulation.</p><p>HPV E6 and E7 act as viral oncoproteins by binding and promoting degradation of tumour suppressor proteins, p53 and retinoblastoma (pRb), respectively<xref ref-type="bibr" rid="b10">10</xref>. These viral oncoproteins are an ideal set of targets for a therapeutic vaccine against CIN2/3 and cervical cancer because these proteins not only induce tumorigenesis but also are constitutively expressed in HPV-infected pre-malignant and malignant cells<xref ref-type="bibr" rid="b10">10</xref>. Since the regression of cervical lesions is associated with the presence of a cellular, but not humoral immune response<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>, a therapeutic vaccine capable of selectively inducing robust E6/E7-specific T-cell immunity is highly desirable. Several attempts at this feat are currently underway with a varying level of success. Subcutaneous immunization with a recombinant vaccinia virus-expressing E6/E7 with interleukin-2 (IL-2) induced the regression of cervical lesions and cleared HPV infection in 10 out of 21 CIN2/3 patients<xref ref-type="bibr" rid="b13">13</xref>. Intrauterus immunization with another recombinant vaccinia virus-expressing bovine papilloma virus E2 elicited the regression of high-grade lesions in 19 out of 34 patients<xref ref-type="bibr" rid="b14">14</xref>. Unfortunately, none of these clinical studies presented whether the vaccine could induce a relevant T-cell immunity and elucidated the mechanistic explanation behind the observed therapeutic effect.</p><p>Induction of an HPV-specific T-cell response was observed with other vaccines, but without any meaningful clinical efficacy for the treatment of high-grade cervical lesions. One study found that subcutaneous immunization with an HPV16 E6/E7 synthetic long-peptide vaccine induced detectable level of HPV-specific interferon-&#x003b3; (IFN-&#x003b3;)-producing T-cell response in all five patients with high-grade cervical dysplasia, but without any reduction in HPV DNA and histological improvement of the cervical lesions<xref ref-type="bibr" rid="b15">15</xref>. This study resembles the past finding with intramuscular injections of an E6/E7 fusion protein mixed together with ISCOMATRIX adjuvant, which induced IFN-&#x003b3; enzyme-linked immunospot (ELISPOT) responses in five out of 15 patients, but without any regression of the cervical lesions<xref ref-type="bibr" rid="b16">16</xref>. In clinical trials with DNA vaccines, intramuscular administration of an E7 DNA vaccine co-expressing HSP70 as a genetic adjuvant revealed measurable T-cell response in eight out of 15 patients with CIN2/3, although a histological regression was shown only in three out of nine patients<xref ref-type="bibr" rid="b17">17</xref>. In a recent study, E6/E7 DNA vaccine delivered via electroporation (EP) induced a significant HPV-specific IFN-&#x003b3;-producing T-cell response in 14 out of 18 subjects, but the clinical response could not be examined because the enroled subjects had their cervical lesions surgically removed before vaccination<xref ref-type="bibr" rid="b18">18</xref>. In case of HPV-associated lower genital epidermal lesions, vaccination with a synthetic long-peptide vaccine induced T-cell response in all subjects and exhibited 47% of complete response rate in patients with vulvar intraepithelial neoplasia 3 (ref. <xref ref-type="bibr" rid="b19">19</xref>). In essence, a highly effective therapeutic HPV vaccine that could induce more potent anti-HPV T-cell response and complete regression of high-grade lesions is yet to be clearly demonstrated in HPV-associated cervical neoplasia.</p><p>Here, we describe a newly developed HPV E6/E7 DNA therapeutic vaccine, designated GX-188, designed to facilitate the processing and presentation of HPV E6/E7 antigens by dendritic cells (DCs) through the co-expression of Fms-like tyrosine kinase-3 ligand (Flt3L). Vaccination with GX-188 by EP (GX-188E) elicits a significant E6/E7-specific T helper (Th) 1-polarized cellular immune response in all nine patients with CIN3. Importantly, GX-188E vaccination significantly induces polyfunctional HPV16-specific CD8 T-cell response in seven out of nine patients, which correlates with HPV clearance and complete resolution of high-grade cervical lesions on cytology and histology. Therefore, we offer an effective therapeutic vaccine strategy for eradicating persistent HPV infections and HPV-induced tumours, together with an important insight into the immune correlates to control persistent viral infection.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>GX-188E vaccination is safe and well tolerated</title><p>GX-188 DNA vaccine is engineered to express E6 and E7 proteins of HPV16 and HPV18 fused to extracellular domain of Flt3L and the signal sequence of tissue plasminogen activator (tpa); the purpose for inclusion of Flt3L and tpa is to promote antigen presentation and trafficking of the fused protein to the secretory pathway, respectively (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The activity of tpa is evident, as the GX-188-induced E6/E7 fusion protein was detected only in the cytoplasmic compartment of transfected cells, whereas E7 protein expressed by the same vector without tpa was found in both cytoplasmic and nuclear compartments (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1a</xref>). The arrangement of HPV E6 and E7 genes in GX-188 was intentionally shuffled, by alternatively placing the amino (N-) and carboxyl-terminal (C-terminal) domains of the two genes, but with an overlapping region of 16 amino acids to minimize the loss of potential T cell-recognizing epitopes at the junction sites (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The gene shuffling was done to prevent the homodimerization of E6 and E7 regions of the fusion protein, which is crucial for their binding and degradation of p53 and pRb tumor suppressor proteins<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. As expected, the E6/E7 fusion protein generated by GX-188 DNA vaccine was unable to degrade p53 and pRb proteins, whereas wild-type E6 and E7 proteins induced their degradation (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1b,c</xref>).</p><p>According to the inclusion and exclusion criteria for this study, nine out of 11 screened patients with CIN3 were enroled (<xref ref-type="table" rid="t1">Table 1</xref>). The screened patients were examined by multiple methods, including colposcopy, cytology, histology and HPV type test, at the visit for screening (VS) time point, 2 weeks before the start of the trial. All participating subjects received three injections of GX-188E, with the last two injections given at 4 and 12 weeks after the first injection (<xref ref-type="fig" rid="f1">Fig. 1b</xref>).</p><p>A total of 49 adverse events (AEs) were recorded during all visits. Twenty-three AEs, including eczema, ecchymosis, vaginal itching, sleepiness, anorexia and dizziness were determined to be unrelated to the vaccination. Nineteen AEs including chills, injection site pain, swelling and hypoaesthesia, were recorded to be associated with GX-188E vaccination (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>). Although the cause of the remaining seven AEs, including headache, rhinitis and fatigue, were unknown, they could be potentially associated with GX-188E vaccination. However, all these AEs were considered to be mild (grade 1) and all patients recovered completely within 3 days after GX-188E vaccination. Since neither severe AEs nor laboratory abnormalities were observed at any given dose (<xref ref-type="supplementary-material" rid="S1">Supplementary Tables 1</xref> and <xref ref-type="supplementary-material" rid="S1">2</xref>), the dose of GX-188E was elevated from 1 to 2&#x02009;mg, and then to 4&#x02009;mg (three patients at each dose) without the enrolment of additional three subjects at each dose level according to 3+3 dose-escalation design of this clinical trial protocol.</p><p>Since it was reported that the administration of Flt3L protein could increase the frequency of white blood cells (WBCs)<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>, we measured the number of WBCs and the level of Flt3L in the blood. A change in the number of WBCs was not observed (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>), which is likely due to no significant upregulation of Flt3L level in the blood on GX-188E vaccination (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). Such a finding is in line with previous reports with incorporated genetic adjuvants in DNA vaccines, such as cytokines and HSP70 (refs <xref ref-type="bibr" rid="b17">17</xref>, <xref ref-type="bibr" rid="b24">24</xref>, <xref ref-type="bibr" rid="b25">25</xref>, <xref ref-type="bibr" rid="b26">26</xref>), implicating that the use of genetic adjuvants in a DNA vaccine is not usually associated with severe AEs.</p><p>To determine the immunological safety of our approach, we investigated whether the enhanced delivery of GX-188E generated anti-DNA antibodies, which are known to be associated with autoimmune disorders<xref ref-type="bibr" rid="b26">26</xref>. The level of antibodies against DNA in the blood of patients with CIN3 was below the detection limit (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 4</xref>), which is comparable to the previous results obtained from subjects immunized with DNA vaccine without EP<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. Taken together, these results indicate that the incorporation of EP and genetic adjuvants is relatively tolerable in clinical trials of DNA vaccines and shows very similar safety profiles observed with the administration of a basic DNA vaccine without EP.</p></sec><sec disp-level="2"><title>GX-188E vaccination induces strong cellular immunity</title><p>To study the cellular immune response induced by GX-188E, an IFN-&#x003b3; ELISPOT assay as described in the Methods was performed before, at VS time point (&#x02212;2 weeks), during, at VT2 (2 weeks) and VT4 time points (8 weeks), and after, at VF1 (20 weeks) and VF2 time points (36 weeks), GX-188E vaccination. Relatively high pre-existing IFN-&#x003b3; ELISPOT response was detected in one patient (A03), whereas the other eight patients displayed weak pre-existing HPV-specific cellular immunity before vaccination. On the basis of the criteria described in the Methods, all subjects exhibited a marked increase in the vaccine-induced E6- and E7-specific IFN-&#x003b3; ELISPOT response compared with the background level before vaccination (<xref ref-type="fig" rid="f2">Fig. 2</xref>). Interestingly, two out of nine patients (A06 and A08) developed a considerably enhanced IFN-&#x003b3; response even after a single immunization (VT2), and additional five patients (A02, A04, A05, A07, and A09) exhibited such an elevated response after two vaccinations (VT4). The remaining two patients (A01 and A03) in the 1-mg dose group displayed an increased IFN-&#x003b3; response after three shots of the GX-188E vaccine (VF1). Taken together, these results suggest that vaccine-induced cellular immune responses became progressively stronger in all patients during GX-188E vaccination. In particular, patient A08 exhibited the highest magnitude of IFN-&#x003b3; ELISPOT response with reactivity up to 3,500 spot-forming unit per 10⁶ peripheral blood mononuclear cells (PBMCs). Interestingly, the response against the E6 antigen was more vigorous than against E7 as determined by the magnitude of response (69&#x02013;89% against E6 versus 11&#x02013;31% against E7 at VF1; <xref ref-type="fig" rid="f2">Fig. 2</xref>).</p><p>The establishment of memory T cells, normally starting to form about 4 weeks after immunization, is usually one of the indispensable factors for protective efficacy of a vaccine<xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>. A relatively high level of IFN-&#x003b3; ELISPOT response was observed in eight out of nine patients at 24 weeks (VF2) following the last vaccination, which, when compared with the responses at 8 weeks (VF1) post vaccination, is decreased for one patient (A03), comparable for three patients (A01, A06, and A09) and increased for four patients (A02, A05, A07, and A08; <xref ref-type="fig" rid="f2">Fig. 2</xref>). Overall, this finding indicates that GX-188E vaccination-induced E6/E7-specfiic memory T-cell response can be maintained for at least 24 weeks post last vaccination. To address whether the IFN-&#x003b3; response to E6/E7 antigens measured by ELISPOT assay was generated mainly by T cells and to determine which subset of T cells played a predominant role, we performed intracellular cytokine staining (ICS) assays for IFN-&#x003b3; at pre- and post-vaccination time points (VS and VF1). As shown in <xref ref-type="fig" rid="f3">Fig. 3</xref>, the vaccination with GX-188E resulted in an increase in HPV16-specific IFN-&#x003b3;⁺ CD4 T-cell responses in all nine patients, while IFN-&#x003b3;⁺ CD8 T-cell response was enhanced in eight out of nine patients. Thus, with the exception of patient A04, GX-188E vaccine elicited activation of both HPV16-specific CD4 and CD8 T cells.</p><p>Since persistent HPV infection impairs Th1 cellular response to HPV, leading to cervical cancer progression<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>, we investigated whether our DNA vaccine could drive the differentiation of HPV-specific CD4 T cells into Th1 effector cells. As expected, the baseline production of common Th1 effector cytokines, such as IFN-&#x003b3;, IL-2 and tumour necrosis factor-&#x003b1; (TNF-&#x003b1;), before vaccination was remarkably low on stimulation with E6/E7 peptides. However, the amounts of these cytokines markedly increased after vaccination in most of the patients (median 49.9-, 13- and 22.9-fold increases for IFN-&#x003b3;, IL-2, and TNF-&#x003b1;, respectively; <xref ref-type="fig" rid="f4">Fig. 4</xref>). Consistent with the IFN-&#x003b3; ELISPOT and ICS data, A08 patient also showed the greatest increase of Th1 cytokine production. On the other hand, Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines were not significantly increased by vaccination, although patient A04 had a slightly increased level in production of an immunosuppressive cytokine, IL-10 (<xref ref-type="fig" rid="f4">Fig. 4</xref>). Taken together with the above IFN-&#x003b3; ELISPOT and ICS analyses, these results suggest that GX-188E vaccination leads to the induction of a strong Th1-polarized HPV-specific cellular immune response.</p></sec><sec disp-level="2"><title>GX-188E vaccine-induced CD8 T cells are polyfunctional</title><p>To determine whether GX-188E vaccination induced multiple aspects of HPV-specific CD8 T-cell functionality, we assessed the ability of CD8 T cells, on stimulation with a pool of HPV16 E6 and E7 peptides, to simultaneously express the following five different effector functions: cytokines IFN-&#x003b3;, IL-2 and TNF-&#x003b1;, a chemokine MIP-1&#x003b2; and the cytotoxic activity as determined by expression of CD107a/b, which is exclusively found during degranulation of cytotoxic T cells<xref ref-type="bibr" rid="b34">34</xref>. When T cells were examined for two effector functions (IFN-&#x003b3; together with IL-2, TNF-&#x003b1;, MIP-1&#x003b2; or CD107a/b), we obtained results similar to that obtained by ICS for only IFN-&#x003b3;. Thus, eight out of nine patients, with exception of A04, displayed an increase in proportions of HPV-specific CD8 T cells with two effector functions after vaccination (VF1) compared with before vaccination (VS) (<xref ref-type="fig" rid="f5">Fig. 5a&#x02013;d</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2b&#x02013;e</xref>). Examination of all five effector functions simultaneously as assessed using Boolean gating revealed even more striking results. Data on post-vaccination T cells from the most responsive patient (A08) are shown as an example in <xref ref-type="fig" rid="f5">Fig. 5e</xref>, and together with the pie chart in <xref ref-type="fig" rid="f5">Fig. 5f</xref>, shows that 87.6% of HPV16-specific CD8 T cells were at least triple-positive and 15% of them had all 5 functions. Similar analysis in the other six patients (A01, A02, A03, A05, A06 and A07) revealed that 7.8&#x02013;46.3% of HPV-specific CD8 T cells had three or more functions (<xref ref-type="fig" rid="f5">Fig. 5f</xref>). In contrast, almost all HPV16-specific CD8 T cells from patient A09 were not polyfunctional (<xref ref-type="fig" rid="f5">Fig. 5f</xref>). It is worth noting that Boolean gating analysis could not be performed with prevaccination T cells from all patients and post-vaccination T cells from patient A04 because of extremely low frequency of responding T cells that displayed effector function. Overall, these results indicate that GX-188E vaccination efficiently induced generation of polyfunctional HPV-specific CD8 T cells in most patients (seven out of nine patients).</p><p>It was reported that optimal expansion of responding T cells on antigen stimulation is essential for providing effective protective immunity by therapeutic vaccination<xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>. Therefore, we examined the proliferative potential of HPV-specific CD8 T cells by stimulating patients&#x02019; PBMCs from pre- (VS) and post- (VF1) vaccination with a pool of HPV16 E6/E7 peptides for 5 days, followed by staining for the expressions of Ki67 and CD38, which serve as a marker of proliferation and activation, respectively<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref>. PBMCs stimulated with medium alone served as a negative control to assess antigen specificity of <italic>in vitro</italic>-expanded CD38⁺Ki67⁺ CD8 T cells. Although one patient (A01) displayed a relatively high pre-existing proliferative level prevaccination (VS), the rest of the patients demonstrated low levels of Ki67⁺CD38⁺ CD8 T cells (<xref ref-type="fig" rid="f6">Fig. 6</xref>). After vaccination, all patients exhibited meaningful improvement in proliferative activity of HPV-specific CD8 T cells. In accordance with the pattern of functional CD8 T cell response (<xref ref-type="fig" rid="f5">Fig. 5</xref>), two patients (A04 and A09) displayed only a minor increase in proliferating CD8 T cell population, whereas the other seven patients displayed a much greater increase of Ki67⁺CD38⁺ CD8 T-cell population, within a range of 3.2- to 21.3-fold increase. Collectively, these results indicate that GX-188E vaccination in CIN3 patients substantially augmented both the expansion and polyfunctionality of HPV-specific CD8 T cells.</p></sec><sec disp-level="2"><title>GX-188E elicits a weak antibody response to E7 protein</title><p>When plasma samples were evaluated for total IgG antibody responses to E6 and E7 by an end point dilution enzyme-linked immunosorbent assay, all patients had barely detectable or undetectable IgG titer to both E6 and E7 proteins at baseline (VS; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>), indicating no meaningful pre-existing E6- and E7-specific IgG antibody responses. Interestingly, the antibody titres to E6 were not induced in any dose cohort patients after vaccination. Three out of nine patients (A05, A07, and A09) generated weak anti-E7 antibody responses following vaccination with antibody titres ranging from 1:8 to 1:256 (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). It is worth noting that T-cell responses to E7 antigens were generally lower than those against E6 antigens (<xref ref-type="fig" rid="f2">Fig. 2</xref>) and measurable antibody titres to E7 proteins were not associated with CD8 T cell responses to E7 antigens.</p></sec><sec disp-level="2"><title>GX-188E vaccination clears HPV infections and lesions</title><p>GX-188E-induced clinical responses were determined by evaluating the patients&#x02019; HPV infection status as well as the cytological and histological changes of their high-grade cervical lesions over the 36-week period of the clinical trial (<xref ref-type="table" rid="t2">Table 2</xref> and <xref ref-type="fig" rid="f1">Fig. 1b</xref>). At baseline (VS), all nine patients had CIN3 with either severe dysplasia (A01, A02, A05, A06, A07 and A08) or carcinoma <italic>in situ</italic> (A03, A04 and A09) according to the histological evaluation of colposcopic-directed biopsy specimens (<xref ref-type="table" rid="t1">Tables 1</xref> and <xref ref-type="table" rid="t2">2</xref>). At 8 weeks post last vaccination (VF1), six out of nine patients were free of lesions&#x02014;two patients from each cohort (A01 and A03 from 1&#x02009;mg cohort, A05 and A06 from 2&#x02009;mg cohort, A07 and A08 from 4&#x02009;mg cohort)&#x02014;indicating dose independency of the response presumably due to saturation dose at 1&#x02009;mg (<xref ref-type="table" rid="t2">Table 2</xref>). Three of these responder patients (A03, A06 and A08) were negative for the intraepithelial lesion based on cytological analysis after the second immunization at week 12 (VT4), while three other patients (A01, A05 and A07) displayed such responses after the third vaccination at week 20 (VF1) and the last responder patient (A02) cleared the lesion at the end of the 36-week trial (VF2). Notably, none of the six early responders displayed any recurrent cervical dysplasia during the remaining period of the trial. In cases of two non-responders, patient A04 was treated by cervical conization at week 24, while patient A09 was monitored without surgery until the end of study per patient&#x02019;s request and remained stable at CIN3 without progressing to invasive carcinoma. Colposcopic, cytological and histological image analysis before vaccination (VS) and at the end of the trial (VF2) more clearly demonstrated the difference in clinical responses to GX-188E between responders and non-responders, as shown by the photographs from representative responder A05 and non-responder A09 patients (<xref ref-type="fig" rid="f7">Fig. 7</xref>).</p><p>HPV16 was identified in the lesions of all nine subjects at the start of the trial, and one patient (A05) was found to be also co-infected with HPV18. At week 12 (VT4), four patients (A01, A03, A06 and A08) and patient A05 showed clearance of HPV16 and HPV18 viruses, respectively (<xref ref-type="table" rid="t2">Table 2</xref>), indicating viral clearance after the second immunization. At week 20 (VF1), HPV DNAs in cervical lesions were cleared in six out of nine patients (A01, A03, A05, A06, A07 and A08) and one more patient (A02) cleared the virus at week 36 (VF2). Since these seven patients also cleared their lesions with the identical kinetics, there was perfect correlation between the clinical and virological responses (<xref ref-type="table" rid="t2">Table 2</xref>). Beside HPV16 and HPV18, two patients (A06 and A07) were found to be co-infected with other high-risk common types of HPV at baseline (VS). In addition, one patient (A05) became infected with the common HPV type in the midst of the trial (VT4). In contrast to A07 patient, A05 and A06 patients cleared co-infected common types of HPV at VF2 and VT4, respectively, presumably due to a bystander effect caused by the elimination of HPV16-infected intraepithelial neoplastic cells.</p><p>It is notable that the three patients (A03, A06 and A08) who cleared their lesions and HPV infection at the early time point (VT4) promptly displayed a relatively high magnitude of HPV-specific polyfunctional CD8 T-cell response (<xref ref-type="table" rid="t2">Table 2</xref> and <xref ref-type="fig" rid="f5">Fig. 5</xref>). In addition, the other four patients (A01, A02, A05 and A07) with a meaningful polyfunctional CD8 T-cell response exhibited the complete resolutions of their lesions and HPV infections after the third vaccination either at week 20 (VF1) or at the end of the trial (VF2; <xref ref-type="table" rid="t2">Table 2</xref> and <xref ref-type="fig" rid="f5">Fig. 5</xref>). In contrast, two non-responder patients (A04 and A09) had almost no polyfunctional CD8 T-cell response. The correlation between the induction of polyfunctional T-cell response and clinical outcome is readily apparent when the individual data from the patients were grouped into non-responders (A04 and A09) and responders (A01, A02, A03, A05, A06, A07 and A08) to generate the polyfunctional profile with three or more functions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). Hence, our results indicate that the clinical efficacy of GX-188E vaccine strongly correlates with the extent of systemic HPV-specific polyfunctional CD8 T-cell response. Overall, GX-188E vaccination led to the clinically and virologically meaningful complete response rate of 78% (seven out of nine patients) (<xref ref-type="table" rid="t2">Table 2</xref>).</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In this study, we have demonstrated the efficacy of a novel HPV E6/E7 DNA therapeutic vaccine, designated GX-188, strategically designed to induce robust type 1 T cell-mediated immunity and to eradicate HPV infection-associated lesions. So far, several types of HPV therapeutic vaccines have been evaluated in patients diagnosed with CIN2/3 lesions using viral vectors<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>, peptides<xref ref-type="bibr" rid="b15">15</xref>, proteins<xref ref-type="bibr" rid="b16">16</xref> or plasmid DNA<xref ref-type="bibr" rid="b17">17</xref>. In contrast to these previous studies that performed excision surgery at weeks 9&#x02013;15 after vaccination, preventing extended observation of histological change, our current trial was designed to assess the clinical outcomes and immune responses at 20 and/or 36 weeks after the first vaccination to investigate the kinetics of immune response and the durability of vaccine efficacy. In this study, viral clearance (four out of nine patients) and cytological recovery (three out of nine patients) were already apparent at week 12 and most of the complete responders (six out of seven patients) cleared the cervical lesions within week 20 post vaccination with durable immunity, that is, without any recurrence of cervical dysplasia or re-emergence of HPV infection during the remaining 16 weeks period of the clinical trial. Collectively, compared with the previous reports, our results appear to be highly novel and clinically meaningful for the following reasons: first, the EP-delivered GX-188 elicited the relatively high and durable HPV-specific Th1-polarized cellular immune responses in eight out of nine CIN3 patients with severe dysplasia or carcinoma <italic>in situ</italic>, and seven out of nine patients (78%) had complete regression of their high-grade lesion and clearance of HPV DNA, which are the highest response rate compared with the previous reports in clinical trials of HPV vaccines. Second, cytological, histological and virological evaluations yielded the same results in the responders, indicating induction of a complete response by GX-188E. Third, the normalization of lesions by GX-188E vaccination was maintained without recurrence of dysplasia during the entire study period of 36 weeks, indicating a sustained therapeutic efficacy of the vaccine. Finally, clinical and virological responses at the cervix were correlated with the magnitude of the polyfunctional CD8 T-cell response in the blood, suggesting that systemic HPV-specific CD8 T-cell responses might serve as a predictive biomarker for determining clinical outcomes of therapeutic vaccine in the setting of high-grade cervical lesions.</p><p>It has been reported that regression of CIN2/3 can occur spontaneously in some patients at a rate ranging from 11 to 30% over &#x02265;1 year to 38% between 9 and 20 weeks after colposcopic biopsy<xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref>. Even when considered in light of these reports, our finding of complete regression in six out of nine patients (67%) at 20 weeks and in seven out of nine patients (78%) at 36 weeks after GX-188E vaccination indicates that vaccine-induced immunity is considerably more effective than natural immunity. However, we cannot rule out the possibility that some of the responders, especially the ones who have responded early, would have spontaneously cleared their lesions. A corollary to this possibility is that the vaccine-induced response rate beyond the maximum rate of spontaneous regression is in the same ballpark as that of landmark vaccine trial in which a synthetic long-peptide vaccination exhibited 47% of complete response rate in patients with another HPV-associated lower genital dysplasia, vulvar intraepithelial neoplasia 3 (ref. <xref ref-type="bibr" rid="b19">19</xref>).</p><p>The induction of strong HPV-specific Th1 and CD8 T-cell immunity by GX-188E vaccination leading to complete clearance of HPV DNA and high-grade cervical lesions observed in this study is likely due to the following reasons: codon optimization of HPV E6/E7 genes<xref ref-type="bibr" rid="b42">42</xref>, intracellular targeting of expressed E6/E7 antigens to secretion pathway by the fused tpa signal sequence<xref ref-type="bibr" rid="b43">43</xref>, enhanced DNA vaccine delivery by EP<xref ref-type="bibr" rid="b44">44</xref> and the utilization of a high-expression vector<xref ref-type="bibr" rid="b45">45</xref>. In addition, since mutations or deletions of the E6/E7 sequence motifs crucial for binding to p53 and pRb could result in loss of their T cell-recognizing epitopes<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>, we instead genetically engineered HPV E6 and E7 genes by shuffling N- and C-terminal domains, with an overlapping region, to maintain T-cell epitopes, while abrogating the oncogenic potential of these proteins. Finally, the Flt3L gene was fused to engineered E6/E7 genes as a built-in genetic adjuvant, since Flt3L is known to induce Th1 immune responses through preferential expansion of CD8&#x003b1;⁺ lymphoid DCs<xref ref-type="bibr" rid="b46">46</xref><xref ref-type="bibr" rid="b47">47</xref><xref ref-type="bibr" rid="b48">48</xref><xref ref-type="bibr" rid="b49">49</xref>. It was reported that bone marrow-derived DCs pulsed with cell lysates containing Flt3L-E7 fusion protein presented E7 antigens through the major histocompatibility complex class I pathway more efficiently than those pulsed with wild-type E7 protein<xref ref-type="bibr" rid="b50">50</xref>, indicating a potential role of Flt3L in facilitating cross-priming. Moreover, Flt3L-fused DNA vaccine elicited the highest antigen-specific IFN-&#x003b3;-secreting T-cell responses compared with other molecules for targeting antigens to DCs, such as CD40L and flagellin<xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b51">51</xref>. In contrast to T-cell responses, GX-188E vaccination induced the mild increase of IgG titres against only E7 protein in three out of nine patients (A05, A07 and A09; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>). These weak antibody responses can be explained by the feature of GX-188 DNA vaccine in which Flt3L-mediated Th1-polarization may inhibit Th2-type responses and antibody production by cross-regulation of Th1/Th2 responses<xref ref-type="bibr" rid="b52">52</xref>. Since persistent HPV infection is known to promote Th2 response, thereby leading to poor T-cell immunity and continual progression of lesion<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref>, our strategy to incorporate Flt3L to HPV DNA vaccine antigen may be an effective approach for inducing Th1-polarized cellular immunity.</p><p>The immunological and clinical outcomes after vaccination are generally variable and may be determined in part by host genetic factors. In the present study, GX-188E vaccine achieved complete response in seven out of nine patients (78%). Among seven responders, six patients carrying human leukocyte antigens (HLA)-A*02 exhibited high polyfunctional CD8 T-cell responses as well as complete regression of CIN3 (<xref ref-type="table" rid="t1">Table 1</xref>). Among the two non-responders, patient A04 with HLA-A*26 and -A*30 did not induce HPV-specific CD8 T-cell responses at all. It was reported that 5&#x02013;15% of vaccine failed to seroconvert by the standard hepatitis B vaccination schedule and this was found to be associated with specific HLA alleles<xref ref-type="bibr" rid="b53">53</xref>. In this regard, it will be of interest to investigate whether specific HLA alleles can be a contributing factor for the impaired induction of HPV-specific CD8 T-cell response by GX-188E vaccination.</p><p>The other non-responder patient (A09) exhibited a comparable magnitude of antigen-specific CD8 T-cell response with two effector functions, but had lower polyfunctional profile of CD8 T cells with three or more effector functions than responder patients, indicating that the polyfunctionality of vaccine-induced CD8 T cells may contribute to clinical outcomes. It is worth noting that the patient A09, who was infected with only HPV16, displayed a higher T-cell response to HPV18 than to HPV16 in IFN-&#x003b3; ELISPOT assay. Thus, this biased response towards HPV18 in the patient A09 may have led to the impaired HPV16-specific polyfunctional CD8 T-cell response. Alternatively, it is possible that local immunity at cervix could be involved in the clearance of lesions. Recently, combined vaccination with a heterologous DNA prime-recombinant vaccinia vector-based boost regimen induced a tissue-localized T-cell response in most patients with CIN2/3, which was suggested to be informative for determining vaccine efficacy<xref ref-type="bibr" rid="b54">54</xref>. In our current trial, complete clearance of lesions was already achieved at the first biopsy after vaccination (at week 20) in most patients, and thus we could not obtain the lesions to assess vaccine-induced local immunity. To address this issue, we are planning to take a biopsy at an earlier time point (week 6&#x02013;8 after first vaccination) in upcoming phase 2 clinical trial. Finally, patient A09 had the largest lesion size with carcinoma <italic>in situ</italic>, probably providing more severe immunosuppressive microenvironment by which the effector function of vaccine-induced systemic CD8 T cells could be thwarted. It was reported that median cell density of stromal Foxp3⁺ regulatory T cells expressing IL-10 and indoleamine 2, 3-dioxygenase appear to increase significantly in the cervix with increasing pathology and cancer<xref ref-type="bibr" rid="b55">55</xref>. Thus, it would be important to develop strategies to neutralize these immunosuppressive factors and/or enhance the vaccine-induced T-cell responses to improve the clinical efficacy of DNA vaccines in patients with larger lesions and cancer.</p><p>Among three CIN3 patients with carcinoma <italic>in situ</italic>, one patient (A03) who had a relatively high pre-existing HPV-specific cellular immune response completely cleared both HPV infection and the high-grade lesions on GX-188E vaccination. Thus, it would be of interest to investigate whether the pre-existing cellular immunity could be an indicator of the clinical outcome of therapeutic vaccine in CIN3 patients with carcinoma <italic>in situ</italic>. Most importantly, all six CIN3 patients with severe dysplasia showed eradication of HPV DNA and regression of their lesion following GX-188E DNA vaccination. Even though the number of patients (n=9) is too small to draw a definite conclusion, our findings may provide an important insight into the key requirements for a therapeutic vaccine to be effective against various persistent infections and chronic diseases. Considering that the current primary treatment option for CIN3 is surgical excision, which is often accompanied with several side effects<xref ref-type="bibr" rid="b8">8</xref>, our non-invasive immunological approach with good safety and excellent clinical efficacy within 12 to 36 weeks could mark one of important milestones for treating CIN3 patients with severe dysplasia or carcinoma <italic>in situ</italic>.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Ethics statement</title><p>The clinical trial protocol was reviewed and approved by the Institutional Review Board at Cheil General Hospital and Women&#x02019;s Healthcare Center (CGH-IRB-2012-35). This study was conducted in accordance with the ethical principles that had their origin in the current Declaration of Helsinki and was consistent with International Conference on Harmonization Good Clinical Practice (IHC GCP) and applicable regulatory requirements. All study participants gave written informed consent before undergoing screening for study eligibility and enrolment. This trial is listed at <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.clinicaltrials.gov">http://www.clinicaltrials.gov</ext-link> (NCT01634503)</p></sec><sec disp-level="2"><title>Study design and patients</title><p>This phase 1 clinical study was conducted as an open label, single center, dose-escalation study at Cheil General Hospital &#x00026; Women&#x02019;s Healthcare Center, Seoul, Korea. The primary end point was to evaluate safety and tolerability in patients with CIN3. The secondary end points included systemic induction of HPV E6- and E7- specific T-cell immune responses measured by IFN-&#x003b3; ELISPOT, and changes of involved lesions and HPV infection status at the uterine cervix. Women aged between 20 and 50 years with histologically and virologically proven HPV16- or HPV18-associated CIN3 were enroled in the study. The CIN3 was confirmed by colposcopy-directed biopsy and HPV16 or HPV18 positivity was determined by PCR. Subjects with hepatitis B virus, hepatitis C virus or human immunodeficiency virus infections, abnormal electrocardiography including arrhythmia, history of severe adverse drug events or severe allergic diseases were excluded. Females who were pregnant or planning to be pregnant were not recruited in the study. Vaccination consisted of a series of three vaccine injections administered intramuscularly to alternating deltoid muscles at weeks 0, 4 and 12. A standard 3+3 dose-escalation scheme was followed and dose levels of 1, 2 and 4&#x02009;mg were tested. At the highest dose, 4&#x02009;mg of GX-188E was split into 2+2&#x02009;mg and injected to the left and right deltoids muscles. For the intramuscular injector, an EP device (TriGrid Delivery System, Ichor medical systems, Inc.) was used to facilitate DNA uptake into cells.</p></sec><sec disp-level="2"><title>HLA typing</title><p>HLA typing was accomplished at Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea. Sequence-based typing (SBT) of HLA was performed by heterozygous amplification followed by sequencing of the complete exons 2, 3 of HLA-A and -B. For locus-specific amplification, primers were used in in-house method. After application by PCR, agarose gel electrophoresis of the PCR products was conducted to assess the quantity and quality. Cycle sequencing reactions using the ABI PRISM BigDye terminator kit (Applied Biosystems, CA, USA) and the automated ABI377 DNA Sequencer (Applied Biosystems, CA, USA) were performed. These data were analysed by using SBT analysis programme (Conexio Genomics, Assign SBT v3.5.1).</p></sec><sec disp-level="2"><title>DNA vaccine</title><p>The study vaccine, pGX-188, contains a plasmid DNA encoding E6 and E7 proteins of HPV serotypes 16 and 18 fused with tpa signal sequence and extracellular domain of Flt3L. Synthetic codon-optimized E6 or E7 genes were fragmented into two parts (C-terminal and N-terminal regions) with a small overlapping sequence (encoding 16 amino acids), and shuffled as shown in <xref ref-type="fig" rid="f1">Fig. 1a</xref>. The fused DNA sequences including tpa, Flt3L and shuffled E6/E7 genes were inserted in high-expression vector, pGX27 (ref. <xref ref-type="bibr" rid="b45">45</xref>), to generate GX-188, which was produced in <italic>E. coli</italic> DH5&#x003b1; under cGMP condition at Althea Technologies, Inc., San Diego, CA.</p></sec><sec disp-level="2"><title>Virological and clinical responses</title><p>The assessments including colposcopy, histology, endocervical cytology and HPV genotyping test were conducted by local laboratory at the trial site. The assessments were performed in compliance with the standardized method or the internal protocol of Cheil General Hospital and Women&#x02019;s Healthcare Center. Responses to treatment were evaluated using virology and histology results at weeks 20 and 36 post GX-188E vaccination.</p><p>For histological evaluation, biopsy samples were taken during screening and two follow-up visits at weeks 20 and 36. Samples were fixed with 10% formaldehyde and 4&#x02013;5&#x02009;&#x003bc;m sections were stained with haematoxylin and eosin. Endocervical samples were collected using cytobrush (Cytyc Corp., Boxborough, MA) during colposcopic examination. This endocervical cytology test was also used in addition to histology for the assessment of GX-188E vaccination. Data from histological and cytological analyses were reviewed independently by at least two pathologists and results were confirmed after discussion with by conference of all pathologists and investigators.</p><p>To evaluate virological response, HPV typing was performed to determine whether subjects were infected by either HPV16 and/or HPV18. Samples were collected from the cervix by using a swab-type device, and total DNAs were extracted using the AccuPrep Genomic DNA Extraction kit (Bioneer Com. Seoul, Korea). HPV detection and genotyping was done by Multiplex-PCR system using the IVD CE marked Seeplex HPV4A ACE Screening kit (Seegene Inc., Seoul, Korea) according to the manufacture&#x02019;s protocol. The Seeplex HPV4A ACE Screening kit can identify HPV16, HPV18, other high-risk types (High risk common: 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and/or 82), HPV6 and HPV11 types at the same time. PCR products were analysed using an automatic MultiNA instrument (Shimadzu Co., Tokyo, Japan). HPV DNA genotyping was double checked in cervical cells using Cheil HPV DNA Chip with real time PCR to compensate the accuracy of HPV genotype as previously described<xref ref-type="bibr" rid="b56">56</xref>.</p></sec><sec disp-level="2"><title><italic>Ex vivo</italic> IFN-&#x003b3; ELISPOT</title><p>Cryopreserved and thawed PBMCs were adapted with OpTmizer CTS medium (Life Technologies) for more than 6&#x02009;h at 37&#x02009;&#x000b0;C, 5% CO₂, and subsequently PBMCs (2 &#x000d7; 10⁵ cells per well) were stimulated with 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 of four different pools of HPV16 and HPV18 E6- or E7-derived peptides (20-mer with 10 amino acids overlapping) for 48&#x02009;h. Phytohaemagglutinin and the medium only served as positive and negative controls, respectively. After stimulation, spots indicating IFN-&#x003b3;-secreting cells were developed according to manufacturer&#x02019;s instructions (BD Bioscience). The number of spots was analysed with an automated ImmunoSpot Analyzer (Cellular Technology Ltd.). The HPV-specific responses were calculated by subtracting the mean number of spots in the medium only control from the mean number of spots in experimental wells, which were expressed as SFUs per 10⁶ PBMCs<xref ref-type="bibr" rid="b57">57</xref>. The assay was performed in triplicate, and the background number of spots was 5.7&#x000b1;2.2 (mean&#x000b1;s.d.). Antigen-specific T-cell responses were considered to be positive when the mean number of spots in the well with the antigen was threefold higher than that of the well with medium control<xref ref-type="bibr" rid="b58">58</xref>. In addition, a post-analysed vaccine-induced response was defined as positive when at least a threefold increase in T-cell frequency was observed after vaccination compared to before vaccination<xref ref-type="bibr" rid="b15">15</xref>.</p></sec><sec disp-level="2"><title>ICS</title><p>Cryopreserved and thawed PBMCs were resuspended in OpTimizer CTS, and rested for more than 6&#x02009;h at 37&#x02009;&#x000b0;C, 5% CO₂. PBMCs were plated in duplicate and stimulated with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1, &#x003b1;-CD3 (positive control, 10&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1, UCHT1, BD Bioscience) or the medium alone (negative control) in the presence of 1&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 of &#x003b1;-CD28 (L293, BD Bioscience) and &#x003b1;-CD49d (L25, BD Bioscience) for 13&#x02009;h. Secretion inhibitors (monensin/brefeldin A, BD Bioscience) were added 90&#x02009;min after initial stimulation. After stimulation, cells were washed with PBS for subsequent immunostaining and polychromatic flow cytometric analysis. Antibodies for staining cells were CD19-APCCy7 (5&#x02009;&#x003bc;l per test, HIB19, Biolegend), CD4-PerCPCy5.5 (5&#x02009;&#x003bc;l per test, RPA-T4, Biolegend), CD8-PECy7 (5&#x02009;&#x003bc;l per test, RPA-T8, BD Bioscience), CD3-BV605 (Bright Violet 605) (5&#x02009;&#x003bc;l per test, UCHT1, Biolegend), CD3-BV500 (5&#x02009;&#x003bc;l per test, UCHT1, BD Horizon), Live/dead-APCCy7 (0.5&#x02009;&#x003bc;l per test, Life technologies), MIP-1&#x003b2;-PE (0.5&#x02009;&#x003bc;l per test, D21-1351, BD Bioscience), IFN-&#x003b3;-APC (5&#x02009;&#x003bc;l per test, 4S.B3, Biolegend), TNF-&#x003b1;-BV421 (5&#x02009;&#x003bc;l per test, MAb11, Biolegend), IL-2-BV711 (5&#x02009;&#x003bc;l per test, 5344.111, BD Horizon), CD107a-FITC (5&#x02009;&#x003bc;l per test, H4A3, BD Bioscience) and CD107b-FITC (5&#x02009;&#x003bc;l per test, H4B4, BD Bioscience). Fluorescence-activated cell sorting analysis was accomplished by Fortessa flow cytomer (BD Bioscience), and the data were analysed using FlowJo software (Tree Star). Boolean gating was used to determine simultaneous cytokine production from CD8 T cells. Analysis of polyfunctionality was performed with SPICE<xref ref-type="bibr" rid="b59">59</xref>. A positive response was defined as detecting at least twice the percentage of cytokine-producing T cells than in the medium only control, and the response should be visible as a clearly distinguishable population of cytokine-producing cells separated from the nonproducing cells. A post-analysed vaccine-induced response was defined as detecting at least a threefold increase in the percentage of antigen-specific cytokine-producing T cells than that at prevaccination<xref ref-type="bibr" rid="b60">60</xref>.</p></sec><sec disp-level="2"><title>Cytokine profile analysis by cytometric bead array</title><p>Cryopreserved and thawed PBMCs (2 &#x000d7; 10⁵ per well) were resuspended in OpTimizer CTS, and rested for more than 6&#x02009;h at 37&#x02009;&#x000b0;C, 5% CO₂, and subsequently PBMCs were plated in duplicate and were stimulated in RPMI 1,640 containing 10% fetal bovine serum, 100&#x02009;U&#x02009;ml^&#x02212;1 penicillin and 100&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 streptomycin with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration of 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 or the medium only as negative control in 96-well plates. Culture supernatants were harvested 48&#x02009;h after the stimulation and cytokines were quantitated by Th1/Th2/Th17 cytometric bead array (CBA) kit (BD Biosciences). According to manufacturer&#x02019;s instructions, the proposed detection limit was 2.5&#x02013;5&#x02009;pg&#x02009;ml^&#x02212;1 (IL-2, IL-4, IL-10, TNF-&#x003b1; and IFN-&#x003b3;) or 19&#x02009;pg&#x02009;ml^&#x02212;1 (IL-17A), and the cutoff value was set at 5&#x02009;pg&#x02009;ml^&#x02212;1 because the standard curve of each cytokine showed linearity starting at this concentration (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>). Positive antigen-specific reaction was defined as a cytokine concentration above the cutoff value and &#x0003e;2 &#x000d7; the concentration of the medium control<xref ref-type="bibr" rid="b60">60</xref>. A post-analysed vaccine-induced response was defined as being at least threefold higher in the cytokine production than that at prevaccination<xref ref-type="bibr" rid="b60">60</xref>.</p></sec><sec disp-level="2"><title>Proliferation assay</title><p>Cryopreserved and thawed PBMCs (1 &#x000d7; 10⁶ cells per well) were adapted with OpTmizer CTS medium (Life technologies) for more than 6&#x02009;h at 37&#x02009;&#x000b0;C, 5% CO₂. PBMCs were plated and stimulated with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration 2&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 in RPMI 1,640 containing 10% fetal bovine serum, 100&#x02009;U&#x02009;ml^&#x02212;1 penicillin and 100&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 streptomycin for 5 days. &#x003b1;-CD3 mAb and the medium alone served as positive and negative controls, respectively. After 3 days, cell cultures were replaced with 100&#x02009;&#x003bc;l of fresh medium. At the end of culture, cells were washed with PBS for subsequent immunostaining and polychromatic flow cytometric analysis. The cells were stained with CD19-FITC (5&#x02009;&#x003bc;l per test, HIB19, Biolegend), CD4-PerCPCy5.5, CD8-PECy7, CD38-BV421 (5&#x02009;&#x003bc;l per test, HIT2, BD Bioscience), CD3-BV605, Ki-67-PE (20&#x02009;&#x003bc;l per test, B56, BD Bioscience), and Live/Dead-APCCy7. Responses at least threefold greater than those of the medium control were considered to be positive. A post-analysed vaccine-induced response was defined as being at least threefold higher in the percentage of antigen-specific proliferating CD8 T cells than that at prevaccination.</p></sec><sec disp-level="2"><title>Statistical analysis</title><p>Descriptive statistics of the safety, pharmacodynamics and pharmacokinetic outcomes was performed using SAS (V9.1) software. Standard and two-tailed paired Student&#x02019;s <italic>t</italic>-test was performed to analyse statistical significance of all quantitative data using Prism 5.0 software (GraphPad).</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>T.J.K., H.-T.J., S.-Y.H., Y.-Y.H., Y.S.S., J.S.P. and Y.C.S. designed study; T.J.K., H.-T.J., H.G.Y., Y.B.S., S.R.H., C.-W.L., S.K., K.S.P., I.-H.L., K.-T.L. and M.S.J. performed experiments and analysed data; T.J.K., H.-T.J., S.-Y.H., Y.B.S., J.-W.W., J.P., C.D.S., Y.S.S., J.S.P. and Y.C.S. wrote the paper.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article</bold>: Kim, T. J. <italic>et al.</italic> Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients. <italic>Nat. Commun.</italic> 5:5317 doi: 10.1038/ncomms6317 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-5, Supplementary Tables 1-4, Supplementary Methods and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6317-s1.pdf"/></supplementary-material></sec>

Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

<p>The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. <italic>De novo</italic> methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.</p>

<contrib contrib-type="author"><name><surname>Gilsbach</surname><given-names>Ralf</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Preissl</surname><given-names>Sebastian</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Gr&#x000fc;ning</surname><given-names>Bj&#x000f6;rn A.</given-names></name><xref ref-type="aff" rid="a3">3</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Schnick</surname><given-names>Tilman</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Burger</surname><given-names>Lukas</given-names></name><xref ref-type="aff" rid="a6">6</xref><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Benes</surname><given-names>Vladimir</given-names></name><xref ref-type="aff" rid="a8">8</xref></contrib><contrib contrib-type="author"><name><surname>W&#x000fc;rch</surname><given-names>Andreas</given-names></name><xref ref-type="aff" rid="a9">9</xref></contrib><contrib contrib-type="author"><name><surname>B&#x000f6;nisch</surname><given-names>Ulrike</given-names></name><xref ref-type="aff" rid="a9">9</xref></contrib><contrib contrib-type="author"><name><surname>G&#x000fc;nther</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Backofen</surname><given-names>Rolf</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Fleischmann</surname><given-names>Bernd K.</given-names></name><xref ref-type="aff" rid="a10">10</xref></contrib><contrib contrib-type="author"><name><surname>Sch&#x000fc;beler</surname><given-names>Dirk</given-names></name><xref ref-type="aff" rid="a6">6</xref><xref ref-type="aff" rid="a11">11</xref></contrib><contrib contrib-type="author"><name><surname>Hein</surname><given-names>Lutz</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a12">12</xref></contrib><aff id="a1"><label>1</label><institution>Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg</institution>, Albertstrasse 25, 79104 Freiburg, <country>Germany</country></aff><aff id="a2"><label>2</label><institution>Hermann Staudinger Graduate School, University of Freiburg</institution>, Albertstrasse 21, 79104 Freiburg, <country>Germany</country></aff><aff id="a3"><label>3</label><institution>Bioinformatics Group, Department of Computer Science, University of Freiburg</institution>, Georges-K&#x000f6;hler-Allee 106, 79110 Freiburg, <country>Germany</country></aff><aff id="a4"><label>4</label><institution>Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences, University of Freiburg</institution>, Hermann-Herder-Strasse 9, 79104 Freiburg, <country>Germany</country></aff><aff id="a5"><label>5</label><institution>University Heart Center Freiburg/Bad Krozingen, Department of Congenital Heart Defects and Paediatric Cardiology, University of Freiburg</institution>, Hugstetter Strasse 55, 79106 Freiburg, <country>Germany</country></aff><aff id="a6"><label>6</label><institution>Friedrich Miescher Institute for Biomedical Research</institution>, Maulbeerstrasse 66, 4058 Basel, <country>Switzerland</country></aff><aff id="a7"><label>7</label><institution>Swiss Institute of Bioinformatics</institution>, Maulbeerstrasse 66, 4058 Basel, <country>Switzerland</country></aff><aff id="a8"><label>8</label><institution>European Molecular Biology Laboratory, Genomics Core Facility</institution>, Meyerhofstra&#x000df;e 1, 69117 Heidelberg, <country>Germany</country></aff><aff id="a9"><label>9</label><institution>Max Planck Institute of Immunobiology and Epigenetics</institution>, St&#x000fc;beweg 51, 79108 Freiburg, <country>Germany</country></aff><aff id="a10"><label>10</label><institution>Institute of Physiology I, Life and Brain Center, University of Bonn</institution>, Sigmund-Freud-Stra&#x000df;e 25, 53127 Bonn, <country>Germany</country></aff><aff id="a11"><label>11</label><institution>University of Basel</institution>, Petersplatz 1, 4003 Basel, <country>Switzerland</country></aff><aff id="a12"><label>12</label><institution>BIOSS Centre for Biological Signalling Studies, University of Freiburg</institution>, Sch&#x000e4;nzlestrasse 18, 79104 Freiburg, <country>Germany</country></aff>

Nature Communications

<p>During its development and postnatal life, the heart has to adapt to diverse physiological and pathophysiological needs. Cardiomyocytes arise from progenitors in early development and mature with limited cell division after birth<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. The transition from pre- to postnatal life demands a number of contractile and metabolic adaptations to facilitate organ growth in the presence of optimal contractile function<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. Due to its limited regenerative capacity, cardiomyocytes respond to these challenges during development and disease with characteristic gene expression programmes. Several epigenetic processes, including microRNAs<xref ref-type="bibr" rid="b6">6</xref>, chromatin and histone proteins<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref> as well as DNA methylation<xref ref-type="bibr" rid="b10">10</xref>, have been implicated as modulators of cardiac gene expression in development and disease.</p><p>DNA methylation is a stable hallmark of cell type identity and is essential for mammalian development<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>. It occurs mainly in palindromic CpG dinucleotides. Whereas most regions of the genome are depleted for CpGs, they are clustered in CpG islands. CpG islands mark 70% of annotated mammalian promoters<xref ref-type="bibr" rid="b13">13</xref>. CpG methylation is essential for proper gene expression, development and genome stability<xref ref-type="bibr" rid="b14">14</xref>. DNA methylation patterns are maintained during cell division by DNA methyltransferase 1 (DNMT1). <italic>De novo</italic> DNA methylation is mediated by DNMT3A and DNMT3B<xref ref-type="bibr" rid="b15">15</xref>. Removal of DNA methylation involves oxidation of 5-methyl-cytosine. The key enzymes for this initial step are the recently discovered ten-eleven translocation enzymes TET1-3 (ref. <xref ref-type="bibr" rid="b16">16</xref>). So far there is limited knowledge about the <italic>in vivo</italic> time course of DNA methylation pattern establishment in cardiomyocytes<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>.</p><p>Here we show that DNA methylation is highly dynamic during cardiomyocyte development, postnatal maturation and disease. Demethylated regions in neonatal and adult cardiomyocytes are localized in cell type-specific enhancer regions and in gene bodies of cardiomyocyte genes. Postnatal DNA demethylation correlates with active histone marks and increased gene expression. Repression of demethylated genes is achieved by polycomb-mediated histone H3K27 trimethylation or by <italic>de novo</italic> methylation by DNA methyltransferases DNMT3A/B. Dynamic DNA methylation is important for the perinatal switch in sarcomere protein isoforms and postnatal cardiomyocyte maturation and adaptation.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Epigenetic characterization of cardiomyocytes</title><p>To adapt to the requirements of the growing organism, cardiomyocytes increase dramatically in size during physiological postnatal growth (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). In response to cardiac injury, for example, during chronic ventricular pressure overload, cardiomyocytes may initiate further pathological growth (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). To provide insight into the dynamics of DNA methylation in development and disease of cardiomyocytes as a prototypical terminally differentiated cell type, we generated DNA methylomes of newborn, adult healthy and adult failing cardiomyocytes.</p><p>Since the heart is a complex tissue with cardiomyocytes contributing only 20&#x02013;30% of the total cell population<xref ref-type="bibr" rid="b19">19</xref>, we purified cardiomyocyte nuclei from murine cardiac tissue. An antibody against pericentriolar material 1 (PCM1)<xref ref-type="bibr" rid="b20">20</xref> was used to isolate cardiomyocyte nuclei using flow cytometry (fluorescence-activated cell sorting (FACS)) or magnetic-assisted nuclei sorting with very high purity (&#x0003e;97%; <xref ref-type="fig" rid="f1">Fig. 1b</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1a&#x02013;c</xref>). To validate the specificity of PCM1 staining, we used transgenic mice with cardiomyocyte-specific histone H2B-mCherry expression (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1d</xref>). Isolated cardiac nuclei of these animals and wild-type mice were stained with PCM1 antibody or isotype control IgG (immunoglobulin G) and were analysed by FACS (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1e&#x02013;g</xref>). The overlap of mCherry and PCM1-positive nuclei was &#x0003e;95% confirming that PCM1 specifically stains cardiomyocyte nuclei (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1g</xref>). Furthermore, a molecular beacon targeting cardiac troponin T type 2 (<italic>Tnnt2</italic>) mRNA stained the major fraction of PCM1-positive nuclei (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1h</xref>). Analysis of different developmental and disease stages revealed that the proportion of cardiomyocyte nuclei decreased from 68.7&#x000b1;0.6% in newborn hearts to 29.7&#x000b1;0.5% in adult healthy hearts and to 19.7&#x000b1;0.7% in adult failing hearts (mean&#x000b1;s.e.m., <italic>n</italic>=6; <xref ref-type="fig" rid="f1">Fig. 1c</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1i&#x02013;k</xref>). DNA from these purified cardiomyocyte nuclei was bisulfite converted and DNA methylomes were obtained at base-pair resolution using deep sequencing. Three independent experiments with high reproducibility (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2</xref>) resulted in high-coverage methylomes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 1</xref>). DNA methylomes from murine embryonic stem (ES) cells<xref ref-type="bibr" rid="b21">21</xref> and from total cardiac tissue<xref ref-type="bibr" rid="b10">10</xref> were used for comparison.</p><p>To gain further insight into the epigenetic landscape, we generated maps for four histone modifications (H3K4me1, H3K4me3, H3K27ac and H3K27me3) from purified cardiomyocyte nuclei (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 2</xref>). To generate matching cardiomyocyte-specific transcriptomes, hearts were digested enzymatically, vital cardiomyocytes were sorted by FACS and cardiomyocyte mRNA was subjected to RNA sequencing.</p></sec><sec disp-level="2"><title>Differential CpG methylation</title><p>Visual inspection of two prototypical cardiomyocyte genes, &#x003b1;- and &#x003b2;-myosin heavy chains (<italic>Myh6</italic> and <italic>Myh7</italic>), revealed a progressive CpG demethylation in cardiomyocytes purified from newborn as compared with adult and failing hearts (<xref ref-type="fig" rid="f1">Fig. 1d</xref>). Almost complete demethylation of gene body CpGs of <italic>Myh6</italic> and <italic>Myh7</italic> was apparent in purified cardiomyocyte nuclei but could not be detected in cardiac tissue<xref ref-type="bibr" rid="b10">10</xref>, which contains several additional cell types (<xref ref-type="fig" rid="f1">Fig. 1d</xref>). To further validate this finding, methylation of CpGs of the cardiomyocyte-specific gene encoding for the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2A, gene <italic>Atp2a2</italic>) was analysed by pyrosequencing (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). <italic>Atp2a2</italic> methylation levels greatly differed between myocyte and non-myocyte cell fractions with intermediate levels in cardiac tissue (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a&#x02013;d</xref>).</p><p>In total, we identified 79,655 differentially methylated regions (DMRs) with an average size of 840&#x02009;bp when comparing adult healthy cardiomyocytes and undifferentiated ES cells (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5</xref>). This amounted to 5% of all CpGs in the mouse genome (<xref ref-type="fig" rid="f1">Fig. 1e</xref>). Ninety percent of these CpGs were hypomethylated and 10% were hypermethylated in cardiomyocytes versus ES cells (<xref ref-type="fig" rid="f1">Fig. 1e</xref>). Remarkably, a substantial number of CpGs were differentially methylated between newborn and adult cardiomyocytes (6,436 DMRs; <xref ref-type="fig" rid="f1">Fig. 1e</xref>). These data indicate that differential CpG methylation was not restricted to cell type specification during embryonic development, but extended well into the postnatal period of cardiomyocytes. Genomic annotation of cardiomyocyte DMRs showed a predominance for intragenic and CpG island-flanking regions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>).</p></sec><sec disp-level="2"><title>Hypomethylated regions with cardiomyocyte enhancer signature</title><p>Previous studies have identified transcription factor-binding sites and histone marks, which are characteristic for enhancers in demethylated regions<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>. This prompted us to analyse the enrichment of transcription factor (TF)-binding motifs in DMRs. Demethylated regions in ES cells (<xref ref-type="fig" rid="f1">Fig. 1f</xref>) were significantly enriched (<italic>P</italic>&#x0003c;10^&#x02212;20, hypergeometric test) for binding sites of stem cell transcription factors including OCT4 (gene symbol <italic>Pou5f1</italic>), NANOG and others (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 1</xref>). In contrast, demethylated regions of adult cardiomyocytes were significantly enriched (<italic>P</italic>&#x0003c;10^&#x02212;50, hypergeometric test) for TF motifs of known cardiac transcription factors including MEF2C, GATA1-4 and others (<xref ref-type="fig" rid="f1">Fig. 1f</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7a&#x02013;c</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 1</xref>). These TF included the recently identified master regulators of cellular identity of ES cells or hearts, respectively<xref ref-type="bibr" rid="b23">23</xref>. In the demethylated state, TF-binding regions were found to be enriched for histone modifications (H3K4me1, H3K27ac), which are typical for active <italic>cis</italic>-regulatory sites<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b24">24</xref> (<xref ref-type="fig" rid="f1">Fig. 1f</xref>). In line with being regulatory, these DMRs were located within distal regulatory regions of genes characteristic for ES cells or cardiomyocytes, respectively (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7d,e</xref>).</p></sec><sec disp-level="2"><title>Genic CpG demethylation affects specific gene programmes</title><p>Since many sites of dynamic DNA methylation occurred within genic regions (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>), we explored this aspect in more detail (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>). At 765 genes, genic DMRs extended to more than 25% of the entire gene body (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>). In 78 cases, gene bodies were demethylated in ES cells, yet they were methylated to a higher degree in cardiomyocytes (<xref ref-type="fig" rid="f2">Fig. 2b</xref>, group I; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). Gene ontology analysis of this group revealed a predominance of genes involved in embryonic pattern specification and morphogenesis (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8</xref>) containing known pluripotency factors like <italic>Pou5f1</italic> and <italic>Nanog</italic> (<xref ref-type="fig" rid="f2">Fig. 2c</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). However, the inverse case with 687 genes, which were demethylated in cardiomyocytes versus ES cells, was much more prevalent (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>, group II). This group was highly enriched in genes involved in cardiac transcriptional regulation, cardiac development, muscle contraction and energy supply (<xref ref-type="fig" rid="f2">Fig. 2d&#x02013;f</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 9</xref>). Genes that were further demethylated during postnatal maturation of cardiomyocytes were involved in cardiac contraction or mitochondrial function (<xref ref-type="fig" rid="f2">Fig. 2e,f</xref>).</p></sec><sec disp-level="2"><title>Genic CpG methylation correlates with gene expression</title><p>To gain more insight into the kinetics of DNA methylation, we analysed the time course of gene body demethylation of the <italic>Atp2a2</italic> gene by pyrosequencing (<xref ref-type="fig" rid="f3">Fig. 3a&#x02013;f</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4</xref>). Demethylation of <italic>Atp2a2</italic> in cardiomyocytes was initiated at the 5&#x02032; end and progressed towards the 3&#x02032; region of the gene (<xref ref-type="fig" rid="f3">Fig. 3a&#x02013;d</xref>). While the 5&#x02032; end of the gene body and upstream TF motifs were already demethylated at birth, the 3&#x02032; region lost its CpG methylation by adulthood (<xref ref-type="fig" rid="f3">Fig. 3b&#x02013;f</xref>). Gene body demethylation correlated with increased expression of <italic>Atp2a2</italic> during postnatal cardiomyocyte maturation (<xref ref-type="fig" rid="f3">Fig. 3g</xref>). Adult failing cardiomyocytes expressed lower levels of <italic>Atp2a2</italic> than adult control myocytes (<xref ref-type="fig" rid="f3">Fig. 3g</xref>) without significant changes in gene body methylation (<xref ref-type="fig" rid="f3">Fig. 3b&#x02013;g</xref>).</p><p>To search for a possible relationship between gene body CpG methylation status and gene expression, we ranked all genes according to their expression level in adult cardiomyocytes and determined the changes in gene expression and DNA methylation after birth (<xref ref-type="fig" rid="f4">Fig. 4a,b</xref>). Those genes that showed the greatest increase in expression after birth (<xref ref-type="fig" rid="f4">Fig. 4a</xref>; expression increase &#x0003e;250 fragments per kilobase of transcript per million reads, FPKM) were most strongly demethylated at their gene bodies during postnatal development (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). Genes with demethylation of gene bodies after birth were involved in several metabolic processes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 10</xref>). Inactive genes showed a higher level of CpG methylation at the promoter and the first exon when compared with expressed genes (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). CpG methylation of the first exon and the remainder of the gene was inversely correlated with gene expression (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). Actively expressed genes were decorated with H3K4me1, H3K27ac and H3K4me3 and they lacked H3K27me3 (<xref ref-type="fig" rid="f4">Fig. 4d&#x02013;g</xref>). In contrast, gene bodies of inactive genes showed abundant H3K27me3 in addition to high levels of CpG methylation (<xref ref-type="fig" rid="f4">Fig. 4c,g</xref>). These genes were grouped into distinct functional categories (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 11</xref>). Taken together, these data demonstrate that a demethylation wave runs through the gene body of actively transcribed genes. This wave is initiated at the 5&#x02032; end of cardiomyocyte genes, becomes further extended until adulthood and correlates with gene expression.</p></sec><sec disp-level="2"><title>Polycomb-mediated silencing of early developmental genes</title><p>The group of genes, which were demethylated in their gene bodies in cardiomyocytes as compared with ES cells (<xref ref-type="fig" rid="f2">Fig. 2a,b</xref>, group II), contained genes that were known to be expressed during cardiac development but showed no significant expression in adult cardiomyocytes. Thus, we asked whether specific histone marks characterize demethylated genes that are inactive in postnatal cardiomyocytes. First, two prototypic genes with either high levels of expression in adult cardiomyocytes (<italic>Tnnt2</italic>) or expression restricted to early cardiac development (<italic>Isl1</italic>) were analysed (<xref ref-type="fig" rid="f5">Fig. 5a,b</xref>). Visual inspection of the <italic>Tnnt2</italic> locus revealed a demethylated gene body in adult cardiomyocytes, which was covered with active chromatin marks H3K27ac, H3K4me3 and H3K4me1 (<xref ref-type="fig" rid="f5">Fig. 5a</xref>). The cardiac differentiation factor <italic>Isl1</italic> gene, which is expressed at embryonic day (E) 8.5<xref ref-type="bibr" rid="b25">25</xref> but not in postnatal cardiomyocytes, remained demethylated until adulthood too. Thus, DNA methylation was not restored on gene inactivation. However, the <italic>Isl1</italic> gene was covered with the repressive chromatin mark H3K27me3 in newborn (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 12</xref>) and adult cardiomyocytes (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). The gain of this repressive mark set by the enzymatically active subunit enhancer of zeste homologue 2 (EZH2) of the polycomb repressive complex 2 (PRC2) was not restricted to this gene but rather typical for demethylated genes that were not expressed (&#x0003c;1 FPKM; <xref ref-type="fig" rid="f2">Fig. 2a</xref>, group II) in adult cardiomyocytes (<xref ref-type="fig" rid="f5">Fig. 5c</xref>, group 1). Affected genes were expressed either in early embryogenesis, such as the transcription factors of the Hox cluster<xref ref-type="bibr" rid="b26">26</xref>, or at early stages of cardiac development, such as <italic>Isl1 and Pitx2</italic> (ref. <xref ref-type="bibr" rid="b27">27</xref>).</p><p>We asked whether the affected genes were already marked by H3K27me3 in developmental stages. Analysis of H3K27me3 in newborn cardiomyocytes as well as reanalysis of previously published data sets<xref ref-type="bibr" rid="b28">28</xref> were performed. These data revealed enrichment of H3K27me3 in embryonic hearts at E12.5 (ref. <xref ref-type="bibr" rid="b28">28</xref>) and in newborn cardiomyocytes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 12a</xref>). In addition, EZH2 was found to be localized at these genes in embryonic hearts at E12.5 (ref. <xref ref-type="bibr" rid="b28">28</xref>) (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 12a</xref>). Recently, the functional importance of PRC2 for proper heart development was shown by cardiac ablation of EZH2 (refs <xref ref-type="bibr" rid="b28">28</xref>, <xref ref-type="bibr" rid="b29">29</xref>). Remarkably, a subset of genes including <italic>Cdkn2a</italic>, <italic>Isl1</italic> and the previously described polycomb target genes <italic>Six1</italic> and <italic>Pax6</italic> (refs <xref ref-type="bibr" rid="b28">28</xref>, <xref ref-type="bibr" rid="b29">29</xref>), which were decorated by H3K27me3, showed increased gene expression on EZH2 ablation<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 12b&#x02013;e</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). Interestingly, these alterations were more evident in E12.5 hearts as compared with right ventricles of adult hearts<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref> (<xref ref-type="supplementary-material" rid="S1">Supplementary Table 3</xref>). In contrast, none of the CpG-demethylated and actively transcribed genes (&#x0003e;250 FPKM; <xref ref-type="fig" rid="f2">Fig. 2a</xref>, group II) were marked by H3K27me3, but all of these genes were decorated with H3K27ac, H3K4me3 and H3K4me1 (<xref ref-type="fig" rid="f5">Fig. 5c</xref>, group 2).</p></sec><sec disp-level="2"><title>Postnatal <italic>de novo</italic> DNA methylation requires DNMT3A/B</title><p>Postnatal maturation of cardiomyocytes was not only accompanied by demethylation of gene bodies but also by <italic>de novo</italic> DNA methylation (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). Overall, 127 genes gained methylation and 313 lost methylation of their gene bodies during the postnatal period (<xref ref-type="fig" rid="f6">Fig. 6a</xref>). These changes in postnatal DNA methylation of gene bodies correlated inversely with gene expression (<xref ref-type="fig" rid="f6">Fig. 6a</xref>; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). Genes that were hypermethylated in cardiomyocytes after birth were involved in myocyte contraction (<xref ref-type="fig" rid="f6">Fig. 6b</xref>), cardiac morphogenesis, cell differentiation and other processes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 11</xref>). Functional adaptation of the heart during the postnatal period includes isoform switches in the expression of sarcomere proteins<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. As an example, we analysed DNA methylation and expression of the fetal and adult troponin I isoforms that are encoded by <italic>Tnni1</italic> and <italic>Tnni3</italic> genes, respectively (<xref ref-type="fig" rid="f6">Fig. 6b&#x02013;d</xref>). The gene body of <italic>Tnni1</italic> was demethylated at birth and it was <italic>de novo</italic> methylated in adult cardiomyocytes (<xref ref-type="fig" rid="f6">Fig. 6b,c</xref>). Postnatal <italic>de novo</italic> DNA methylation correlated with repression of <italic>Tnni1</italic> expression (<xref ref-type="fig" rid="f6">Fig. 6c</xref>). In contrast, the adult troponin I isoform <italic>Tnni3</italic> was demethylated and turned on in its expression after birth (<xref ref-type="fig" rid="f6">Fig. 6b,d</xref>). To test whether postnatal DNA methylation and gene repression were mediated by <italic>de novo</italic> DNA methyltransferases 3A/B, mice with cardiac myocyte-specific deletion of the <italic>Dnmt3a</italic> and <italic>Dnmt3b</italic> genes were generated and analysed (<xref ref-type="fig" rid="f6">Fig. 6c,d</xref>). Ablation of DNMT3A/B expression completely prevented postnatal <italic>de novo</italic> methylation of <italic>Tnni1</italic> and partially relieved repression of this gene (<xref ref-type="fig" rid="f6">Fig. 6c</xref>). Similarly, postnatal DNA methylation of the sarcomere components <italic>Tpm2</italic> and the glucose transporter GLUT1 gene (<italic>Slc2a1</italic>) was mediated by DNMT3A/B (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 13</xref>). Thus, isoform switching of sarcomere proteins during postnatal life was accompanied by <italic>de novo</italic> methylation of fetal genes and demethylation of adult isoforms (<xref ref-type="fig" rid="f6">Fig. 6b</xref>).</p><p>As postnatal cardiomyocyte maturation is accompanied by cell cycle arrest<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>, we analysed postnatal CpG methylation dynamics of all genes, which were contained in the gene ontology group &#x02018;cell cycle&#x02019; (GO:0007049; <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14</xref>). Twenty-one out of these 1,279 genes in this group were differentially methylated during postnatal development (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14a</xref>). Most of the differentially methylated cell cycle-associated genes showed opposite changes of DNA methylation and RNA expression after birth (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14b</xref>) as further illustrated for <italic>Ccnd3</italic> and <italic>Bmp7</italic> (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14c,d</xref>). Both genes showed their highest expression levels at E14.5. Postnatal <italic>de novo</italic> DNA methylation of <italic>Ccnd3</italic> and <italic>Bmp7</italic> was absent in cardiomyocytes from <italic>Dnmt3a/b</italic>-deficient mice. However, in these cases, <italic>Dnmt3a/b</italic> ablation did not affect gene expression (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 14c,d</xref>).</p><p>Analysis of expression of the group of cell cycle-associated genes revealed that 502 genes were differentially expressed in adult versus newborn cardiomyocytes (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 15a</xref>). Thus, we asked whether polycomb-mediated gene silencing is involved in the regulation of these cell cycle genes. However, postnatally repressed cell cycles genes were not decorated with the repressive mark H3K7me3 and did not show increased genic CpG methylation (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 15b</xref>).</p></sec><sec disp-level="2"><title>DNA methylation changes after chronic pressure overload</title><p>To explore how the cardiac methylome changes in heart disease, we analysed differential CpG methylation of cardiomyocyte nuclei that were purified from failing murine hearts after chronic left ventricular pressure overload (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 16a&#x02013;e</xref>). Compared with developmental alterations, the observed changes in CpG methylation were smaller in scale (<xref ref-type="fig" rid="f7">Fig. 7a,b</xref>). We therefore adjusted the cutoff criteria for DMR detection to 20% differential methylation. Using these less-stringent criteria, we identified 5,346 disease-associated DMRs (<xref ref-type="fig" rid="f7">Fig. 7c</xref>). These DMRs were mostly intergenic (<xref ref-type="fig" rid="f7">Fig. 7d</xref>). Disease-associated DMRs (5.9%) showed an overlap with enhancer or promoter regions marked by H3K27ac, H3K4me1 and H3K4me3 in adult cardiomyocytes (<xref ref-type="fig" rid="f7">Fig. 7e</xref>). Disease-associated DMRs overlapping with postnatal DMRs were adjacent to genes involved in cardiac muscle cell development, cardiac morphogenesis and energy metabolism (<xref ref-type="fig" rid="f7">Fig. 7f</xref>), indicating adaptation of DNA methylation in disease-relevant regions. Methylation levels of these disease-associated DMRs partially resembled the newborn CpG methylation pattern (<xref ref-type="fig" rid="f7">Fig. 7g</xref>).</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>The present study resolves the DNA methylome of cardiomyocytes at base-pair resolution from embryonic development to postnatal maturation and disease. For this purpose, cardiomyocyte nuclei were highly purified from frozen heart tissue by FACS and magnetic-assisted sorting. One of the most intriguing features of the cardiomyocyte DNA methylome is a demethylation wave running through gene bodies of cardiomyocyte genes, which orchestrates with <italic>de novo</italic> DNA methylation and activation of polycomb marks to shape the epigenome of maturing cardiomyocytes after birth (<xref ref-type="fig" rid="f8">Fig. 8</xref>).</p><p>Bisulfite sequencing has uncovered distinct patterns of DNA methylation in different genomic contexts, including promoters, gene bodies and regulatory elements<xref ref-type="bibr" rid="b12">12</xref>. We identified several distinct features of DNA methylation also in cardiomyocytes. When compared with ES cells as a model for an undifferentiated, pluripotent cell type, cardiomyocytes contained short regions of low DNA methylation, which showed characteristics of <italic>cis</italic>-regulatory elements. Tissue-specific DMRs were previously identified for a large number of human and murine cell and tissue types<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b22">22</xref>. Demethylated regions contained binding motifs for tissue-specific transcription factors and contained histone marks that are characteristic for poised (H3K4me1) or active (H3K27ac) enhancers<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b22">22</xref>. Our data demonstrate that demethylation of enhancers was already established in newborn cardiomyocytes and contained cardiac-specific transcription factor motifs that are characteristic for super-enhancers of the cardiac lineage<xref ref-type="bibr" rid="b23">23</xref>. These cardiomyocyte-specific sites were characterized by H3K4me1 marks and part of them also contained H3K27ac, thus identifying them as poised or active enhancers<xref ref-type="bibr" rid="b24">24</xref>, respectively. Previous studies have suggested that transcription factor activity could be a key factor for active DNA demethylation of enhancer regions<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b32">32</xref>. Thus, the DNA methylomes presented in this study provide comprehensive enhancer maps of cardiomyocytes during development and maturation, which extends the previous studies in cardiac tissues.</p><p>As a second characteristic feature of the cardiomyocyte DNA methylome, we identified large demethylated regions that covered entire gene bodies of large cardiomyocyte genes, for example, the myosin heavy-chain locus or the <italic>Atp2a2</italic> gene (<xref ref-type="fig" rid="f8">Fig. 8</xref>, upper trace). Some of the longest demethylated regions were identified in the genes encoding for the cardiac ryanodine receptor (<italic>Ryr2</italic>, 180&#x02009;kbp), the &#x003b1;_1C-subunit of the L-type Ca²⁺ channel (<italic>Cacna1c</italic>, 162&#x02009;kbp) or titin (<italic>Ttn</italic>, 121&#x02009;kbp; <xref ref-type="supplementary-material" rid="S1">Supplementary Data 2</xref>). The significance of DNA methylation in gene bodies is still only partially understood<xref ref-type="bibr" rid="b12">12</xref> and studies have shown both positive or negative correlations between genic DNA methylation and gene expression. In neurons<xref ref-type="bibr" rid="b33">33</xref>, hematopoietic stem cells<xref ref-type="bibr" rid="b34">34</xref> and other lineages<xref ref-type="bibr" rid="b35">35</xref>, demethylation of regions containing the transcription start and gene bodies was identified in highly expressed genes. In contrast, a positive correlation between CpG methylation and gene expression was identified in human ES cells<xref ref-type="bibr" rid="b36">36</xref>, mouse oocytes<xref ref-type="bibr" rid="b37">37</xref> or B lymphocytes<xref ref-type="bibr" rid="b38">38</xref>. In the present study, gene bodies were demethylated starting at the 5&#x02032; end and extending towards the 3&#x02032; region during postnatal maturation of cardiomyocytes. Genic demethylation correlated well with active histone marks and increased expression of cardiomyocyte genes.</p><p>However, not all genes that were demethylated in cardiomyocytes versus ES cells were also expressed in postnatal cardiomyocytes. More than one hundred genes were DNA demethylated and decorated by trimethylation of H3K27 (<xref ref-type="fig" rid="f8">Fig. 8</xref>, middle trace). This histone mark is associated with polycomb-mediated gene repression, and EZH2 is one of the core proteins of the PRC2 in the heart<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. Deficiency of EZH2 in cardiomyocytes caused lethal heart malformations<xref ref-type="bibr" rid="b28">28</xref> or postnatal cardiomyopathy<xref ref-type="bibr" rid="b29">29</xref>, depending on the time of EZH2 ablation. Binding of EZH2 and H3K27me3 could be identifed at E12.5 and H3K27me3 marks persisted at the same loci in newborn and adult cardiomyocytes. Demethylated genes that were transiently expressed during embryonic development, for example, <italic>Isl1</italic> (ref. <xref ref-type="bibr" rid="b25">25</xref>) and <italic>Pitx2</italic> (ref. <xref ref-type="bibr" rid="b27">27</xref>), were stably repressed by H3K27me3. However, deletion of <italic>Ezh2</italic> led to reactivation of <italic>Isl1</italic>, <italic>Pitx2</italic> and other developmental genes<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. It is interesting to note that H3K27me3 was specifically associated with repressed and demethylated genes in cardiomyocytes. This finding is supported by previous studies that have detected H3K27me3-mediated gene repression in genomic regions lacking DNA methylation<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref>. Most recently, it was shown that polycomb may selectively be recruited to unmethylated DNA sequences and transcriptional activity or CpG methylation prevented polycomb binding and function<xref ref-type="bibr" rid="b39">39</xref>.</p><p>As a second mechanism for repression of demethylated cardiomyocyte genes, we identified <italic>de novo</italic> CpG methylation mediated by DNA methyltransferases 3A/B (<xref ref-type="fig" rid="f8">Fig. 8</xref>, lower trace). Genes that were hypo- or hypermethylated in cardiomyocytes after birth were involved in multiple physiological processes including the transition from fetal to adult sarcomeric gene expression. To adapt to the specific demands of pre- versus postnatal life, several protein isoforms of the sarcomere are switched during the perinatal phase by an unknown mechanism<xref ref-type="bibr" rid="b5">5</xref>. Indeed, we found the slow skeletal troponin I isoform (<italic>Tnni1</italic>) to be repressed by postnatal DNA methylation, which was mediated by DNMT3A/B. Cardiomyocyte-specific ablation of DNMT3A/B completely prevented <italic>Tnni1</italic> methylation after birth and partially reactivated gene expression. The perinatal switch from fetal to adult sarcomere protein isoforms has been associated with important aspects of myocyte development and contractile function<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. In particular, the decrease in Ca²⁺ sensitivity of the sarcomere has been linked with the transition from slow skeletal (<italic>Tnni1</italic>) to cardiac troponin I (<italic>Tnni3</italic>)<xref ref-type="bibr" rid="b4">4</xref>. In addition to the fetal sarcomere isoforms, genes involved in the fetal to adult transition of energy metabolism<xref ref-type="bibr" rid="b3">3</xref> and several other process were differentially methylated in their gene body after birth. These findings suggest that dynamic gene methylation may play an important role in cardiomyocyte switching and maturation after birth.</p><p>To determine possible alterations in the DNA methylome during cardiac disease, we have subjected mice to 3 weeks of cardiac pressure overload. Constriction of the aortic arch resulted in ventricular hypertrophy, interstitial fibrosis and pulmonary oedema, thus partially resembling the phenotype of chronic human heart failure. While DNA methylation in failing cardiomyocytes returned to a fetal methylation pattern, the observed changes were less promiment and abundant compared with physiological postnatal myocyte maturation. Thus, future studies will be important to determine whether the human cardiomyocyte DNA methylome shows similar features during development and in disease. Recent studies have investigated DNA methylation in human cardiac tissue biopsies from patients with chronic heart failure of different etiologies<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. Both studies described distinct signatures of DNA methylation in failing versus non-failing hearts. The present study describes a path to assign such alterations to a specific cell type in the heart. Future studies will be important to uncover the DNA methylomes of other cardiac cell types, for example, fibroblasts, endothelial cells, immune cells and so on, to determine their epigenetic contribution to cardiac development and disease.</p><p>The present study establishes DNA methylation in cardiomyocytes as dynamic and reversible during development, postnatal maturation and disease. Future studies unravelling the signals and mechanisms involved in shaping the cardiac DNA methylome will be essential for a better understanding of cardiac myocyte biology.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Animal procedures</title><p>All animal procedures were approved by the responsible animal care committee (Regierungspr&#x000e4;sidium Freiburg, Germany) and they conformed to the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences, 2011. Mice with cardiac myocyte-specific ablation of DNMT3A/B expression were generated by crossing <italic>Dnmt3a</italic>^{<italic>flox</italic>} and <italic>Dnmt3b</italic>^{<italic>flox</italic>} mice<xref ref-type="bibr" rid="b40">40</xref> with mice expressing cre recombinase under control of the cardiac <italic>Mlc2a</italic> promoter<xref ref-type="bibr" rid="b41">41</xref>. Male mice of the indicated ages were dissected to obtain cardiac tissues. To induce cardiac pressure overload by transverse aortic constriction for 3 weeks, male mice (8 weeks old) were anaesthetized with 2% (vol) isoflurane in oxygen. After thoracotomy, a 7.0 silk suture was placed around a 27-G hypodermic needle to constrict the transverse aorta<xref ref-type="bibr" rid="b42">42</xref>.</p></sec><sec disp-level="2"><title>ES cells</title><p>Murine ES cells, which were originally derived from blastocysts of mixed 129&#x02014;C57BL6 background<xref ref-type="bibr" rid="b21">21</xref>, were maintained in ES cell medium (DMEM, 15% fetal calf serum (FCS), non-essential amino acids, 10&#x02009;ng&#x02009;ml^&#x02212;1 leukaemia inhibiting factor, 0.1% &#x003b2;-mercaptoethanol) on gelatine-coated plates<xref ref-type="bibr" rid="b21">21</xref>.</p></sec><sec disp-level="2"><title>Echocardiography</title><p>Echocardiography was performed using a Vivid 7 Dimension (GE Healthcare, Munich, Germany) echocardiograph equipped with a 14-MHz transducer<xref ref-type="bibr" rid="b42">42</xref>. Parasternal short-axis views were used for M-mode analysis. Enddiastolic and endsystolic left ventricular inner diameters (LVIDd, LVIDs) were measured and fractional shortening (FS) and ejection fraction (EF) were calculated using the following equations: FS=(LVIDd&#x02014;LVIDs)/LVID, EF=(LVIDd³&#x02212;LVIDs³)/LVIDd³ (ref. <xref ref-type="bibr" rid="b42">42</xref>).</p></sec><sec disp-level="2"><title>Histology</title><p>Hearts were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), embedded in paraffin, cut into 3&#x02009;&#x003bc;m slices and stained with haematoxylin&#x02013;eosin, Sirius-red or fluorescent wheat germ agglutinin (WGA; Alexa Fluor 488 conjugate, Invitrogen, Karlsruhe, Germany). Nuclei were counterstained with propidium iodide (Sigma, Munich, Germany). For immunohistochemical staining, hearts were snap frozen, embedded in optimum cutting temperature (OCT) medium (Tissue-Tek), cut into 3&#x02009;&#x003bc;m slices and stained with an antibody against PCM1 (1:1,000, HPA023370, Sigma) in combination with a Cy3-labelled anti-rabbit antibody (1:1,000, Invitrogen). Glycocalyx was stained with WGA (Alexa Fluor 488 conjugate, Invitrogen) and nuclei were counterstained with DAPI (4&#x02032;,6-diamidino-2-phenylindole, Invitrogen).</p></sec><sec disp-level="2"><title>Flow cytometric analysis and sorting of cardiomyocyte nuclei</title><p>Cardiomyocyte nuclei were isolated from cardiac tissue<xref ref-type="bibr" rid="b20">20</xref> and were stained by an antibody directed against PCM1 (1:1,000, HPA023370, Sigma) and an Alexa488-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) or a molecular beacon targeting mRNA<xref ref-type="bibr" rid="b43">43</xref> of <italic>Tnnt2</italic> (<xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>; 50&#x02009;nM) for 30&#x02009;min. Nuclei were identified by 7-aminoactinomycin D (7-AAD) (1:500, Invitrogen). For analysis of &#x003b1;MHC-H2B-mCherry transgenic mice, an Alexa647-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) and for nuclei-staining DAPI (1:1,000, Invitrogen) was used. Nuclei were analysed or sorted by flow cytometry (CyFlow Space, Partec, M&#x000fc;nster, Germany; BD Influx cell sorter, BD Biosciences, Heidelberg, Germany; Bio-Rad S3 cell sorter, Bio-Rad Laboratories, Munich, Germany).</p></sec><sec disp-level="2"><title>Magnetic-assisted sorting</title><p>Magnetic-assisted cell sorting (MACS) of myocyte nuclei was performed after isolation and staining of nuclei with a PCM1 antibody (1:1,000, HPA023370, Sigma). Nuclei were centrifuged and resuspended in 80&#x02009;&#x003bc;l MACS buffer. Anti-rabbit IgG MicroBeads (20&#x02009;&#x003bc;l; Miltenyi, Bergisch Gladbach, Germany) were added and incubated for 15&#x02009;min at 4&#x02009;&#x000b0;C. MACS buffer (900&#x02009;&#x003bc;l) was added and the nuclei suspension was applied to an M column (Miltenyi), washed with 3&#x02009;ml MACS buffer and eluted with 1&#x02009;ml MACS buffer after removal of the magnet. The eluate was applied to another M column, washed again and eluted in 1&#x02009;ml elution buffer (1&#x02009;mM EDTA in PBS). Sorting efficiency was determined by staining aliquots before and after cell separation with an Alexa488-labelled secondary anti-rabbit antibody (1:1,000, Invitrogen) and by flow cytometric analysis (CyFlow Space, Partec).</p></sec><sec disp-level="2"><title>Isolation of cardiomyocytes for transcriptome analysis</title><p>Adult and neonatal hearts were dissociated to obtain a single-cell suspension<xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref>. Adult hearts were dissociated by retrograde perfusion with Tyrode&#x02019;s solution supplemented with 25&#x02009;mM butanedione monoxime, 2&#x02009;mM CaCl₂, 0.8&#x02009;mg&#x02009;ml^&#x02212;1 collagenase B (Roche, Mannheim, Germany), 0.4&#x02009;mg&#x02009;ml^&#x02212;1 hyaluronidase (Sigma) and 3&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 trypsin (Sigma) for 12&#x02009;min. Hearts were gently dissected and enzymatic digestion was stopped by addition of Tyrode&#x02019;s buffer supplemented with 5% FCS, 25&#x02009;mM butanedione monoxime and 2&#x02009;mM CaCl₂. Newborn hearts were dissociated by several rounds of digestion with trypsin (Gibco) in Hank&#x02019;s Balanced Salt Solution (HBSS, Life Technologies). Digestion was stopped by resuspension in HBSS containing 4% FCS. Cell suspensions of adult and neonatal hearts were kept at 4&#x02009;&#x000b0;C until sorting. Sorting of cardiomyocytes from cardiac cell suspensions was carried out at 4&#x02009;&#x000b0;C using a Bio-Rad S3 cell sorter. Cardiomyocytes and non-cardiomyocytes were visualized using the cell-permeable nucleid acid stain Vybrant DyeCycle Ruby (Life Technologies). Non-viable cells were identified by counterstaining with the cell-impermeable nucleid acid stains Sytox Green or 7-AAD (Life Technologies). Cardiomyocytes were discriminated from other cardiac cells by high forward scatter (FSC) signals. FSC pulse width was used to exclude doublets from sorting. Gates were selected to sort viable cardiomyocytes directly into RLT lysis buffer (Qiagen, Hilden, Germany). To confirm the gating strategy, small aliquots were permeabilized with 0.1% saponin (Sigma) and stained with antibodies against cardiac troponin I (ab47003, Abcam) and &#x003b1;-actinin (At7811, Sigma) in combination with secondary Alexa Fluor-coupled antibodies (Life Technologies).</p></sec><sec disp-level="2"><title>External data sets</title><p>Previously published MethylC-seq data from murine ES cells (GSE30206)<xref ref-type="bibr" rid="b21">21</xref> and murine adult heart (GSM1051154)<xref ref-type="bibr" rid="b10">10</xref> were downloaded, mapped and analysed in the same way as the methylomes generated in this study. Previously published chromatin immunoprecipitation-sequencing (ChIP-seq) data for H3K27ac (GSM851290) and H3K4me1 (GSM851287) for embryonic heart (E14.5)<xref ref-type="bibr" rid="b46">46</xref> and ES cells (GSM594577 and GSM594579)<xref ref-type="bibr" rid="b24">24</xref> were reanalysed. ChIP-seq data for H3K27me3 and EZH2 for E12.5 hearts (GSE29994) were reanalyzed<xref ref-type="bibr" rid="b28">28</xref>. RNA-seq data of <italic>Ezh2</italic> mutant mice and corresponding controls were reanalyzed (GSE29992)<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>.</p></sec><sec disp-level="2"><title>Whole-genome bisulfite sequencing</title><p>MethylC-seq was performed as described<xref ref-type="bibr" rid="b36">36</xref>. In brief, 1&#x02009;&#x003bc;g of genomic DNA was extracted from purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen). DNA-seq libraries were prepared with methylation adapters (ILMN ME-100-0010, Illumina, San Diego, USA), bisulfite conversion was carried out with the EZ DNA methylation kit (D5001, Zymo Research, Freiburg, Germany) for 16&#x02009;h and final PCR was performed with the KAPA Uracil+ system (16 cycles, KAPAbiosystems KK2801). All libraries were sequenced on a paired-end 101-bp sequencing run (Illumina HiSeq 2000).</p></sec><sec disp-level="2"><title>CpG methylation analysis</title><p>All bioinformatic tools used in this study were integrated into the Galaxy web server<xref ref-type="bibr" rid="b47">47</xref><xref ref-type="bibr" rid="b48">48</xref><xref ref-type="bibr" rid="b49">49</xref>. The generated data format is compliant with the BED file format. Sequencing reads were trimmed (Phred quality score&#x000b1;20) and mapped using Bismark<xref ref-type="bibr" rid="b50">50</xref> (bowtie 1 (ref. <xref ref-type="bibr" rid="b51">51</xref>), mouse genome assembly mm9) and PCR duplicates were removed with SAMtools<xref ref-type="bibr" rid="b52">52</xref>. For further analysis, we developed a set of bioinformatic tools (Methtools; <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/bgruening/methtools">https://github.com/bgruening/methtools</ext-link>), including differential methylation analysis. Results for the sense and antisense strand were combined after calling of CpG methylation values. Only CpGs with a minimal coverage of 4 were included in further analysis steps. In case of ES cell data<xref ref-type="bibr" rid="b21">21</xref>, SNPs between C57BL/6J and 129S5 mouse strains were filtered. Methylation values were smoothed using a running average of three consecutive CpGs. For determination of DMRs of two methylomes (DMRs), data sets were intersected to exclude CpGs determined in only one data set using Bedtools<xref ref-type="bibr" rid="b53">53</xref>. A sliding window approach was used to identify DMRs. Regions displaying a mean CpG methylation delta &#x02265;40% over five CpGs between methylomes were selected as DMRs. Additional exclusion criteria were a minimal difference of 10% for each individual CpG and a maximal distance of 1,000&#x02009;bp between adjacent CpGs. Selected regions were extended stepwise until they failed to fulfil the cutoff criteria and were then reported as DMRs. In case of disease-associated DMRs, a mean delta of 20% or greater was accepted. Differential gene body methylation between ES cells and adult cardiomyocytes was identified by DMRs covering &#x0003e;25% of a gene body with a minimal DMR length of 1&#x02009;kbp. For comparison of newborn and adult cardiomyocytes, the cutoff criterion was &#x0003e;5% of the gene body. Genes smaller than 500&#x02009;bp were excluded from all analyses. The Integrative Genomics Viewer<xref ref-type="bibr" rid="b54">54</xref>, Bioconductor<xref ref-type="bibr" rid="b55">55</xref> and deepTools<xref ref-type="bibr" rid="b56">56</xref> were used for visualization.</p></sec><sec disp-level="2"><title>Motif analysis and binding site prediction</title><p>Location of transcription factor-binding sites and motif enrichments within DMRs were determined by the Homer tool<xref ref-type="bibr" rid="b57">57</xref> using default parameters. The known motifs used in this study were derived from the Homer tool and were validated by ChIP-seq. Motifs lacking this experimental proof were omitted. HAND1/HAND2 motifs were derived from JASPAR.</p></sec><sec disp-level="2"><title>Pyrosequencing</title><p>Genomic DNA (500&#x02009;ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). The concentration of converted DNA was adjusted to 5&#x02009;ng&#x02009;&#x003bc;l^&#x02212;1 and DNA was amplified with the PyroMark PCR Kit (Qiagen) using a three-primer approach with a universal biotinylated primer<xref ref-type="bibr" rid="b58">58</xref>. PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument<xref ref-type="bibr" rid="b59">59</xref> and quantified with the PyroMark Q24 software (Qiagen). For each assay, a standard curve of 0, 25, 50, 75 and 100% methylated DNA was measured. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the CpG methyltransferase M.Sssl (NEB, Frankfurt, Germany) was used to generate methylated DNA. Primer sequences are listed in <xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>.</p></sec><sec disp-level="2"><title>Annotation of <italic>cis</italic>-regulatory regions</title><p>Prediction of functional significance of regions was performed using the GREAT tool<xref ref-type="bibr" rid="b60">60</xref>. The basal gene regulatory domain was defined as the region starting 5&#x02009;kbp upstream and ending 1&#x02009;kbp downstream of the TSS. The gene regulatory domain was extended in both directions up to 50&#x02009;kbp to the nearest gene&#x02019;s basal domain.</p></sec><sec disp-level="2"><title>Gene ontology analysis</title><p>ClueGO<xref ref-type="bibr" rid="b61">61</xref> was used to find over-represented GO terms in the categories &#x02018;biological process&#x02019;, &#x02018;cellular component&#x02019; and &#x02018;molecular function&#x02019;. Benjamini&#x02013;Hochberg correction was performed for multiple testing-controlled <italic>P</italic> values. Significantly enriched terms were functionally grouped and visualized. The GO term connectivity threshold was adjusted to &#x0003e;0.3 (kappa value). The highest significant term of the group was displayed as leading term.</p></sec><sec disp-level="2"><title>CpG island and genomic annotation</title><p>Random sequences from MethylC-seq-assessable genomic regions were selected to analyse genome representation of genomic features and CpG islands. Genomic feature annotation was performed using Homer tools<xref ref-type="bibr" rid="b57">57</xref>. CpG island annotation<xref ref-type="bibr" rid="b62">62</xref> and UCSC gene prediction annotation<xref ref-type="bibr" rid="b63">63</xref> were downloaded from UCSC.</p></sec><sec disp-level="2"><title>Gene expression analysis</title><p>Polyadenylated RNA was isolated from 500&#x02009;ng of total RNA with magnetic beads (NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB). Libraries were constructed (NEBNext Ultra RNA Library Prep Kit for Illumina, NEB) and size selection was performed with AMPure XP Beads (Beckman Coulter, Krefeld, Germany). Thirteen PCR cycles were used for library amplification and were sequenced on a HiSeq 2500 (50&#x02009;bp, Illumina, San Diego, USA). RNA-seq data were mapped to the mm9 genome assembly using Tophat<xref ref-type="bibr" rid="b64">64</xref>. FPKM values were calculated by Cufflinks<xref ref-type="bibr" rid="b65">65</xref> as an estimate of transcript expression. Differential gene expression was calculated using Cuffdiff<xref ref-type="bibr" rid="b65">65</xref>. The analysis was performed with default parameters and the UCSC gene annotation<xref ref-type="bibr" rid="b63">63</xref> as a reference. Quantitative real-time PCR was carried out as described<xref ref-type="bibr" rid="b66">66</xref>. Primer sequences are listed in <xref ref-type="supplementary-material" rid="S1">Supplementary Data 3</xref>.</p></sec><sec disp-level="2"><title>ChIP-sequencing</title><p>ChIP-seq was performed as described with modifications<xref ref-type="bibr" rid="b67">67</xref>. Lysis buffers for preparation of nuclei were supplemented with 10&#x02009;mM sodium butyrate (Sigma). MACS-purified nuclei were fixed with 1% freshly prepared paraformaldehyde (Sigma) for 2&#x02009;min at room temperature. Fixation was stopped by adding glycine to a final concentration of 125&#x02009;mM. Nuclei were washed three times with PBS and were lysed in SDS buffer (50&#x02009;mM Tris&#x02013;HCl, pH 8.0, 10&#x02009;mM EDTA, 1% SDS) supplemented with protease inhibitors (Roche Complete Protease Inhibitor Cocktail, Roche). Chromatin was sheared using a Bioruptor (Diagenode, Liege, Belgium) with 30 high-energy cycles (30&#x02009;s on/30&#x02009;s off) to an average size of 200&#x02013;400&#x02009;bp. Lysates were diluted 1:10 with buffer (22&#x02009;mM Tris&#x02013;HCl, pH 8.0, 165&#x02009;mM NaCl, 2.2&#x02009;mM EDTA, 1.1% Triton X-100) and were precleared with 0.1% bovine serum albumin for 2&#x02009;h at 4&#x02009;&#x000b0;C. Antibodies were preincubated with Protein A Dynabeads (Invitrogen) for 2&#x02009;h at 4&#x02009;&#x000b0;C. For immunoprecipitation, either 5&#x02009;&#x003bc;g of chromatin was incubated with antibodies directed against H3K4me3 (2&#x02009;&#x003bc;g, pAb-003-050, Diagenode) and H3K27ac (2&#x02009;&#x003bc;g, ab4729, Abcam, Cambridge, UK) overnight at 4&#x02009;&#x000b0;C or 2.5&#x02009;&#x003bc;g of chromatin was incubated with an anti-H3K4me1 antibody (1&#x02009;&#x003bc;g, ab8895, Abcam) or an anti-H3K27me3 antibody (1&#x02009;&#x003bc;g, pAb-069-050, Diagenode). Immunocomplexes were washed for 10&#x02009;min each with buffer 1 (20&#x02009;mM Tris&#x02013;HCl, pH 8.0, 150&#x02009;mM NaCl, 2&#x02009;mM EDTA, 1% Triton X-100, 0.1% SDS), buffer 2 (20&#x02009;mM Tris&#x02013;HCl, pH 8.0, 500&#x02009;mM NaCl, 2&#x02009;mM EDTA, 1% Triton X-100, 0.1% SDS), buffer 3 (20&#x02009;mM Tris&#x02013;HCl, pH 8.0, 250&#x02009;mM LiCl, 2&#x02009;mM EDTA, 1% IGEPAL CA-630, 1% sodium deoxycholate) and three times with TE buffer (20&#x02009;mM Tris&#x02013;HCl, pH 8.0, 2&#x02009;mM EDTA) at 4&#x02009;&#x000b0;C. Elution from beads was carried out by shaking in elution buffer (100&#x02009;mM NaHCO₃, 1% SDS) at 1,400&#x02009;r.p.m. at room temperature for 1&#x02009;h. DNA was treated with 50&#x02009;ng&#x02009;&#x003bc;l^&#x02212;1 RNAse A (Sigma) for 30&#x02009;min at 37&#x02009;&#x000b0;C and crosslinks were reversed by adding 0.5&#x02009;&#x003bc;g&#x02009;&#x003bc;l^&#x02212;1 proteinase K (AppliChem, Darmstadt, Germany) and incubation at 65&#x02009;&#x000b0;C for 5&#x02009;h. DNA was purified using phenol/chloroform followed by ethanol precipitation at &#x02212;20&#x02009;&#x000b0;C overnight. DNA was quantified using Quant-iT PicoGreen dsDNA Reagent (Invitrogen). Sequencing libraries were prepared from 5&#x02009;ng DNA with the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB) with 15 PCR cycles and sequenced on a HiSeq 2500 (50&#x02009;bp, Illumina, San Diego, USA).</p></sec><sec disp-level="2"><title>ChIP-seq data analysis</title><p>ChIP-seq reads were mapped to the mm9 genome assembly by Bowtie2 (ref. <xref ref-type="bibr" rid="b68">68</xref>). For read coverage of input and histone modifications, the genome was binned. The number of reads per bin was counted and normalized to the total number of mappable reads (reads per kilobase per million mapped reads, RPKM). The enrichment of histone modifications in a given genomic region was calculated as the log2 ratio of ChIP RPKM and input RPKM. Peak calling was performed using MACS2 (ref. <xref ref-type="bibr" rid="b69">69</xref>).</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>R.G. and S.P. designed and performed the experiments. T.S. performed surgery and physiology. V.B. and U.B. performed sequencing and A.W. performed FACS. B.A.G. developed bioinformatic tools and B.A.G., R.G. and S.P. performed computational analysis. L.B., S.G., R.B. and B.K.F. interpreted the data and edited the manuscript. L.H. conceived the project and L.H., D.S., R.G. and S.P. wrote the manuscript.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>Accession codes:</bold> DNA methylomes, ChIP-Seq and RNA-seq files have been submitted to the NCBI BioSample databases under accessions SRP033288 (MethylC-seq), SRP033385 (ChIP-seq) and SRP033386 (RNA-seq).</p><p><bold>How to cite this article:</bold> Gilsbach, R. <italic>et al.</italic> Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease. <italic>Nat. Commun.</italic> 5:5288 doi: 10.1038/ncomms6288 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-16, Supplementary Tables 1-3 and Supplementary References</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6288-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Data 1</title><p>Analysis of transcription factor (TF) enrichment of hypo- and hypermethylated DMRs in adult cardiomyocytes as compared with ES cells.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6288-s2.xlsx"/></supplementary-material><supplementary-material id="d33e30" content-type="local-data"><caption><title>Supplementary Data 2</title><p>Life of genes with differential gene body methylation</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6288-s3.xlsx"/></supplementary-material><supplementary-material id="d33e36" content-type="local-data"><caption><title>Supplementary Data 3</title><p>Primer sequences.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6288-s4.xlsx"/></supplementary-material></sec>

Presynaptic GABAergic inhibition regulated by BDNF contributes to neuropathic pain induction

<p>The gate control theory proposes the importance of both pre- and post-synaptic inhibition in processing pain signal in the spinal cord. However, although postsynaptic disinhibition caused by brain-derived neurotrophic factor (BDNF) has been proved as a crucial mechanism underlying neuropathic pain, the function of presynaptic inhibition in acute and neuropathic pain remains elusive. Here we show that a transient shift in the reversal potential (<italic>E</italic>_GABA) together with a decline in the conductance of presynaptic GABA_A receptor result in a reduction of presynaptic inhibition after nerve injury. BDNF mimics, whereas blockade of BDNF signalling reverses, the alteration in GABA_A receptor function and the neuropathic pain syndrome. Finally, genetic disruption of presynaptic inhibition leads to spontaneous development of behavioural hypersensitivity, which cannot be further sensitized by nerve lesions or BDNF. Our results reveal a novel effect of BDNF on presynaptic GABAergic inhibition after nerve injury and may represent new strategy for treating neuropathic pain.</p>

<contrib contrib-type="author"><name><surname>Chen</surname><given-names>Jeremy Tsung-chieh</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Da</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Campanelli</surname><given-names>Dario</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Frattini</surname><given-names>Flavia</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Mayer</surname><given-names>Florian</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Luming</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kuner</surname><given-names>Rohini</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Heppenstall</surname><given-names>Paul A.</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Knipper</surname><given-names>Marlies</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Hu</surname><given-names>Jing</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Centre for Integrative Neuroscience</institution>, Otfried-Mueller-Strasse 25, 72076 T&#x000fc;bingen, <country>Germany</country></aff><aff id="a2"><label>2</label><institution>Hearing Research Centre</institution>, Elfriede Aulhornstrasse 5, 72076 T&#x000fc;bingen, <country>Germany</country></aff><aff id="a3"><label>3</label><institution>Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research</institution>, 72076 T&#x000fc;bingen, <country>Germany</country></aff><aff id="a4"><label>4</label><institution>Pharmacology Institute, University of Heidelberg</institution>, Im Neuenheimer Feld 584, 69120 Heidelberg, <country>Germany</country></aff><aff id="a5"><label>5</label><institution>Mouse Biology Unit, European Molecular Biology Laboratory (EMBL)</institution>, Via Ramarini 32, 00016 Monterotondo, <country>Italy</country></aff>

Nature Communications

<p>It has been almost half a century since the gate control theory of pain was first proposed by Melzack and Wall<xref ref-type="bibr" rid="b1">1</xref>. This theory suggests that nociceptive sensory information transmitted to the brain is normally under strong pre- and postsynaptic inhibitory control in the spinal cord. The gate control theory further predicts that under pathological conditions a disruption of the spinal inhibition efficacy may contribute to chronic pain development. Both pre- and postsynaptic inhibitions are controlled by local inhibitory interneurons and inhibitory descending fibres<xref ref-type="bibr" rid="b2">2</xref>. GABA_A receptors (GABA_AR) located in primary afferent terminals of nociceptors cause reduction in transmitter release, thus modulating the afferent input from dorsal root ganglion (DRG) neurons into nociceptive-specific projection neurons (presynaptic inhibition), whereas postsynaptic GABA_AR in the spinal cord neurons directly reduce their excitability and therefore control the output (postsynaptic inhibition). Although there is growing evidence indicating that spinal inhibition contribute to physiological and pathological pain sensation<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>, the precise contribution of different mode of inhibition (pre versus post) in various pain modalities remains elusive. This is mainly due to the difficulty to selectively manipulate pre- or postsynaptic inhibition<xref ref-type="bibr" rid="b9">9</xref>.</p><p>Unlike neurons in adult central nervous system, under physiological conditions, DRG neurons have a rather high <italic>E</italic>_GABA, which determines a depolarizing effect by GABA_AR activation. This primary afferent depolarization (PAD) is involved in the presynaptic inhibitory control of pain possibly through inactivation of voltage-gated sodium or calcium channels and through activation of a shunting conductance, which ultimately inhibits the propagation of action potential into presynapse, thus the transmitter release<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b10">10</xref>. It has been proposed that under pathological conditions, such as tissue or nerve injury, a depolarizing shift of <italic>E</italic>_GABA in DRG neurons, which may result in an enhanced PAD sufficient to directly evoke an action potential, would lead to an excitatory effect of presynaptic GABA_AR activation and cause touch-evoked pain<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>. Indeed, after nerve injury, a depolarizing shift of <italic>E</italic>_GABA in DRG neurons has been observed<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. However, it remains unclear whether this shift switches presynaptic inhibition into excitation and contributes to neuropathic pain.</p><p>Recently, the loss of postsynaptic inhibition caused by brain-derived neurotrophic factor (BDNF) has been shown as an important substrate for neuropathic pain<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>. Peripheral tissue or nerve damage increases the expression of BDNF in DRG and the spinal cord<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. A growing body of evidence has demonstrated that BDNF binding to its high-affinity receptor, TrkB receptor, is involved in both induction and maintenance of neuropathic pain<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>. BDNF released from activated microglia in the spinal cord dorsal horn binding to TrkB receptors on nociceptive-specific projection neurons causes downregulation of the neuronal potassium-chloride cotransporter KCC2, impairing Cl^&#x02212; homeostasis and leading to a depolarizing shift in <italic>E</italic>_GABA. This shift diminishes the postsynaptic inhibition mediated by GABA_AR. Both presynaptic primary afferent fibres and postsynaptic neurons in dorsal horn of the spinal cord have been shown to express full-length TrkB receptor<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>. However, although the diminishing effect of postsynaptic BDNF-TrkB signalling after injury on postsynaptic inhibition has been well characterized, the consequence of BDNF binding to presynaptic TrkB receptors and the effect on presynaptic inhibition remains elusive.</p><p>Here we show that immediately after nerve injury there is a transient depolarizing shift in <italic>E</italic>_GABA and a reduction in the conductance of presynaptic GABA_AR (<italic>G</italic>_GABA) in DRG neurons, together leading to a reduction of presynaptic inhibition but not a switch to excitation. Two-photon imaging of calcium signal explicitly in primary afferent terminals in the spinal cord confirms the reduction of GABA-mediated presynaptic inhibition. Exogenous BDNF mimics the alteration in both GABA_AR function and the neuropathic pain syndrome, whereas blocking BDNF and its receptor TrkB signalling reverse the change of GABA_AR function and the reduced nociceptive threshold after nerve injury. To specifically determine the function of presynaptic inhibition in pain behaviour, we engineered mice deficient in GABA_AR in primary nociceptors. These mice display increased sensitivity to both mechanical and thermal stimuli. Unlike the wild-type (WT) littermates, the thermal hypersensitivity in these mice cannot be further sensitized by nerve lesions or BDNF. Our results provide the first <italic>in vivo</italic> evidence that presynaptic inhibition is required for setting the sensitivity of pain behaviour under physiological condition and indicate that after nerve injury BDNF-modulated loss of presynaptic inhibition, but not switch to excitation, is crucial for the neuropathic pain initiation.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Transient disinhibition in DRG neurons after nerve injury</title><p>To investigate the alteration of GABA_AR after nerve injury, we first induced neuropathic pain in mice by making chronic constriction injury (CCI) to the sciatic nerve (<xref ref-type="fig" rid="f1">Fig. 1a,b</xref>). We observed that both mechanical and thermal hypersensitivity had reached the peak effect 2 days post injury (d.p.i.) and persisted for at least 4 weeks tested. Therefore, we decided to first measure the <italic>E</italic>_GABA of DRG neurons prepared from 2 d.p.i. or intact animals by performing gramicidin-perforated patch recordings. We analysed the data separately for large (&#x0003e;25&#x02009;&#x003bc;m diameter, putative mechanoreceptors) and small (&#x02264;25&#x02009;&#x003bc;m diameter, putative nociceptors) neurons (<xref ref-type="fig" rid="f1">Fig. 1c&#x02013;f</xref>)<xref ref-type="bibr" rid="b23">23</xref>. Responses to exogenous GABA (&#x003b3;-aminobutyric acid) application showed that <italic>E</italic>_GABA was &#x02212;37.5&#x000b1;2.4&#x02009;mV in control large neurons (diameter: 29.0&#x000b1;0.6&#x02009;&#x003bc;m; <italic>n</italic>=20) and &#x02212;35.2&#x000b1;2.5&#x02009;mV in small neurons (diameter: 21.4&#x000b1;0.8&#x02009;&#x003bc;m, <italic>n</italic>=15). Two days post nerve injury, a significant depolarization shift of <italic>E</italic>_GABA was observed in both groups of neurons (<xref ref-type="fig" rid="f1">Fig. 1d,e</xref>). In large neurons (29.8&#x000b1;0.8&#x02009;&#x003bc;m, <italic>n</italic>=15) <italic>E</italic>_GABA shifted to &#x02212;28.3&#x000b1;3.4&#x02009;mV (<italic>P</italic>&#x0003c;0.05) and in small neurons, which often respond to noxious stimuli (20.4&#x000b1;0.7&#x02009;&#x003bc;m, <italic>n</italic>=17), <italic>E</italic>_GABA was &#x02212;22.7&#x000b1;2.4&#x02009;mV (<italic>P</italic>&#x0003c;0.01). To determine whether this shift persists as long as the neuropathic pain syndrome, we further measured the <italic>E</italic>_GABA at different time points after CCI. Surprisingly, we found this depolarizing shift occurred immediately (2&#x02009;h) after nerve injury but already started to recover 7 d.p.i. and reached control levels at 21 d.p.i., although the neuropathic pain symptoms still persisted (<xref ref-type="fig" rid="f1">Fig. 1e</xref>). Thus, after nerve injury, the <italic>E</italic>_GABA in both small and large neurons underwent a transient depolarizing shift, which might result in an enhanced PAD sufficient to directly evoke an action potential, indicating a transient reduction of GABA_AR-mediated presynaptic inhibition or even a switch to excitation.</p><p>To confirm the <italic>E</italic>_GABA shift after nerve injury is due to the change of Cl^&#x02212; homeostasis in DRG neurons, we performed two-photon Cl^&#x02212; imaging in acutely isolated DRG from transgenic mouse carrying a chloride sensor Clomeleon. Two days after nerve injury, a decrease in Clomeleon ratio (yellow fluorescent protein (YFP)/cyan flurescent protein (CFP)), corresponding to an increase in intracellular chloride, was observed in both large and small neurons (<italic>P</italic>&#x0003c;0.0001 for both, <xref ref-type="fig" rid="f2">Fig. 2a,b</xref>).</p><p>Previous reports have indicated that the Cl^&#x02212; homeostasis in DRG neurons is mainly due to the sodium potassium chloride co-transporter (NKCC1)<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref>; we therefore examined the effect of injury on DRG NKCC1 expression. We first analysed the change of NKCC1 protein level in whole DRG. Immunoblotting result showed a trend but not significant increase of NKCC1 expression (<xref ref-type="supplementary-material" rid="S1">Supplementary Figs 1 and 2</xref>). NKCC1 activity is largely determined by the amount of NKCC1 proteins present on the plasma membrane and post-translational modifications. Therefore, we next quantified the NKCC1 membrane proteins in DRG neurons by performing immunohistochemistry and found the cell surface NKCC1 level was significantly higher in DRG neurons 2 days after injury than in control (<xref ref-type="fig" rid="f2">Fig. 2c,d</xref>). This result demonstrates that NKCC1 is responsible for the increase in [Cl^&#x02212;]_i induced by nerve injury.</p><p>In vertebrate primary afferent axons, activation of presynaptic GABA_AR causes depolarization and increases membrane conductance (shunting). It has been shown that either PAD or shunting is sufficient to produce inhibition<xref ref-type="bibr" rid="b26">26</xref>. To examine the change of shunting effect, we calculated the peak <italic>G</italic>_GABA from the plot of GABA-induced peak current versus holding potential (<xref ref-type="fig" rid="f1">Fig. 1d,f</xref>). The GABA-mediated conductance in large neurons (12.0&#x000b1;2.0&#x02009;ns, <italic>n</italic>=20) was significantly higher than in small neurons (4.8&#x000b1;0.8&#x02009;ns, <italic>n</italic>=15, <italic>P</italic>&#x0003c;0.005) taken from naive animals. Two days after injury, there was a significant reduction in both large (6.1&#x000b1;0.9&#x02009;ns, <italic>n</italic>=15, <italic>P</italic>&#x0003c;0.05) and small neurons (2.5&#x000b1;0.3&#x02009;ns, <italic>n</italic>=17, <italic>P</italic>&#x0003c;0.05) (<xref ref-type="fig" rid="f1">Fig. 1f</xref>). This result is consistent with previous reports that GABA-mediated current strength decreased and messenger RNA of GABA_AR was significantly downregulated in DRG neurons after injury<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. Similar to the time course of <italic>E</italic>_GABA shift, significant reduction of GABA-mediated conductance was only detected 2 days after injury and started to recover from 1 week on (<xref ref-type="fig" rid="f1">Fig. 1f</xref>), indicating a transient reduction of shunting-mediated presynaptic inhibition.</p><p>Taken together, the early transient shift of <italic>E</italic>_GABA and reduction of <italic>G</italic>_GABA, both lead to the same alteration of GABA_AR function after nerve injury, that is, a loss of inhibitory effect. It has been reported that after nerve injury, the shift of <italic>E</italic>_GABA could switch postsynaptic inhibition into excitation<xref ref-type="bibr" rid="b16">16</xref>. This raised the question here whether the depolarizing shift of <italic>E</italic>_GABA in presynaptic primary sensory neurons would also lead to excitatory effect, while the <italic>G</italic>_GABA is reduced. To address this, we first performed Ca²⁺ imaging of Fura-2-AM-loaded DRG neurons from intact and 2 d.p.i. animals. Exogenous GABA caused an intracellular Ca²⁺ concentration increase in 35% (<italic>n</italic>=37) of large neurons and 43% (<italic>n</italic>=207) of small neurons. Two days after nerve injury, unlike the postsynaptic GABA_AR activation<xref ref-type="bibr" rid="b16">16</xref>, this proportion did not increase (<xref ref-type="fig" rid="f2">Fig. 2e&#x02013;g</xref>, <italic>P</italic>&#x0003e;0.05 for both large and small neurons). This result suggests that after nerve injury the GABA_AR activation did not induce Ca²⁺ influx in more neurons and become excitatory. This is mostly because accompanying the depolarizing shift of <italic>E</italic>_GABA there is a reduction of <italic>G</italic>_GABA, which might limit the increase of PAD. We further explored this idea more directly by performing gramicidin-perforated patch recordings under current clamp mode. Administration of exogenous GABA induced a similar depolarization in DRG neurons from both intact and injured animals (<xref ref-type="fig" rid="f2">Fig. 2h</xref>). In addition, only in a few neurons (control: 3 of 14 large and 2 of 20 small neurons; 2 d.p.i.: 0 of 8 large and 1 of 14 small neurons) could an action potential be directly evoked by GABA application, indicating a non-excitatory effect of GABA_AR activation. Thus, our results here showed that after nerve injury, unlike postsynaptic disinhibition, presynaptic inhibition is not switched to excitation but rather abolished via reduction of the shunting effect. The similar phenomena has been reported by Wei and colleagues (2013) in a trigeminal neuropathic pain model where they observed a disinhibition and excitation effect on pre- and postsynaptic GABA actions, respectively<xref ref-type="bibr" rid="b29">29</xref>.</p></sec><sec disp-level="2"><title>Loss of presynaptic inhibition after injury</title><p>Although cultured DRG neurons have long been used as a model in evaluating the pathogenic mechanisms of peripheral neuropathies<xref ref-type="bibr" rid="b30">30</xref>, does the shift of <italic>E</italic>_GABA and reduction of <italic>G</italic>_GABA on the soma of DRG neurons we observed here really represent what happens in the primary afferent central terminal, where presynaptic inhibition locates, after nerve injury? To answer this question, we double stained spinal cord sections with antibodies to &#x003b1;1-GABA_AR and calcitonin gene-related peptide and found that indeed 2 days after nerve injury there is a significant reduction of presynaptic GABA_AR expression (<xref ref-type="fig" rid="f3">Fig. 3a&#x02013;g</xref>). Furthermore, we have checked the NKCC1 expression level on the primary afferent central terminal after nerve injury. Both immunoblotting and immunohistochemical staining results showed a trend but not significant increase of NKCC1 (<xref ref-type="fig" rid="f3">Fig. 3h&#x02013;l</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). It has been shown that a nerve injury induced increase of the phosphorylated NKCC1 in DRG accounts for the intracellular Cl^&#x02212; accumulation<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b31">31</xref>. This might explain why we could not detect an increase of overall NKCC1 expression level in either whole DRG or the spinal cord. To further confirm the involvement of NKCC1 in the hypersensitive behaviour developed after nerve injury, we have treated 2, 14 and 21 d.p.i. animals with either saline or NKCC1 inhibitor bumetanide. Administration of bumetanide significantly increased the paw withdrawal latency time in 2 d.p.i. mice. This anti-hyperalgesic effect of bumetanide became much less profound in 14 d.p.i. mice and was completely absent in 21 d.p.i. mice, confirming that a transient upregulation of NKCC1 activity after nerve injury contributes to neuropathic pain initiation (<xref ref-type="fig" rid="f3">Fig. 3m&#x02013;o</xref>).</p><p>The final consequence of presynaptic inhibition is to reduce the calcium influx, thus the transmitter release from presynapses. To investigate the functional change of presynaptic GABA_AR in the spinal cord after nerve injury, we sought to perform two-photon calcium imaging specifically in the primary afferent terminals. To target genetically encoded calcium sensors to primary afferents, we crossed mice carrying the floxed <italic>GCaMP3</italic> allele (Ai38)<xref ref-type="bibr" rid="b32">32</xref> to a mouse line expressing Cre recombinase from the locus of the sensory neuron-specific gene <italic>Advillin</italic> (advillin-Cre)<xref ref-type="bibr" rid="b33">33</xref> (<xref ref-type="fig" rid="f4">Fig. 4a,b</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4a,b</xref>). To record the calcium transients in the primary afferent terminals, we performed two-photon imaging on the spinal cord slices taken from control or 2 d.p.i. mouse. Direct application depolarizing concentration of KCl to the spinal cord slice elicited robust fluorescence increases in the primary afferent terminals from both control and 2 d.p.i. mice (<xref ref-type="fig" rid="f4">Fig. 4c,d,g,h,o&#x02013;r</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Movies 1 and 2</xref>). Next, we applied GABA to test whether GABA alone could be excitatory and induce calcium transient. In either the control or the 2 d.p.i. spinal cord slices, we did not observe much excitatory effect of GABA (control: 5.0&#x000b1;2.1%, 712 regions of interest (ROIs) from 4 slices from 4 animals; injury: 2.3&#x000b1;1.2%, 566 ROIs from 5 slices from 5 animals; <italic>P</italic>&#x0003e;0.05; <xref ref-type="fig" rid="f4">Fig. 4e,i,o&#x02013;r,u</xref>). Consistent with our finding on the cultured DRG neurons, this result suggests that after nerve injury the activation of presynaptic GABA_AR alone does not become excitatory, not sufficient enough to directly induce Ca²⁺ influx, thus the transmitter release from presynapses. Finally, to test the inhibitory effect of GABA on presynaptic activity, we applied GABA together with depolarizing concentration of KCl. GABA exhibited an inhibitory effect on KCl-induced fluorescence increase in only 11% of the ROIs from injured animals, in contrast with 57% of the ROIs from control mice (control: 1,205 ROIs from 5 slices from 5 animals; injury: 1,115 ROIs from 6 slices from 6 animals; <italic>P</italic>&#x0003c;0.0001; <xref ref-type="fig" rid="f4">Fig. 4f,j,o&#x02013;r,v&#x02013;w</xref>), indicating a substantial loss of presynaptic inhibition after nerve injury.</p></sec><sec disp-level="2"><title>Abnormal pain behaviour in <italic>sns- &#x003b2;3</italic> ^{&#x02212;<italic>/</italic>&#x02212;}mouse</title><p>Although we have shown that after nerve injury there is an early transient shift of <italic>E</italic>_GABA and reduction of <italic>G</italic>_GABA leading to a reduced presynaptic inhibition, its relevance to the induction of neuropathic pain behaviour remains unclear. To address the functional importance of GABA_AR-mediated presynaptic inhibition <italic>in vivo</italic> and determine whether presynaptic disinhibition is involved in neuropathic pain induction, we generated conditional nociceptor-specific &#x003b2;3-GABA_AR knockout mice (<italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;}) by crossing mice carrying the floxed <italic>Gabrb3</italic> allele (Gabrb3^{<italic>fl/fl</italic>}) to a mouse line expressing Cre recombinase under the control of the nociceptor-specific sodium channel Na_v1.8 promotor (SNS-Cre)<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b34">34</xref> (<xref ref-type="fig" rid="f5">Fig. 5a</xref>).</p><p>To quantify changes in GABA_AR &#x003b2;3 subunit expression, we performed quantitative reverse transcriptase&#x02013;PCR in lumbar DRG and the spinal cord. Significant reduction of &#x003b2;3 subunit expression was observed in DRG but not in the spinal cord when <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice were compared with their WT littermates (<xref ref-type="fig" rid="f5">Fig. 5d</xref>). To confirm that conditional genetic deletion of &#x003b2;3 subunit attenuated GABA_AR function in DRG neurons, whole cell voltage-clamp recordings were performed on DRG neurons isolated from <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice or their WT littermates. Exogenous GABA-mediated currents were significantly reduced in small DRG neurons from <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice compared with WT mice (large neurons: &#x02212;375&#x000b1;60&#x02009;pA, <italic>n</italic>=15 versus &#x02212;542&#x000b1;67&#x02009;pA, <italic>n</italic>=16; <italic>P</italic>&#x0003e;0.05; small neurons: &#x02212;173&#x000b1;37&#x02009;pA, <italic>n</italic>=29 versus &#x02212;492&#x000b1;141&#x02009;pA, <italic>n</italic>=14; <italic>P</italic>&#x0003c;0.01), demonstrating a functional loss of GABA_AR in DRG neurons isolated from <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice (<xref ref-type="fig" rid="f5">Fig. 5b,c</xref>).</p><p>Next we analysed pain behaviour in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice. We first examined acute heat and mechanical nocifensive reflex. <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} adult male mice exhibited a hypersensitive phenotype to both noxious heat (Hargreaves test) and mechanical stimulation (Dynamic plantar and von Frey test) compared with their WT male littermates (<xref ref-type="fig" rid="f5">Fig. 5e&#x02013;g</xref>), indicating that the presynaptic GABA_AR is required to control both heat and mechanical pain signal processing under healthy condition. In contrast, acute sensitivity to cold stimulus is not affected in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice as demonstrated by cold plantar test (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5a</xref>). This is, to our knowledge, the first <italic>in vivo</italic> evidence showing that presynaptic inhibition plays a major role in the processing of noxious signals<xref ref-type="bibr" rid="b2">2</xref>. By contrast, no significant difference was observed in the hot plate test between WT and <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5b</xref>), which is consistent with observations in global <italic>&#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice<xref ref-type="bibr" rid="b35">35</xref>. Hot plate test is considered to be supraspinally integrated responses. The lack of difference might be due to the adjustment of pain perception at the cognitive level during development.</p><p>Both pre- and postsynaptic disinhibition have been proposed to be involved in neuropathic pain initiation and maintenance<xref ref-type="bibr" rid="b2">2</xref>. If the hypersensitivity developed after injury is due to mechanisms including postsynaptic disinhibition other than loss of presynaptic inhibition, we should expect the same extent of increase in heat or mechanical sensitivity in the <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice as in WT mice. Strikingly, when tested with CCI pain model, the heat sensitivity in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice did not increase, while the injury produced profound thermal hypersensitivity in WT littermates (<xref ref-type="fig" rid="f5">Fig. 5i</xref>). After nerve injury, the paw withdrawal latency in WT littermates decreased to the level of <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice. To test the possibility that the loss of thermal hypersentivity development in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice after injury is due to the floor effect, we administered bicuculline or saline to the <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice and their WT littermates. Bicuculline administration dramatically increased the heat sensitivity in both WT and <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice (<xref ref-type="fig" rid="f5">Fig. 5h</xref>), excluding the possibility of floor effect in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice. This indicates that loss of presynaptic inhibition is sufficient to induce thermal hypersensitivity after nerve injury. Dynamic mechanical hypersensitivity was also significantly reduced in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice when compared with their WT littermates (<xref ref-type="fig" rid="f5">Fig. 5j</xref>).</p><p>The switch of presynaptic inhibition to excitation has been proposed as one of the mechanisms underlying touch-evoked pain<xref ref-type="bibr" rid="b12">12</xref>. To test this type of mechanical hypersentivity, we performed a von Frey filament test. After nerve injury, both <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice and their WT littermates showed a significant reduction in the mechanical threshold for evoking a nociceptive withdrawal reflex (<xref ref-type="fig" rid="f5">Fig. 5k</xref>). Despite the fact that <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice are more sensitive to mechanical stimuli under healthy conditions (<xref ref-type="fig" rid="f5">Fig. 5g</xref>), they developed the same extent of static mechanical allodynia as their WT littermates after nerve injury. Together with our two-photon calcium imaging result on the primary afferents, these findings demonstrate that nociceptor-specific presynaptic GABA_AR-mediated inhibition does not become excitatory and contribute to static mechanical allodynia after nerve injury. Factoring this together with the transient change of GABA_AR function after nerve injury, we conclude that loss of presynaptic inhibition is required for the generation but not for the maintenance of thermal and mechanical hyperalgesia induced by nerve injury, while postsynaptic disinhibition contributes mostly to the development of static mechanical allodynia.</p></sec><sec disp-level="2"><title>Presynaptic disinhibition regulated by BDNF</title><p>We next asked how presynaptic GABA_AR function is controlled after nerve injury. BDNF is upregulated in both activated microglia and DRG neurons after nerve injury<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b36">36</xref>. If the depolarizing shift of <italic>E</italic>_GABA and decrease in <italic>G</italic>_GABA is due to BDNF<xref ref-type="bibr" rid="b37">37</xref>, an exogenous BDNF treatment on DRG neurons from a naive mouse should have the same effect. To test this possibility, we incubated control DRG neurons with BDNF. <italic>E</italic>_GABA of cultured DRG neurons incubated with BDNF (50&#x02009;ng&#x02009;ml^&#x02212;1) overnight was significantly more depolarized than that of control neurons (large neuron: &#x02212;24.9&#x000b1;2.7&#x02009;mV, 30.8&#x000b1;0.8&#x02009;&#x003bc;m, <italic>P</italic>&#x0003c;0.005, <italic>n</italic>=23; small neurons: &#x02212;20.0&#x000b1;3.7&#x02009;mV, 20.1&#x000b1;0.7&#x02009;&#x003bc;m, <italic>P</italic>&#x0003c;0.005, <italic>n</italic>=14) (<xref ref-type="fig" rid="f1">Figs 1c</xref> and <xref ref-type="fig" rid="f6">6a,b</xref>). In addition, a significant reduction of the conductance in large and small neurons was also observed (large neuron: 6.2&#x000b1;0.9&#x02009;nS, <italic>P</italic>&#x0003c;0.05; <italic>n</italic>=23; small neurons: 2.7&#x000b1;0.3&#x02009;nS, <italic>P</italic>&#x0003c;0.05, <italic>n</italic>=14) (<xref ref-type="fig" rid="f6">Fig. 6a,c</xref>). Similar to what we found in cultured DRG neurons after injury, BDNF did not increase the GABA-mediated calcium influx (<xref ref-type="fig" rid="f6">Fig. 6d</xref>). Thus, exogenous BDNF produced a similar effect as nerve injury on both <italic>E</italic>_GABA and <italic>G</italic>_GABA in both large and small neurons. We then carried out two-photon imaging experiments on BDNF-treated spinal cord slices (<xref ref-type="fig" rid="f4">Fig. 4k&#x02013;n,s&#x02013;w</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Movie 3</xref>). A significant loss of inhibitory effect of GABA on KCl-induced fluorescence increase was observed, while GABA alone did not show any excitatory effect (<xref ref-type="fig" rid="f4">Fig. 4s&#x02013;w</xref>), confirming that BDNF is sufficient to cause loss of presynaptic inhibition but not switch to excitation after nerve injury.</p><p>If the altered GABA_AR function after nerve injury is due to increased BDNF level in DRG and the spinal cord, a pharmacological blockade of BDNF signalling should be able to reverse the change of GABA_AR function and the reduced nociceptive threshold after nerve injury. To examine this, we administered a BDNF-sequestering fusion protein (TrkB-Fc) intrathecally to the mice that had developed thermal hypersensitivity 2 days after CCI. A single injection of TrkB-Fc could significantly reverse the decrease in paw withdrawal latency, while saline produced no change (<xref ref-type="fig" rid="f6">Fig. 6e</xref>). To determine whether BDNF-TrkB signalling is necessary for the nerve injury-induced change of presynaptic GABA_AR function, we tested the effects of a TrkB-Fc and a Trk receptor inhibitor K252a on <italic>E</italic>_GABA and <italic>G</italic>_GABA in cultured large and small DRG neurons taken from 2 d.p.i. animals. Indeed, treatment with TrkB-Fc or K252a could completely reverse the <italic>E</italic>_GABA and <italic>G</italic>_GABA changes in both large and small neurons taken from injured animals (<xref ref-type="fig" rid="f6">Fig. 6b,c</xref>). These findings indicate that BDNF-trkB signalling on DRG neurons is essential for the alteration in GABA_AR function after nerve injury, which leads to neuropathic pain induction.</p><p>It has also been shown that blocking BDNF-TrkB signalling after injury by intrathecally administering anti-TrkB or TrkB-Fc could acutely reverse the shift of post-synaptic <italic>E</italic>_GABA in spinal neurons and the neuropathic pain symptoms<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b38">38</xref>. However, both pre- and postsynaptic GABA_ARs are localized in the spinal cord and could be affected by intrathecal drug application. This raises the question as to whether the reduction in hyperalgesia behaviour is due to the reversal of pre- and/or postsynaptic disinhibition.</p><p>To discriminate the <italic>in vivo</italic> effect of BDNF on presynaptic inhibition from postsyatptic inhibition, we administered BDNF intrathecally to the <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} adult male mice or their WT male littermates (<italic>n</italic>=6 mice per group). A large body of evidence has demonstrated the similarities between long-lasting thermal or mechanical hypersensitivity induced by nerve injury and single intrathecal BDNF injection<xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref>. Indeed, we observed that BDNF could cause an increase in both thermal and mechanical sensitivity almost immediately after injection (0.5&#x02009;h) and persist for at least 6&#x02009;h of testing in WT mice (<xref ref-type="fig" rid="f6">Fig. 6f,g</xref>). Remarkably, thermal hypersensitivity could not be further enhanced by BDNF in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice (<xref ref-type="fig" rid="f6">Fig. 6f</xref>), indicating that BDNF acts to remove nociceptor-specific presynaptic GABA_AR-mediated inhibition to induce heat hypersensitivity. In contrast to the heat hypersensitivity, static mechanical allodynia development was reserved in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice after BDNF injection (<xref ref-type="fig" rid="f6">Fig. 6g</xref>), suggesting a presynaptic inhibition independent mechanism, most probably through postsynaptic disinhibition regulated by BDNF.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Here we have demonstrated that presynaptic inhibition is required for setting the pain sensitivity under physiological condition and its regulation by BDNF after nerve injury is essential for neuropathic pain initiation. Two distinct inhibitory mechanisms are employed in the spinal cord to control pain signal processing: presynaptic inhibition of the primary afferents and postsynaptic inhibition of the dorsal horn projection neurons. Enormous evidence has shown that blockade of spinal GABA-mediated inhibition produced an increase in nociceptive reactions to noxious stimuli and painful sensations to innocuous stimuli<xref ref-type="bibr" rid="b41">41</xref><xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref><xref ref-type="bibr" rid="b46">46</xref>. However, none of these studies made a clear distinction between pre- and postsynaptic inhibition. Zeilhofer&#x02019;s laboratory has first applied a genetic approach to selectively knock out presynaptic GABA_AR &#x003b1;2 subunit on spinal nociceptor terminals (<italic>sns- &#x003b1;2</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice), to address the contribution of presynaptic inhibition to spinal pain control<xref ref-type="bibr" rid="b9">9</xref>. Surprisingly, GABAergic membrane currents recorded from nociceptive DRG neurons are the same between WT and <italic>sns- &#x003b1;2</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice. Furthermore, <italic>sns- &#x003b1;2</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice exhibited normal response thresholds to thermal and mechanical stimulation, and developed normal inflammatory and neuropathic pain. This is most probably due to the upregulation of other GABA_AR subunits. Here we have generated a new mouse line in which GABA_AR &#x003b2;3 subunit is selectively knocked out in primary nociceptors (<italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice). The amplitude of GABA_AR current is significantly reduced in <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice compared with WT mice, demonstrating a functional loss of presynaptic GABA_ARs. Remarkably, <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice exhibited a hypersensitive phenotype to both noxious heat and mechanical stimulation compared with WT mice. To our knowledge, this is the first <italic>in vivo</italic> evidence being able to dissect the relative contribution of presynaptic inhibition in controlling both heat and mechanical pain signal processing under healthy condition.</p><p>The capacity for neuronal inhibition by GABA_AR activation is critically dependent on [Cl^&#x02013;]_i. An increased activity of NKCC1 and a depolarizing shift of <italic>E</italic>_GABA in DRG neurons have been observed after nerve injury in several studies<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b31">31</xref>. However, the net effect of the depolarizing shift of <italic>E</italic>_GABA remains unclear. It has been suggested that the shifted <italic>E</italic>_GABA might produce an enhanced PAD, which can increase excitability of primary nociceptors (hyperalgesia) or even become sufficient enough to directly evoke an action potential, thus transforming an inhibitory process into an excitatory one (tactile allodynia)<xref ref-type="bibr" rid="b12">12</xref>. Here we found that in parallel with the shift of <italic>E</italic>_GABA, there is a reduction of <italic>G</italic>_GABA, together leading to a loss of presynaptic inhibition, but not a switch to excitation, accounting for thermal hypersensitivity but not static mechanical hypersensitivity symptoms after nerve injury. Indeed, when we specifically knocked out the presynaptic inhibition in nociceptors, the <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice developed normal static mechanical hypersensitivity but no thermal hypersensitivity after nerve injury. Surprisingly, we found that the changes in <italic>E</italic>_GABA and <italic>G</italic>_GABA in DRG neurons are rather transient and not maintained at later stages, while persistent neuropathic pain symptoms are still present. Administration of bumetanide achieved anti-hyperalgesic effect in mice 2 days or 14 days after nerve injury but not in mice 21 days after injury, indicating that this transient modulation of presynaptic inhibition is mostly required for the initiation of hypersensitivity, but does not relate to the maintenance of pain in which increased excitability of projecting central areas may play a key role. This observation is strongly in agreement with a NKCC1 transient upregulation after nerve injury reported by M&#x000f3;dol <italic>et al.</italic><xref ref-type="bibr" rid="b31">31</xref></p><p>Postsynaptic disinhibition regulated by BDNF through KCC2, thus unmasking the low-threshold input onto nociceptive-specific spinal projection neurons, has been considered as a major mechanism underlying neuropathic pain conditions, such as mechanical allodynia<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b47">47</xref><xref ref-type="bibr" rid="b48">48</xref>. In primary sensory neurons, TrkB expression was primarily observed in large diameter neurons. However, BDNF has been shown to be essentially required for the postnatal survival of nociceptors and TrkB expression has also been detected in some small and medium-size neurons<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref><xref ref-type="bibr" rid="b51">51</xref><xref ref-type="bibr" rid="b52">52</xref><xref ref-type="bibr" rid="b53">53</xref>. Ultrastructural evidence from Salio <italic>et al.</italic><xref ref-type="bibr" rid="b22">22</xref> showed that TrkB receptor is expressed at axonal terminals of primary afferent fibres in lamina II where most nociceptive fibres terminate. Furthermore, BDNF has been reported to act on these presynaptic TrkB receptors in lamina II and lead to an increase of the frequency of glutamatergic excitatory postsynaptic potentials in spinal dorsal horn of complete Freund's adjuvant-treated rats<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b54">54</xref>. Here we report that BDNF binding to these presynaptic TrkB receptors produce both a transient depolarizing shift of <italic>E</italic>_GABA and a reduction of <italic>G</italic>_GABA in primary sensory neurons after nerve injury, leading to a transient presynaptic disinhibition and consequently to the development of thermal hypersensitivity, while postsynaptic disinhibition regulated by BDNF initiates static mechanical hypesensitivity. Our data reveals a novel functional role of BDNF in regulating presynaptic inhibition after nerve injury and open new possibilities for developing more precise and prompt therapeutic strategies for neuropathic pain treatment.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Animals</title><p>For all experiments, adult male mice (aged 8&#x02013;10 weeks) were used. All mice were housed under a 12-h:12-h light/dark cycle. All animal experiments were done according to the German Animal Protection Law.</p></sec><sec disp-level="2"><title>Generation of <italic>Advillin- GCaMP3</italic> and <italic>sns- &#x003b2;3</italic> ^{&#x02212;<italic>/</italic>&#x02212;} mice</title><p>Sensory neuron-specific <italic>Advillin- GCaMP3</italic> mice were generated by crossing advillin-Cre<xref ref-type="bibr" rid="b33">33</xref> transgenic mice with floxed <italic>GCaMP3</italic> mice<xref ref-type="bibr" rid="b32">32</xref>. Nociceptor-specific <italic>sns- &#x003b2;3</italic>^{&#x02212;<italic>/</italic>&#x02212;} mice were generated by crossing sns-cre transgenic mice<xref ref-type="bibr" rid="b34">34</xref> with floxed &#x003b2;3 mice (<italic>&#x003b2;3</italic>^{<italic>fl/fl</italic>}; JacksonLabs#008310 (ref. <xref ref-type="bibr" rid="b55">55</xref>)). In all experiments, adult male littermates were strictly used to control for genetic effects of the background. Mouse genotype was verified by PCR using primers as follows: Gabrb3 Forward: 5&#x02032;-ATTCGCCTGAGACCCGACT-3&#x02032;; Reverse: 5&#x02032;-GTTCATCCCCACGCAGAC-3&#x02032;. SNS Forward: 5&#x02032;-GAAAGCAGCCATGTCCAATTTACTGACCGTAC-3&#x02032;; Reverse: 5&#x02032;-GCGCGCCTGAAGATATAGAAGA-3&#x02032;. Advillin Forward: 5&#x02032;- GCACTGATTTCGACCAGGTT-3&#x02032;; Reverse: 5&#x02032;-GAGTCATCCTTAGCGCCGTA-3&#x02032;. GCaMP3 Forward: 5&#x02032;-CTT CAA GAT CCG CCA CAA CAT CG-3&#x02032;; Reverse: 5&#x02032;-TTG AAG AAG ATG GTG CGC TCC TG-3&#x02032;.</p></sec><sec disp-level="2"><title>Real-time quantitative PCR</title><p>Total RNA was extracted from DRGs using peqGOLD TriFast (peqlab) following the manufacturer&#x02019;s protocol. RNA was determined with a peqlab NanoDrop ND-1000. Complementary DNA was synthesized from 5&#x02009;&#x003bc;g of total RNA using the SuperScript TM II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) with oligo(dT) primers, according to the manufacturer&#x02019;s protocol. One microlitre of 1:5 dilution cDNA was used in quantitative reverse transcriptase&#x02013;PCR experiments using ABsolute QPCR SYBR Green ROX Mix (Thermo Scientific) on an ABI 7500 Real Time PCR System (ABS/Life Technologies). Primers used were as follows: Gabrb3 Forward: 5&#x02032;-GCCAGCATCGACATGGTTTC-3&#x02032;; Reverse: 5&#x02032;-GCGGATCATGCGGTTTTTCA-3&#x02032;. GAPDH Forward: 5&#x02032;-ACCCTGTTGCTGTAGCCGTATCA-3; Reverse: 5&#x02032;-TCAACAGCAACTCCCACTCTCCA-3.</p></sec><sec disp-level="2"><title>Induction of CCI and nociceptive tests</title><p>In deeply anaesthetized mice (isoflurane), four loose silk ligatures (4/0) were placed (with ~0.5&#x02009;mm spacing) around the sciatic nerve at the level of the right mid-thigh. Ligatures were tied until they elicited a brief twitch in the respective hind limb<xref ref-type="bibr" rid="b56">56</xref>. Dynamic mechanical and thermal hypersensitivity were assessed by dynamic plantar and Hargreaves apparatus (Ugo Basile, Italy)<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b56">56</xref>. Static mechanical hypersentivity was measured using von Frey filaments(Stoelting Europe, Ireland)<xref ref-type="bibr" rid="b17">17</xref>. The withdrawal threshold was measured for each mouse before taking DRG for culture. All experiments were performed blind to the genotype of the mice.</p></sec><sec disp-level="2"><title>Cold plantar test</title><p>The recording of cold-induced withdrawal latency was modified from previous report<xref ref-type="bibr" rid="b57">57</xref>: animals were habituated on metal platform with open grid of square holes (5 &#x000d7; 5&#x02009;mm). Dry ice powder was packed into a 10-ml syringe and compressed into a flattened, dense pellet. The hind paws of mice were targeted by the tip of modified syringe and the withdrawal latency was measured with a stopwatch.</p></sec><sec disp-level="2"><title>DRG neurons culture</title><p>L4 and L5 DRGs were dissected and collected in a 1.0-ml tube of PBS on ice. Ganglia were cleaned, enzymatically treated and mechanically dispersed<xref ref-type="bibr" rid="b58">58</xref>. The isolated neurons were seeded on poly-<sc>L</sc>-lysine and laminin-coated coverslips. Electrophysiology experiments began 12&#x02009;h after plating.</p></sec><sec disp-level="2"><title>Electrophysiology</title><p>Recordings were made from DRG neurons using fire-polished glass electrodes with a resistance of 3&#x02013;7&#x02009;M&#x003a9;. Extracellular solution contained the following (mM) 150 NaCl, 1 MgCl₂, 2 CaCl₂, 5 KCl, 10 glucose and 10 HEPES (pH 7.4), and the internal solution for filled electrodes contained the following (mM): 140 CsCl or 140KCl, 5 EGTA and 10 HEPES (pH 7.3). Membrane current and voltage were amplified and acquired using EPC-10 amplifier sampled at 10&#x02009;kHz. For the perforated-patch recording, 0.1% Lucifer Yellow and 50&#x02013;100&#x02009;&#x003bc;g&#x02009;ml^&#x02212;1 gramicidin were included in the internal solution<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b58">58</xref>. The formation of gramicidin perforation were monitored by the HEKA Patchmaster programme and the measurement was started until serious resistance dropped to &#x0003c;40&#x02009;M&#x003a9;. GABA (1&#x02009;mM)-induced currents were done at various holding potentials (&#x02212;60, &#x02212;40, &#x02212;20, 0 and 20&#x02009;mV) gradually. Data were analysed with Fitmaster software (HEKA) Electronik GmbH, Germany) and the reversal potential of GABA-induced current (<italic>E</italic>_GABA) was calculated and fitted from the GABA-induced current amplitudes at a series of holding potentials.</p></sec><sec disp-level="2"><title>Immunmofluorescence</title><p>The DRG and spinal cords have been isolated and fixed in 2% paraformaldehyde in PBS pH 7.4 overnight and then in 25% sucrose-hanks in PBS overnight, cryo-sectioned at 10&#x02009;&#x003bc;m and mounted on SuperFrost*/plus microscope slides and stored at &#x02013;20&#x02009;&#x000b0;C. The sections were permeablized 3&#x02009;min with 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) in PBS, blocked 30&#x02009;min in 1% BSA (Sigma) in PBS and incubated overnight at 4&#x02009;&#x000b0;C with primary antibody anti-NKCC1 diluted 1:500 (goat, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GABA_ARalpha1 diluted 1:500 (Guinea pig, Synaptic Systems, G&#x000f6;ttingen, Germany) or anti-calcitonin gene-related peptide diluted 1:400 (Rabbit, Merck-Millipore). On the sequent day, the sections were washed in PBS and incubated 1&#x02009;h at room temperature with anti-guinea pig AlexaFluor488-conjugated secondary antibody diluted 1:500 (Life Technologies, Carlsbad, CA, USA) and anti-rabbit Cy3-conjugated secondary antibody diluted 1:1,500 (Jackson Immunoresearch, West Grove, PA, USA), then washed again and mounted with &#x02018;VectashieldTM Mounting Medium with DAPI&#x02019; (Vector Labs, Burlingame, CA, USA). The sections were viewed using an Olympus BX61 microscope equipped with epi-fluorescence illumination. Images were acquired using an Olympus XM10 charge-coupled device monochrome camera and analysed with cellSens Dimension software (OSIS). To increase spatial resolution, slices were imaged over a distance of ~10&#x02009;&#x003bc;m within an image-stack along the <italic>z</italic> axis (<italic>z</italic>-stack, stacks distance: 0.49&#x02009;&#x003bc;M) followed by three-dimensional deconvolution using cellSens Dimension built-in algorithm. The acquired images were analysed using the free software ImageJ (NIH, Bethesda, MD; USA.). To analyse the fluorescence intensity of the GABA_AR alpha 1 staining, the background was reduced in every picture with the rolling ball algorithm and therefore the red, green and blue channel have been separated. The red staining (on the red channel) has been used to determine the ROI. The ROI was applied on the green channel and the green staining was analysed using the Analyze Particles built-in plugin of Image J.</p></sec><sec disp-level="2"><title>Western blotting</title><p>Proteins were isolated and pooled from six DRGs (L4 and L5 from three mice), homogenized with pestle after freeze and thaw in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma-Aldrich). Next, SDS&#x02013;PAGE and western blotting were performed using the &#x02018;XCell II SureLock Mini-Cell and XCell II Blot Module&#x02019; (Invitrogen), according to the manufacturer&#x02019;s instructions. Thirty micrograms of proteins were loaded for each lane on a 4&#x02013;12% Tris-glycine gel (Invitrogen), resolved and transferred onto polyvinylidene difluoride membrane. On blocking 1&#x02009;h at room temperature with 5% milk powder (AppliChem, Darmstadt, Germany) diluted in double distillate water, the blotted proteins were incubated overnight at 4&#x02009;&#x000b0;C with goat polyclonal anti-NKCC1 antibody diluted 1: 300 (Santa Cruz Biotechnology) and mouse monoclonal anti-GAPDH antibody diluted 1:10,000 (Abcam, Cambridge, UK). Next, the membrane was incubated 2&#x02009;h at room temperature with the secondary antibodies ECL peroxidase-labelled anti-goat or mouse antibody (1:2,000; Amersham Biosciences, Freiburg, Germany). Labelled proteins were detected by chemiluminescence using the ECL Prime Western Blotting Detection Reagents (Amersham Biosciences) on X-ray films (AGFA, Mortsel, Belgium). The X-ray films were scanned as a grayscale picture and the bands intensity were analysed using the open source software ImageJ and calculated as expression relative to the housekeeping gene (<italic>GAPDH</italic>). Every lane was analysed with the plot analysis tool of ImageJ and the area of the peak corresponding to the band was used as intensity value.</p></sec><sec disp-level="2"><title>Calcium imaging</title><p>Briefly, fluorescence microscopy was done on an Observer A1 inverted microscope (Zeiss, Germany) using a 25 &#x000d7; 0.8 numerical aperture water immersion objective and a 175&#x02009;W Xenon lamp as a light source. Before imaging, DRG neurons were incubated with 4&#x02009;&#x003bc;M Fura-2 at 37&#x02009;&#x000b0;C for 30&#x02013;40&#x02009;min and washed with extracellular solution at 37&#x02009;&#x000b0;C for another 30&#x02009;min. Excitation light was passed either through a 340-BP 30 filter or a 387-BP 16 filter. Two filters were switched by an ultra-high speed wavelength switcher Lambda DG-4 (Sutter, Novato, CA). Emissions elicited from both excitation wavelengths were passed through a 510-BP 90 filter and collected by a charge-coupled device camera (Zeiss). Different solutions were applied by multi barrel perfusion system (WAS02, DITEL, Prague). AxioVision software (Zeiss) was used to record image data. After background subtraction in each channel (<italic>B</italic>₃₄₀, <italic>B</italic>₃₈₀), the ratio (<italic>R</italic>) of fluorescence elicited by two excitation light, <italic>B</italic>₃₄₀ and <italic>B</italic>₃₈₀, was calculated: <inline-formula id="d33e2230"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2231" xlink:href="ncomms6331-m1.jpg"/></inline-formula><xref ref-type="bibr" rid="b59">59</xref>. Data weres analysed by using AxioVision and Matlab (MathWorks, Natick, Massachusetts).</p></sec><sec disp-level="2"><title>Two-photon chloride imaging</title><p>Transgenic mice expressing Clomeleon, a ratiometric Cl^&#x02212; indicator, under the control of the Thy1 promoter were used<xref ref-type="bibr" rid="b60">60</xref>. L4 and L5 DRG were removed and stored in oxygenized Ringer solution (124&#x02009;mM NaCl, 1.25&#x02009;mM NaH₂PO₄, 26&#x02009;mM NaHCO₃, 3&#x02009;mM KCl, 2&#x02009;mM MgCl₂, 2&#x02009;mM CaCl₂, 10&#x02009;mM glucose) at room temperature. A commercial 2p setup (purchased from LaVision, Germany) consisting of an upright microscope (Olympus BX51WI) and imaging software ImSpector Pro (LaVision, Germany) was used for ratiometric Cl^&#x02212; imaging. A 20 &#x000d7; 1.0 numerical aperture water immersion lens (Plan-APOCHROMAT, Zeiss) was used for the experiment. Fluorescence was elicited using a Ti: Sapphire laser (Mai Tai HP DeepSee, Spectra-physics, Mountain View, CA) tuned to 870&#x02009;nm. CFP signals (filter: 480&#x02009;BP 36) and YFP signals (filter: 537 BP 42) were separated by a beamsplitter (500 LP) and recorded simultaneously. Time series of images or <italic>z</italic>-stacks were acquired from Clomeleon-positive cells in whole DRG. The data were analysed offline with ImageJ and Matlab.</p></sec><sec disp-level="2"><title>Spinal cord slice preparation</title><p><italic>Advillin-GCaMP3</italic> mice that express the fluorescent calcium indicator protein, GCaMP3, exclusively in sensory neurons were used for presynaptic imaging in spinal cord slice. Control, injury and BDNF group animals were anaesthetized with ketamin:xylazine:saline (1.5:0.75:7.75). The lumbosacral spinal cord innervated by L4 and L5 spinal nerve was isolated. After removal of pia mater, the spinal cord was placed in cold Prep-Ringer solution (2&#x02013;4&#x02009;&#x000b0;C; 87&#x02009;mM NaCl, 1.25&#x02009;mM NaH₂PO₄, 25&#x02009;mM NaHCO₃, 2.5&#x02009;mM KCl, 7&#x02009;mM MgCl₂, 0.5&#x02009;mM CaCl₂, 25&#x02009;mM glucose, 75&#x02009;mM sucrose). Next, it was embedded in 2% low melting agarose (Bio-Rad Laboratoried, CA 94547). A 300-&#x003bc;m transverse slice was cut from the embedded spinal cord on a vibrating blade microtome (Leica VT1200, Leica, Germany). The spinal cord slice was stored on a cellulose nitrate filter (Sartorius Stedim Biotech GmbH, Germany) perfused with Ringer solution saturated with 95% O₂ and 5% CO₂ at room temperature following 45&#x02013;60&#x02009;min perfusion at 33&#x02013;34&#x02009;&#x000b0;C for slice recovery.</p></sec><sec disp-level="2"><title>Calcium imaging with <italic>Advillin-GCaMP3</italic> using 2p excitation</title><p>Two-photon imaging setup is same as for chloride imaging experiments, except the laser was tuned to 920&#x02009;nm. GCaMP3 signal (filter: 562 BP 40) was reflected by a beamsplitter (560 LP) and recorded. Time series of images were acquired from the spinal cord dorsal horn. Superficial layer of dorsal horn (not deeper than 100&#x02009;&#x003bc;m from the spinal cord dorsal outline) was imaged. Scanning frequency was 1&#x02009;Hz. Spinal cord slice was transferred to recording chamber of the 2p setup and perfused with oxygenized (95% O₂, 5% CO₂) Ringer solution. A high KCl Ringer solution was used to stimulate neurons. It contains 87&#x02009;mM NaCl, 1.25&#x02009;mM NaH₂PO₄, 26&#x02009;mM NaHCO₃, 40&#x02009;mM KCl, 2&#x02009;mM MgCl₂, 2&#x02009;mM CaCl₂, 10&#x02009;mM glucose. To avoid the effect of activation of glutamate receptor, 10&#x02009;&#x003bc;M NMDA (<italic>N</italic>-methyl-<sc>D</sc>-aspartate) antagonist (RS)-CPP (Tocris Bioscience, UK) and 10&#x02009;&#x003bc;M AMPA (&#x003b1;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) antagonist CNQX (abcamBiochemicals, UK) were added into high KCl Ringer solution. Ringer solution with 1&#x02009;mM GABA was used to investigate neuron response to GABA. Short time perfusion with Ringer solution with GABA followed by high KCl solution with CPP, CNQX and GABA was used to investigate GABA&#x02019;s influence on neuron excitation.</p></sec><sec disp-level="2"><title>Analysis of imaging data</title><p>GCaMP3 responses were analysed offline with ImageJ and Matlab. Owing to the variance of the time period required for diffusion of the applied chemicals, each recording trial was split into individual pieces according to chemical stimulations in ImageJ for later analysis in Matlab. Each piece contains 300 image frames. In each analysis, the whole recording field was segmented into 5 &#x000d7; 5&#x02009;&#x003bc;m square-shaped ROIs and the same ROI settings were applied to different chemical stimulation recording pieces from the same trial. The average pixel value (<italic>F</italic>) in each ROI was calculated. The baseline fluorescence intensity, <italic>F</italic>₀, was measured as the average of <italic>F</italic> during the first 30 image frames in each recording piece. The rest 270 frames were used for response measurement. When the max <italic>F</italic>, <italic>F</italic>_max, during response frames was bigger than <inline-formula id="d33e2489"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2490" xlink:href="ncomms6331-m2.jpg"/></inline-formula>, a ROI was considered to have a positive response. <inline-formula id="d33e2492"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2493" xlink:href="ncomms6331-m3.jpg"/></inline-formula> is the s.d. of <italic>F</italic>₀. <italic>F</italic>_max was converted to &#x00394;<italic>F</italic> using the formula &#x00394;<italic>F</italic>=<italic>F</italic>_max&#x02212;<italic>F</italic>₀. In a given ROI, GABA is considered to have inhibitory effect on high KCl-induced response when <inline-formula id="d33e2529"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2530" xlink:href="ncomms6331-m4.jpg"/></inline-formula>. When <inline-formula id="d33e2532"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2533" xlink:href="ncomms6331-m5.jpg"/></inline-formula> or <inline-formula id="d33e2535"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e2536" xlink:href="ncomms6331-m6.jpg"/></inline-formula>, GABA effect is considered to be excitatory or no effect, respectively. In GABA-alone stimulation piece, only ROIs showing positive responses in KCl stimulation piece from the same trial were used for analysis.</p></sec><sec disp-level="2"><title>Intrathecal injection</title><p>The mice were anaesthetized under 3% of isoflurance for induction and 1&#x02013;2% for maintenance. Intrathecal injection was performed using a winged infusion set connected to a 50-&#x003bc;l Hamilton syringe. BDNF (50&#x02009;ng&#x02009;kg^&#x02212;1, in 5&#x02009;&#x003bc;l 0.9% NaCl) was injected into the intervertebral space of lumbar region between L5 and L6 level of the spinal cord. A reflexive flick of the tail was considered as a sign of the accuracy of each injection<xref ref-type="bibr" rid="b40">40</xref>. Behavioural testing was assessed by Hargreaves apparatus as above.</p></sec><sec disp-level="2"><title>Statistics</title><p>Statistical analysis was done using the Graphpad prism (GraphPad Software Inc., San Diego, CA) suite of programmes. The sample size was justified by significance testing, taking into account available number of neurons per <italic>in vitro</italic> experiment or mice from same litters. All means are expressed as mean&#x000b1;s.e.m.</p></sec></sec><sec disp-level="1"><title>Authors contributions</title><p>J.T.-C. performed electrophysiological. J.T.-C., F.F. and F.M. did behavioural experiments. D.G. carried out calcium imaging and two-photon imaging experiments with J.T.-c.C. D.C. performed immunofluorescence and molecular biological experiments with M.K. L.Z. carried out real-time PCR. R.K. provided the sns-cre transgenic mice. P.A.H. provided the advillin-cre transgenic mice. J.H. planned experimental studies and wrote the paper.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> Chen, J. T.-c. <italic>et al.</italic> Presynaptic GABAergic inhibition regulated by BDNF contributes to neuropathic pain induction. <italic>Nat. Commun.</italic> 5:5331 doi: 10.1038/ncomms6331 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Figures</title><p>Supplementary Figures 1-4.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6331-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Movie 1</title><p>Example of time frame images in spinal cord slice from Advillin-GCaMP3 mouse without injury. Slice was challenged by high KCl, GABA and KCl+GABA in sequential.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6331-s2.avi"/></supplementary-material><supplementary-material id="d33e30" content-type="local-data"><caption><title>Supplementary Movie 2</title><p>Example of time frame images in spinal cord slice from Advillin-GCaMP3 mouse 2 days after CCI injury. Slice was challenged by high KCl, GABA and KCl+GABA in sequential.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6331-s3.avi"/></supplementary-material><supplementary-material id="d33e36" content-type="local-data"><caption><title>Supplementary Movie 3</title><p>Example of time frame images in spinal cord slice from Advillin-GCaMP3 mouse after BDNF treatment. Slice was challenged by high KCl, GABA and KCl+GABA in sequential.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6331-s4.avi"/></supplementary-material></sec>

Dietary Agents and Phytochemicals in the Prevention and Treatment of Experimental Ulcerative Colitis

<p>Inflammatory bowel diseases (IBDs), consisting mainly of ulcerative colitis (UC) and Crohn's disease (CD), are important immune-mediated diseases of the gastrointestinal tract. The etiology of the disease includes environmental and genetic factors. Its management presents a constant challenge for gastroenterologists and conventional surgeon. 5-Amninosalicylates, antibiotics, steroids, and immune modulators have been used to reduce the symptoms and for maintenance of remission. Unfortunately, long-term usage of these agents has been found to lead to severe toxicities, which are deterrent to the users. Pre-clinical studies carried out in the recent past have shown that certain dietary agents, spices, oils, and dietary phytochemicals that are consumed regularly possess beneficial effects in preventing/ameliorating UC. For the first time, this review addresses the use of these dietary agents and spices in the treatment and prevention of IBD and also emphasizes on the mechanisms responsible for their effects.</p>

<contrib contrib-type="author"><name><surname>Saxena</surname><given-names>Arpit</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="fn">&#x02020;</xref></contrib><contrib contrib-type="author"><name><surname>Kaur</surname><given-names>Kamaljeet</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="fn">&#x02020;</xref></contrib><contrib contrib-type="author"><name><surname>Hegde</surname><given-names>Shweta</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kalekhan</surname><given-names>Faizan M</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Baliga</surname><given-names>Manjeshwar Shrinath</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Fayad</surname><given-names>Raja</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Inflammatory bowel disease (IBD), manifested clinically by bloody diarrhea, abdominal cramps, and pain, is an immunologically mediated relapsing and chronic disease that affects the intestinal mucosa.[<xref rid="ref1" ref-type="bibr">1</xref>] Patients with IBD are also at a higher risk to develop colorectal cancer, when compared to the average population.[<xref rid="ref2" ref-type="bibr">2</xref>] Crohn's disease (CD) and ulcerative colitis (UC) represent the two most common forms of IBD.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] These diseases mimic each other in symptoms and some mucosal pathology, but differ sufficiently to be considered as independent ailments.[<xref rid="ref1" ref-type="bibr">1</xref>] The etiology and the exact disease mechanisms remain unknown despite much effort and research.[<xref rid="ref2" ref-type="bibr">2</xref>] It is well known that the incidence of IBD is high in the countries of North America, and northern and western Europe, while it is low in Africa, eastern Europe, South America, Asia, and the Pacific region. Conversely, recent studies indicate that the incidence has stabilized or slightly increased in countries with a high prevalence previously, while it is on the rise in countries with a low incidence previously. Jointly, these reports indicate that IBD could be a global health problem in the future and understanding its pathogenesis and developing affordable safe treatment is important.[<xref rid="ref3" ref-type="bibr">3</xref>]</p></sec><sec id="sec1-2"><title>CURRENT STATUS OF KNOWLEDGE</title><sec id="sec2-1"><title>Molecular events responsible for UC</title><p>From a clinical perspective, the central sign of UC is inflammation of the mucosal lining of the colon and mechanistically, this end event is a result of interplay between various molecular constituents of the cells. The inflammatory pathway in UC involves ubiquitous expression of proinflammatory eukaryotic transcription factors [activator protein (AP)-1 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#x003ba;B)],[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] which leads to the production of pro-inflammatory cytokines like tumor necrosis factor (TNF)-&#x003b1;, interleukin (IL)-6, IL-1&#x003b2;, with T-helper (Th)-17 cytokines such as IL-23 and IL-17 predominating, and a concomitant decrease in the anti-inflammatory cytokines and proteins.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] This surge in pro-inflammatory cytokines is followed by an increase in the production of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), myeloperoxidase,[<xref rid="ref8" ref-type="bibr">8</xref>] and signal transducer and activator of transcription[<xref rid="ref9" ref-type="bibr">9</xref>] -3,[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] which further increases inflammation and leads to oxidative stress[<xref rid="ref10" ref-type="bibr">10</xref>] and a concomitant decrease in the level of antioxidants.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>] All these events lead to an increase in cell inflammation, infiltration of the immune cells, especially the neutrophils, and culminate into epithelial cell damage and colonic barrier dysfunction. UC is also an established risk for colon cancer, which is caused due to the repeated cycle of inflammation leading to spontaneous mutation in the DNA repair mechanism, oncogenes, and tumor suppressor genes like p53.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] Another factor responsible for the development of UC is the change in the constituent, number, and activity of the colon microflora, as studies with germ-free mice have conclusively shown less or no inflammation developing in chemical and genetic models of colitis.</p></sec><sec id="sec2-2"><title>Conventional treatments in UC</title><p>Chemotherapy has been the mainstay of treatment of UC; in the event of a mild disease, anti-inflammatory drugs such as sulfasalazine and 5-aminosalicylic acid are given, while in severe and chronic cases, treatment with rectal and systemic corticosteroid and immunosuppressant is administered.[<xref rid="ref12" ref-type="bibr">12</xref>] In most cases, the benefits are restricted to the reduction of inflammation and its complications.[<xref rid="ref1" ref-type="bibr">1</xref>] However, in extreme conditions, surgery is the last solution to the patient's condition. Conversely, regular intake of these medications is unsafe as they may have severe side effects such as gastric ulcers, Cushing's habitus, hyperglycemia, muscle weakness, fragile skin, purple striae, flaring up of latent infections, delayed wound healing, cataract, osteoporosis, glaucoma and hypothalamic pituitary axis suppression with corticosteroids, and an increased risk of opportunistic infections and development of lymphomas.[<xref rid="ref1" ref-type="bibr">1</xref>] In addition, some refractory condition can lead to severe morbidity and decrease in the quality of life.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Recently, biologics such as anti&#x02013;TNF-&#x003b1;, anti&#x02013;alpha-4 integrin, as well as Peroxisome proliferator-activated receptor gamma (PPAR&#x003b3;) ligand and probiotic therapy are being used, but their long-term benefits are unknown.[<xref rid="ref1" ref-type="bibr">1</xref>] Importantly, reports suggest that the long-term use of biologics, especially infliximab, adalimumab, and certolizumab, may increase the risk of infections and malignancies, especially non-Hodgkin's lymphoma.[<xref rid="ref13" ref-type="bibr">13</xref>] Drugs that block leukocyte adhesion such as natalizumab, those that target cytokines, like IL- 12/23, and antibodies inhibiting T-cell signaling, such as IL-6 receptor antibodies, are also being studied. However, these drugs also have a number of contraindications and side effects, especially when used in combination with classical immunosuppressive drugs. The major effects are opportunistic infections, malignancies, and diverse complications like injection/infusion reactions and autoimmunity, and contraindications such as heart failure and acute infectious diseases.[<xref rid="ref14" ref-type="bibr">14</xref>]</p><p>The repeated relapses, surgery phobia, severe morbidity, and derisory response to conventional drugs make the patient to resort to unconventional treatments with a hope to decrease the symptoms of the disease and concomitantly perk up the quality of life.[<xref rid="ref15" ref-type="bibr">15</xref>] Recent reports indicate that at least 40% of IBD patients have used complementary and alternative medicines, and that the botanicals constitute a major share of all these alternatives.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><p>Results from preclinical studies suggest the beneficial effect of medicinal plants including <italic>Aloe vera</italic> gel (&#x08606;&#x08588; L&#x000fa; Hu&#x000ec;), <italic>Boswellia serrata</italic> (&#x04e73;&#x09999; R&#x001d4; Xi&#x00101;ng), <italic>Cassia fistula, Lepidium sativum, Bunium persicum, Plantago ovata, Pistacia lentiscus, Bunium persicum, Solanum nigrum, Commiphora mukul, Commiphora myrrha, Ocimum basilicum</italic> (&#x07f85;&#x052d2; Lu&#x000f3; L&#x000e8;), <italic>Linum usitatissimum, Dracaena cinnabari, Plantago major, Lallemantia royleana, and Allium porrum</italic>, which have been used since time immemorial in the various systems of traditional and folk medicine.[<xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>]</p><p>From human perspective, it is always desirable to consume dietary agents that also possess medicinal value as their regular use can be achieved easily and regularly. This aspect was very well recognized by Hippocrates, the Father of Medicine, who proclaimed almost 25 centuries ago, <italic>&#x0201c;Let food be thy medicine and medicine be thy food.&#x0201d;</italic> To further substantiate the importance and relevance of this adage, observations from around the world clearly indicate that the incidence of diet-related diseases is progressively increasing due to greater availability of hypercaloric food and a sedentary lifestyle, which cause low-grade inflammation in the individual.[<xref rid="ref18" ref-type="bibr">18</xref>]</p><p>Recent reports also suggest that the functional foods and nutraceuticals rich in polyphenols and antioxidants are beneficial due to their intrinsic ability to scavenge free radicals, induce anti-inflammatory responses, maintaining a homeostatic regulation of the gut microbiota, and activate the intestinal T regulatory cells.[<xref rid="ref18" ref-type="bibr">18</xref>] All these properties are extremely beneficial in the prevention and mitigation of the IBD. Studies have shown that the dietary agents like apple, bilberry, black raspberry, cocoa, bael, green tea (&#x07da0;&#x08336; L&#x001dc; Ch&#x000e1;); spices like garlic (&#x05927;&#x0849c; D&#x000e0; Su&#x000e0;n), Malabar tamarind, saffron (&#x0756a;&#x07d05;&#x082b1; F&#x00101;n H&#x000f3;ng Hu&#x00101;), fenugreek, ginger (&#x0751f;&#x08591; Sh&#x00113;ng Ji&#x00101;ng), turmeric (&#x08591;&#x09ec3; Ji&#x00101;ng Hu&#x000e1;ng); oil of olive; nutraceuticals like grape seed polyphenols; and the dietary phytochemicals like resveratrol, ellagic acid, zerumbone, quercetin, kaempferol, rutoside, and rutin are consumed regularly and are commonly used. They will be addressed in detail by emphasizing on the mechanism of action.</p></sec></sec><sec id="sec1-3"><title>DIETARY AGENTS WITH ANTI-IBD EFFECTS</title><sec id="sec2-3"><title>Apple</title><p>Apple, known as <italic>Malus malus</italic>, belongs to the family Rosaceae and is an important dietary agent.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref>] It has occupied a prime position in the dietary and nutritional requirements of humans and epidemiological studies have linked its consumption with reduced risk of certain cancers, cardiovascular diseases, asthma, and diabetes.[<xref rid="ref21" ref-type="bibr">21</xref>] Apple is a good source of several flavonoids and certain phytochemicals including quercetin glycosides, catechin, epicatechin, procyanidin, cyanidin-3-galactoside, coumaric acid, chlorogenic acid, gallic acid, and phloridzin.[<xref rid="ref22" ref-type="bibr">22</xref>] Recently, D&#x02019;Argenio <italic>et al</italic>. found that rectal administration of apple polyphenols protected rats from 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis by decreasing the transcription and protein levels of COX-2, TNF-&#x003b1;, calpain, as well as tissue transglutaminase. Thus, polyphenolic compounds obtained from apple may serve as potential therapeutic agents for UC patients.[<xref rid="ref23" ref-type="bibr">23</xref>]</p></sec><sec id="sec2-4"><title>Bilberry</title><p>Bilberry, also known as blaeberry (a Scottish name meaning blueberry), mountain bilberry, whinberry, whortleberry, whortles, myrtle whortleberry, tracleberry, and huckleberry, is a plant indigenous to Europe and has long been consumed in jams, pies, cobblers, and cakes.[<xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref>] Its scientific name is <italic>Vaccinium myrtillus</italic> L. and it belongs to the family Ericaceae. Phytochemical studies have shown it to contain the flavonoids like hyperoside, isoquercitrin, quercitrin, and astragaline and the anthocyanins like myrtillin, malvidin, cyanidin, delphinidin, and tannins.[<xref rid="ref26" ref-type="bibr">26</xref>]</p><p>Bilberry has been used to treat ocular disorders and is believed to be useful in improving night vision, prevent the development and progression of cataracts, treat diabetic retinopathy and macular degeneration, and prevent glaucoma.[<xref rid="ref26" ref-type="bibr">26</xref><xref rid="ref27" ref-type="bibr">27</xref>] With respect to its use in UC, an open pilot trial by Biedermann and coworkers with 13 individuals with UC has shown that the daily intake of a standardized bilberry preparation caused 63.4% remission and 90.9% response in the volunteers. Intake of the bilberry preparation decreased the total Mayo score and the histologic Riley index.[<xref rid="ref28" ref-type="bibr">28</xref>] However, an increase in disease activity was observed after cessation of bilberry intake, clearly indicating the beneficial effects of bilberries in UC, but only when taken regularly.</p></sec><sec id="sec2-5"><title>Black raspberry</title><p>The fruits of the perennial shrub <italic>Rubus coreanus</italic> Miquel, colloquially known as black raspberry, have been an important dietary and medicinal agent in traditional medicine.[<xref rid="ref29" ref-type="bibr">29</xref><xref rid="ref30" ref-type="bibr">30</xref><xref rid="ref31" ref-type="bibr">31</xref>] The plants grow in Far East Asian countries, namely South Korea, Japan, and China.[<xref rid="ref32" ref-type="bibr">32</xref>] The anthocyanin fraction of black raspberry has been found to be protective against esophageal and colorectal cancer.[<xref rid="ref31" ref-type="bibr">31</xref>] Montrose and coworkers investigated the protective effects of freeze-dried black raspberry powder on dextran sodium sulfate (DSS)&#x02013;induced UC in C57BL/6J mice and observed it to be effective in ameliorating the clinical conditions.[<xref rid="ref33" ref-type="bibr">33</xref>] The mice fed with black raspberry powder showed better maintenance of body mass and reduction in colonic shortening and ulceration. They had reduced levels of plasma prostaglandin E2 (PGE2), while the levels of nitric oxide (NO) were unaltered.[<xref rid="ref33" ref-type="bibr">33</xref>] Feeding black raspberry powder suppressed the tissue levels of COX-2 and key pro-inflammatory cytokines like TNF-&#x003b1; and IL-1&#x003b2;.[<xref rid="ref33" ref-type="bibr">33</xref>] Mechanistic studies within the colonic tissue showed decreased levels of phospho-I&#x003ba;B&#x003b1; indicating that black raspberry powder modulated the NF-&#x003ba;B, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events. With respect to phytochemicals, experiments have also shown that ellagic acid [<xref ref-type="fig" rid="F1">Figure 1</xref>], which is present in raspberries and other berries like blackberries, cranberries, strawberries, and wolfberries, possesses concentration-dependent protective effects against the DSS-induced colitis in rats.[<xref rid="ref34" ref-type="bibr">34</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Chemical structures of some phytochemicals effective against UC</p></caption><graphic xlink:href="JTCM-4-203-g001"/></fig></sec><sec id="sec2-6"><title>Cocoa</title><p>Cocoa, known as <italic>Theobroma cacao</italic>, is a small evergreen tree native to South America.[<xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref>] Its seeds are used to make cocoa powder and chocolate, and are widely used in confectionery industry.[<xref rid="ref37" ref-type="bibr">37</xref>] Furthermore, the seeds also contain polyphenols and flavonoids that possess several health benefits.[<xref rid="ref38" ref-type="bibr">38</xref>] And&#x000fa;jar <italic>et al</italic>. found the polyphenol-enriched cocoa extract containing epicatechin, procyanidin B2, catechin, and procyanidin B1 to possess anti-inflammatory properties against DSS-induced colitis in mice. This effect was manifested by reduction in inflammation, crypt damage, and leukocyte infiltration in the mucosa due to decrease in the production of NO, COX-2, phospho-STAT-3 (pSTAT-3), pSTAT1&#x003b1;, and NF-&#x003ba;B p65. Similar results have been found <italic>in vitro</italic> using RAW 264.7 cells, indicating that cocoa extract is effective in ameliorating DSS-induced colitis and the effect may be mediated by the inhibition of transcription factor NF-&#x003ba;B in intestinal cells.[<xref rid="ref39" ref-type="bibr">39</xref>] A clinical trial by Monagas <italic>et al</italic>.[<xref rid="ref40" ref-type="bibr">40</xref>] concluded that cocoa polyphenol intake modulated inflammatory mediators in patients with a high risk of cardiovascular disease and could be beneficial against atherosclerosis.[<xref rid="ref40" ref-type="bibr">40</xref>] These anti-inflammatory effects may contribute to the overall benefits of cocoa consumption against atherosclerosis. However, a study by P&#x000e9;rez-Berezo <italic>et al</italic>. showed that cocoa may be effective in reducing oxidative stress by downregulating serum TNF-&#x003b1; and iNOS activity in the colon or by increasing reduced glutathione (GSH), but this is not enough to reverse the DSS-induced colitis in the mice model. They concluded that cocoa intake may decrease colon cell infiltration and inflammation to a certain extent, but not completely.[<xref rid="ref41" ref-type="bibr">41</xref>] Therefore, it becomes imperative to perform more experimental and clinical studies to get a verdict for the use of cocoa in the treatment of colitis before its usage as a complimentary medicine.</p></sec><sec id="sec2-7"><title>Bael</title><p><italic>Aegle marmelos</italic>, commonly known as holy fruit, Bengal quince, Indian quince, golden bael, or bilva, is arguably one of the most important plants in ancient India.[<xref rid="ref42" ref-type="bibr">42</xref>] The plants are indigenous to India, and contain tannins, some essential oils like caryophyllene, citral, sterols, and/or triterpenoids, flavonoids like rutin and coumarins, including aegeline, marmesin, phlobatannins, flavon-3-ols, leucoanthocyanins, and anthocyanins[<xref rid="ref42" ref-type="bibr">42</xref><xref rid="ref43" ref-type="bibr">43</xref>] [<xref ref-type="fig" rid="F1">Figure 1</xref>]. In Ayurveda, the Indian traditional system of medicine, bael is utilized for its ability in the treatment of various diseases including diarrhea, dysentery, and dyspeptic symptoms.[<xref rid="ref44" ref-type="bibr">44</xref>] With regard to IBD, recent studies by Behera <italic>et al</italic>. have shown bael to be effective in ameliorating acetic acid-induced UC and indomethacin-induced enterocolitis in Wistar albino rats. The investigators observed that oral administration of the Bael fruit extract caused a significant decrease in disease activity index, macroscopic score, and microscopic scores. Mechanistic studies showed that administering Bael caused reduction in mast cell degranulation and malondialdehyde (MDA) levels and increased superoxide dismutase (SOD) activity.[<xref rid="ref45" ref-type="bibr">45</xref>]</p></sec><sec id="sec2-8"><title>Green tea (&#x07da0;&#x08336; L&#x001dc; Ch&#x000e1;)</title><p>Tea (<italic>Camellia sinensis</italic>), a plant native to China and Southeast Asia, is today the most commonly used botanical globally.[<xref rid="ref46" ref-type="bibr">46</xref><xref rid="ref47" ref-type="bibr">47</xref>] It is the second most widely consumed beverage after water, and may be consumed as green tea (unfermented), oolong tea (partially fermented), and black tea (fully fermented). Many studies carried out in the past three decades have shown that green tea possesses myriad benefits owing to its polyphenol content.[<xref rid="ref48" ref-type="bibr">48</xref><xref rid="ref49" ref-type="bibr">49</xref><xref rid="ref50" ref-type="bibr">50</xref>] The active compounds of green tea are the catechins [(&#x02013;)-epicatechin (EC), (&#x02013;)-epigallocatechin (EGC), (&#x02013;)-epicatechin-3-gallate (ECG), and (&#x02013;)-epigallocatechin-3-gallate (EGCG)] [<xref ref-type="fig" rid="F1">Figure 1</xref>], proanthocyanidins, flavonols (kaempferol, quercetin, and myricitin in the form of glycosides), gallic acids, and theanine.[<xref rid="ref51" ref-type="bibr">51</xref>]</p><p>Animal studies have proved that use of green tea polyphenol (GrTP) ameliorated the symptoms associated with colitis induced by DSS,[<xref rid="ref52" ref-type="bibr">52</xref>] dinitrobenzene sulfonic acid (DNBS),[<xref rid="ref53" ref-type="bibr">53</xref>] and in IL-2 deficient mice.[<xref rid="ref54" ref-type="bibr">54</xref>] These include bloody diarrhea, weight loss, wet colon weights,[<xref rid="ref55" ref-type="bibr">55</xref>] colonic damage, hemorrhage, ulcers, edema, and neutrophil infiltration.[<xref rid="ref55" ref-type="bibr">55</xref>] Experiments also revealed that GrTP prevented acute colitis and inflammation-associated colon carcinogenesis in male ICR mice.[<xref rid="ref56" ref-type="bibr">56</xref>] Furthermore, the phytochemical EGCG reduced DSS-[<xref rid="ref57" ref-type="bibr">57</xref>] and TNBS-induced colitis in rats[<xref rid="ref58" ref-type="bibr">58</xref>] and mice.[<xref rid="ref55" ref-type="bibr">55</xref>] Moreover, treatment with green tea had significantly less impact on the hematocrit,[<xref rid="ref52" ref-type="bibr">52</xref><xref rid="ref54" ref-type="bibr">54</xref>] serum amyloid A,[<xref rid="ref52" ref-type="bibr">52</xref><xref rid="ref54" ref-type="bibr">54</xref>] and blood GSH[<xref rid="ref52" ref-type="bibr">52</xref>] in animals after colitis induction, indicating an improvement in the animal health with the antioxidant administration.</p><p>Further, GrTP decreased spontaneous interferon-gamma (IFN-&#x003b3;) and TNF-&#x003b1; secretion from the colon explant in IL-2 deficient animals when compared to their control counterparts,[<xref rid="ref54" ref-type="bibr">54</xref>] and reduced the levels of TNF-&#x003b1;,[<xref rid="ref52" ref-type="bibr">52</xref><xref rid="ref53" ref-type="bibr">53</xref><xref rid="ref55" ref-type="bibr">55</xref>] IL-6, IL-10, and the keratinocyte-derived chemokine,[<xref rid="ref55" ref-type="bibr">55</xref>] which further reflects reduction of inflammation by GrTP administration. Administration of the polyphenol EGCG also led to a decrease in the levels of COX-2,[<xref rid="ref58" ref-type="bibr">58</xref>] myeloperoxidase,[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref53" ref-type="bibr">53</xref><xref rid="ref55" ref-type="bibr">55</xref>] and intercellular adhesion molecule-1 (ICAM)-1,[<xref rid="ref53" ref-type="bibr">53</xref>] and rectified the distorted actin cytoskeleton in the colonic tissue.[<xref rid="ref52" ref-type="bibr">52</xref>] These beneficial effects of EGCG were associated with a significant reduction of NF-&#x003ba;B and AP-1 activation.[<xref rid="ref55" ref-type="bibr">55</xref>] The phytochemicals EGCG and Polyphenon E also attenuated inflammation-induced colon cancer caused by DSS and azoxymethane (AOM) and the protective effects were mediated by reducing COX-2 and the mRNA expression levels of TNF-&#x003b1;, IFN-&#x003b3;, IL-6, IL-12, and IL-18 in the colonic mucosa.[<xref rid="ref59" ref-type="bibr">59</xref>] Further, treatment with a combination of EGCG and piperine reduced body weight loss, improved the clinical course, and increased the overall survival, when compared to untreated groups.[<xref rid="ref57" ref-type="bibr">57</xref>] The attenuated colitis was associated with lower histological damage and colon insult, reduction in lipid peroxidation, decreased levels of MPO, and a concomitant increase in the levels of antioxidant enzymes [SOD and glutathione peroxidase (GPx)] in the colonic tissue.[<xref rid="ref57" ref-type="bibr">57</xref>] A recent study by Oz <italic>et al</italic>.[<xref rid="ref60" ref-type="bibr">60</xref>] has shown the protective effect of different dosages of GrTP (0.25%, 0.5%, and 1%), EGCG (0.12%, 0.25%, and 0.5%), and a single dosage of sulfasalazine (50 mg/kg) in experimental colitis model. They found that GrTP and EGCG improved hematocrit values, as compared to sulfasalazine which caused anemia. Also, low dose of EGCG reduced colonic pathological lesions and normalized global antioxidant ratio, but was least beneficial in inhibiting reduction of leptin levels. However, GrTP partially protected animals against weight loss and elevated TNF-&#x003b1;. This study concluded that low-dose EGCG and GrTP may become potential therapeutic or additive agents in the treatment of IBD; however, clinical trials are warranted to prove this.[<xref rid="ref60" ref-type="bibr">60</xref>]</p><p>However, seminal studies by Inoue <italic>et al</italic>.[<xref rid="ref61" ref-type="bibr">61</xref>] have provided evidence that oral treatment of 1% GrTP to both normal and colitic animals induced nephrotoxicity in the ICR mice. The investigators observed that 1% GrTP given to colitic mice significantly increased their kidney weight and increased the levels of serum creatinine and thiobarbituric acid reactive substances (TBARS), but decreased the expression of heme oxygenase-1 (HO-1), NAD (P) H: Quinone oxidoreductase 1 (NQO1), and heat-shock protein (HSP) 90 in both kidney and liver, as compared to the colitic mice treated with the standard diet.[<xref rid="ref61" ref-type="bibr">61</xref>] Antioxidant enzymes&#x02019; mRNA expression and HSPs, such as HO-1, HSP27, and HSP90, were significantly down-regulated in the colitic mice receiving 1% GrTPs.[<xref rid="ref61" ref-type="bibr">61</xref>] It is noteworthy that these results clearly indicate that high-dose GrTP diet disrupts kidney functions through the reduction of antioxidant enzymes and HSP expression in both treated and untreated control ICR mice.[<xref rid="ref61" ref-type="bibr">61</xref>] In clinical studies, green tea has been shown to provide antioxidant protection in metabolic syndrome[<xref rid="ref62" ref-type="bibr">62</xref>] and has shown a positive effect on blood pressure, insulin resistance, inflammation, and oxidative stress in patients with obesity-related hypertension.[<xref rid="ref63" ref-type="bibr">63</xref>]</p></sec><sec id="sec2-9"><title>Grapes</title><p>In the history of mankind, grapes (<italic>Vitis vinifera</italic>) have been one of the most important fruits due to their importance in winery.[<xref rid="ref64" ref-type="bibr">64</xref>] The major constituents of grape are epicatechin gallate; procyanidin dimers, trimers, tetramers; catechin; epicatechin; gallic acid; procyanidin pentamers, hexamers, and heptamers and their gallates; resveratrol; phenolics; flavonoids; and anthocyanins [<xref ref-type="fig" rid="F2">Figure 2</xref>].[<xref rid="ref65" ref-type="bibr">65</xref>] Grapes possess anti-inflammatory, anti-aging, potent antioxidant, antimutagenic, antidiabetic, hepatoprotective, cardioprotective, nephroprotective, neuroprotective, and anti-carcinogenic properties.[<xref rid="ref65" ref-type="bibr">65</xref>] Pre-clinical studies provided evidence that polyphenols from grape seed are effective in reducing TNBS-[<xref rid="ref66" ref-type="bibr">66</xref><xref rid="ref67" ref-type="bibr">67</xref>] and DSS-induced colitis in rats,[<xref rid="ref68" ref-type="bibr">68</xref>] with the protective effects being equal to those of the clinically used drug.[<xref rid="ref66" ref-type="bibr">66</xref>]</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Some phytochemicals present in grape seed</p></caption><graphic xlink:href="JTCM-4-203-g002"/></fig><p>Administering grape seed proanthocyanidin extract (GSPE) reduced the macroscopic and microscopic damage scores and changes in weight/length ratio (mg/mm) of colon segments, when compared with standard diet in rats.[<xref rid="ref66" ref-type="bibr">66</xref><xref rid="ref67" ref-type="bibr">67</xref>] Studies have also shown that when compared to DSS-treated controls, GSPE significantly decreased ileal villus height and mucosal thickness toward the values of normal controls.[<xref rid="ref68" ref-type="bibr">68</xref>] In addition, GSPE significantly reduced the histological severity score only in the proximal colon and failed to prevent crypt damage of both proximal and distal colonic regions of the DSS-treated rats.[<xref rid="ref68" ref-type="bibr">68</xref>]</p><p>Mechanistic studies revealed that the levels of MDO[<xref rid="ref66" ref-type="bibr">66</xref><xref rid="ref67" ref-type="bibr">67</xref>] and NO,[<xref rid="ref67" ref-type="bibr">67</xref>] activities of MPO[<xref rid="ref66" ref-type="bibr">66</xref><xref rid="ref67" ref-type="bibr">67</xref>] and iNOS,[<xref rid="ref67" ref-type="bibr">67</xref>] and the levels of inflammatory cytokines like IL-1&#x003b2;[<xref rid="ref66" ref-type="bibr">66</xref>] were reduced in the colon tissues and serum of the GSPE-treated rats as compared to colitis control group.[<xref rid="ref66" ref-type="bibr">66</xref>] Furthermore, grape seed polyphenol treatment was associated with notably increased SOD and GPx activities and also the glutathione level[<xref rid="ref67" ref-type="bibr">67</xref><xref rid="ref69" ref-type="bibr">69</xref>] of colon tissues and serum of rats. The levels of IL-2 and IL-4[<xref rid="ref66" ref-type="bibr">66</xref>] were also found to be significantly increased. GSPE significantly reduced the expression levels of TNF-&#x003b1;, phospho-Inhibitor of nuclear factor kappa-B kinase subunit alpha/beta (p-IKK&#x003b1;/&#x003b2;), and 9phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (p-I&#x003ba;B&#x003b1;), and the translocation of NF-&#x003ba;B in the colon mucosa. Administration of GSPE did not negatively impact metabolic parameters, nor did it induce any deleterious gastrointestinal side effects in healthy animals.[<xref rid="ref68" ref-type="bibr">68</xref>] Thus, it can be concluded that GSPE exerts a protective effect on colitic rats by modulating the inflammatory response locally and systemically, and promotes tissue repair to improve colonic oxidative stress, at least in part by modulating the NF-&#x003ba;B signaling pathway.[<xref rid="ref69" ref-type="bibr">69</xref>]</p></sec><sec id="sec2-10"><title>Olive oil</title><p>The oil extracted from the fruits of the olive tree (<italic>Olea europaea</italic>) is one of the highly investigated dietary agents in recent times, and studies suggest that it possesses a wide range of therapeutic applications.[<xref rid="ref70" ref-type="bibr">70</xref><xref rid="ref71" ref-type="bibr">71</xref><xref rid="ref72" ref-type="bibr">72</xref>] The trees are native to the Mediterranean basin and parts of Asia Minor, and are mentioned in the ancient literature from Biblical and Roman times to Greek mythology. Olive oil is a major component of the Mediterranean diet and many beneficial effects of this diet have been attributed to the ample use of the oil. The chief active components of olive oil include oleic acid, phenolic constituents, and squalene. The main phenolics include hydroxytyrosol, tyrosol, and oleuropein, which occur in highest levels in virgin olive oil and have demonstrated antioxidant activity.[<xref rid="ref72" ref-type="bibr">72</xref>]</p><p>Seminal studies by S&#x000e1;nchez-Fidalgo and coworkers have shown that the olive oil attenuates DSS-induced acute UC[<xref rid="ref73" ref-type="bibr">73</xref><xref rid="ref74" ref-type="bibr">74</xref><xref rid="ref75" ref-type="bibr">75</xref>] and DSS-colitis-associated colon carcinogenesis in mice.[<xref rid="ref76" ref-type="bibr">76</xref>] The active phytochemical hydroxytyrosol was also shown to be effective in reducing the DSS-induced damage.[<xref rid="ref74" ref-type="bibr">74</xref>] The investigators observed that administering diets enriched with extra virgin olive oil (EVOO) significantly reduced the DSS-induced mortality by nearly 50%, attenuated the clinical and histological signs of damage, and improved the disease activity index.[<xref rid="ref74" ref-type="bibr">74</xref>]</p><p>Mechanistic studies have shown that administering EVOO reduces the damage in acute colitis model by alleviating the oxidative stress. It prevents the degradation of I&#x003ba;B&#x003b1;, deactivates PPAR&#x003b3;, down-regulates the expression of iNOS, COX-2, monocyte chemoattractant protein-1 (MCP-1), and TNF-&#x003b1;, and activates p38 mitogen-activated protein kinases (MAPKs) in the colonic mucosa.[<xref rid="ref73" ref-type="bibr">73</xref><xref rid="ref74" ref-type="bibr">74</xref>] Observations from the DSS-colitis-associated carcinogenesis experiments also suggest that the feeding EVOO reduced the incidence and multiplicity of dysplastic lesions due to reduced &#x003b2;-catenin and decreased levels of COX-2, iNOS, and other proinflammatory cytokines.[<xref rid="ref76" ref-type="bibr">76</xref>]</p><sec id="sec3-1"><title>Agaricus blazei</title><p><italic>Agaricus blazei</italic> Murill, an edible mushroom indigenous to Brazil, is a rich source of a variety of &#x003b2;-glucans, proteoglucans, glycoproteins, saponins, tannins, cerebrosides, polysaccharides, and steroids. It is one of the most important edible and culinary medicinal species.[<xref rid="ref77" ref-type="bibr">77</xref><xref rid="ref78" ref-type="bibr">78</xref>] It is cultivated commercially for the health food market and is shown to possess a range of medicinal properties against diseases like cancer[<xref rid="ref79" ref-type="bibr">79</xref><xref rid="ref80" ref-type="bibr">80</xref>] and chronic hepatitis.[<xref rid="ref81" ref-type="bibr">81</xref>] Preclinical studies have shown it to possess anti-inflammatory effects against a range of ulcerogens.[<xref rid="ref78" ref-type="bibr">78</xref>] With respect to its beneficial effects in the treatment of IBD, a recent clinical study has shown that consumption of 60 ml/day of immunomodulatory extract (AndoSan^&#x02122;) at a concentration of 340 g/l or 20.4 g/day for 12 days caused reduction in the levels of pro-inflammatory cytokines like IL-1&#x003b2; and IL-6 and chemokines like IL-8, Macrophage inflammatory protein (MIP-1&#x003b2;), Monocyte chemoattractant protein-1 (MCP-1), Granulocyte macrophage colony-stimulating factor (GM-CSF), and Granulocyte colony-stimulating factor (G-CSF) in UC and CD patients, indicating a reduction in inflammation. Levels of fecal calprotectin were also reduced in UC patients, with an overall reduction in IBD pathology.[<xref rid="ref82" ref-type="bibr">82</xref>]</p></sec></sec><sec id="sec2-11"><title>Indian gooseberry</title><p>Indian gooseberry or amla, scientifically known as <italic>Emblica officinalis</italic> Gaertn. or <italic>Phyllanthus emblica</italic> Linn., is an indigenous plant of India.[<xref rid="ref83" ref-type="bibr">83</xref>] The fruits are a rich source of ascorbic acid and the juice prepared from the ripe fruits is an excellent coolant in the hot summer season.[<xref rid="ref84" ref-type="bibr">84</xref>] The fruits also contain gallic acid, ellagic acid, chebulinic acid, chebulagic acid, emblicanin-A, emblicanin-B, punigluconin, pedunculagin, ellagitannin, trigalloyl glucose, chebulagic acid, corilagin, quercetin, kaempferol 3-<italic>O</italic>-&#x003b1;-l (6&#x02019;&#x02019; methyl) rhamnopyranoside, and kaempferol 3-<italic>O</italic>-&#x003b1;-l (6&#x02019;&#x02019; ethyl) rhamnopyranoside.[<xref rid="ref84" ref-type="bibr">84</xref>]</p><p>Amla is arguably the most important medicinal botanic in the traditional Indian system of medicine, the Ayurveda. Studies have shown it to possess antibacterial, antifungal, antiviral, free radical scavenging, anti-mutagenic, anti-inflammatory, cardioprotective, gastroprotective, hepatoprotective, nephroprotective, neuroprotective, and anticancer properties.[<xref rid="ref84" ref-type="bibr">84</xref>] A recent study by Deshmukh <italic>et al</italic>.[<xref rid="ref93" ref-type="bibr">93</xref>] has shown that the methanolic extract of <italic>E. officinalis</italic> (200 mg/kg) was effective in ameliorating the severity of acetic acid-induced colitis in rats.[<xref rid="ref85" ref-type="bibr">85</xref>] It reduced colon weight/length ratio, colon insult, and macroscopic scores for inflammation, in addition to lactate dehydrogenase (LDH), indicating that amla protected against the inflammogen and possessed cytoprotective effects.[<xref rid="ref85" ref-type="bibr">85</xref>]</p></sec></sec><sec id="sec1-4"><title>SPICES WITH ANTI-IBD EFFECTS</title><p>Spices, which are termed as aromatic vegetable substances, in the whole, broken, or ground form, and whose significant function in food is seasoning rather than nutrition, are an important constituent of the Indian curries.[<xref rid="ref86" ref-type="bibr">86</xref><xref rid="ref87" ref-type="bibr">87</xref>] In addition to their organoleptic properties, spices are also useful in prolonging the shelf life of foods by preventing rancidity through their free radical scavenging effects and also by imparting antimicrobial activities.[<xref rid="ref86" ref-type="bibr">86</xref><xref rid="ref87" ref-type="bibr">87</xref>] Historical reports also support the fact that ancient physicians like Charaka, Sushrutha, Hippocrates, and Dioscorides used spices extensively in their practice.[<xref rid="ref87" ref-type="bibr">87</xref>] Additionally, most of the spices also possess medicinal benefits and are extensively used to treat various gastrointestinal ailments.[<xref rid="ref86" ref-type="bibr">86</xref><xref rid="ref87" ref-type="bibr">87</xref>] In the following section, the beneficial effects of spices like garlic, saffron, Malabar tamarind, fenugreek, ginger, and turmeric [<xref ref-type="fig" rid="F3">Figure 3</xref>] will be addressed in detail.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Indian medicinal plants reported to afford protection against inflammatory bowel disease in experimental studies</p></caption><graphic xlink:href="JTCM-4-203-g003"/></fig><sec id="sec2-12"><title>Garlic (&#x05927;&#x0849c; D&#x000e0; Su&#x000e0;n)</title><p>Garlic, scientifically known as <italic>Allium sativum</italic> and a member of the Liliaceae family, is highly regarded throughout the world for both its medicinal and culinary value.[<xref rid="ref88" ref-type="bibr">88</xref>] The plant is indigenous to Asia and historical documents suggest that the early men of medicine such as Hippocrates, Pliny, and Aristotle used this botanical for its numerous therapeutic uses, and it is regularly used in various traditional and folk medicines.[<xref rid="ref88" ref-type="bibr">88</xref>] In traditional medicines, garlic, both raw and aged, is used as a natural antiviral, antibacterial, and antifungal agent, to suppress common cough,[<xref rid="ref89" ref-type="bibr">89</xref>] to treat gastrointestinal disorders,[<xref rid="ref90" ref-type="bibr">90</xref>] and to act as a cardioprotective agent.[<xref rid="ref91" ref-type="bibr">91</xref><xref rid="ref92" ref-type="bibr">92</xref>] Different types of antioxidants are present in different garlic preparations, including water- and lipid-soluble organosulfur compounds like S-allylcysteine and S-allylmercaptocysteine[<xref rid="ref93" ref-type="bibr">93</xref>] and non-metals like selenium and phytoalexin-like allixin, which are responsible for the protective effect of garlic in several disease models.[<xref rid="ref94" ref-type="bibr">94</xref><xref rid="ref95" ref-type="bibr">95</xref>] Rats fed garlic (0.25 g/kg b. wt.) orally for 4 weeks and 3 days during acetic acid&#x02013;induced colitis showed a significant reduction in colon weight. Garlic administration restored the levels of GSH and antioxidant enzymes with a concomitant decrease in lipid peroxidation levels, as compared to placebo-treated colitis groups. Also, garlic treatment in the presence of the amino acid l-arginine (625 mg/kg b. wt.) mitigated the changes in both colon weight and colon tissue contents of lipid peroxidation and GSH.[<xref rid="ref92" ref-type="bibr">92</xref>]</p></sec><sec id="sec2-13"><title>Ginger (&#x0751f;&#x08591; Sh&#x00112;ng Ji&#x00101;ng)</title><p>Ginger, a plant native to the northeast region of India, is one of the world's most important culinary and medicinal agents in various alternative systems of medicines.[<xref rid="ref96" ref-type="bibr">96</xref><xref rid="ref97" ref-type="bibr">97</xref><xref rid="ref98" ref-type="bibr">98</xref>] Some of the important bioactive components of ginger extract include 10-gingerol, 8-gingerol, 6-gingerol, and 6-shogaol, with 6-gingerol being antiproliferative.[<xref rid="ref99" ref-type="bibr">99</xref>] It has been documented to treat cold, headaches, nausea, stomach upset, diarrhea, and helps digestion, treats arthritis, rheumatological conditions, and muscular discomfort, and acts as a carminative and antiflatulent.[<xref rid="ref100" ref-type="bibr">100</xref>] Scientific studies have shown that ginger possesses antimicrobial, antischistosomal, anti-inflammatory, antipyretic, antioxidative, hypoglycemic, hepatoprotective, diuretic, and hypocholesterolemic effects.[<xref rid="ref101" ref-type="bibr">101</xref>]</p><p>Preclinical studies have shown that pretreatment with ginger extract ameliorated the acetic acid&#x02013;induced edematous inflammation in the colon by significantly attenuating the extent and severity of edema, necrosis, and inflammatory cell infiltration in the mucosa.[<xref rid="ref102" ref-type="bibr">102</xref>] The activity of colonic MPO and levels of lipid peroxides, protein carbonyl content, TNF-&#x003b1;, and PGE2 were also decreased. Administering ginger restored the levels of GSH, catalase (CAT), and SOD. The protective effect of highest doses of ginger was comparable to that of the standard sulfasalazine.[<xref rid="ref102" ref-type="bibr">102</xref>]</p><p>In addition, studies have also shown that zerumbone (a sesquiterpenoid) [<xref ref-type="fig" rid="F1">Figure 1</xref>], a minor constituent of <italic>Zingiber officinale</italic> but a major component of <italic>Zingiber zerumbet</italic>, mitigated the DSS-induced acute colitis in ICR mice.[<xref rid="ref103" ref-type="bibr">103</xref>] Oral feeding of zerumbone reduced the inflammatory biomarkers (IL-1&#x003b1;, IL-1&#x003b2;, TNF-&#x003b1;, PGE2, and PGF2&#x003b1;) in the colonic mucosa and suppressed DSS-induced colitis. Nimesulide, a selective COX-2 inhibitor, suppressed the histological changes induced by DSS without affecting inflammatory biomarkers; but when combined with zerumbone, it enhanced the protective effects.[<xref rid="ref103" ref-type="bibr">103</xref>]</p></sec><sec id="sec2-14"><title>Saffron (&#x0756a;&#x07d05;&#x082b1; F&#x00101;n H&#x000f3;ng Hu&#x00101;)</title><p>Saffron, scientifically known as Crocus sativus L. and belonging to the family Iridaceae, is a perennial stemless herb widely cultivated in Iran, Pakistan, India, and Greece.[<xref rid="ref104" ref-type="bibr">104</xref><xref rid="ref105" ref-type="bibr">105</xref><xref rid="ref106" ref-type="bibr">106</xref>] The stigmas are the most important plant part and are dried and sold as saffron.[<xref rid="ref106" ref-type="bibr">106</xref>] Saffron has been used to treat depression, cancer, and cardiac ailments. It is used in various traditional and folk systems of medicine in the Arabian countries and in the Indian subcontinent.[<xref rid="ref106" ref-type="bibr">106</xref><xref rid="ref107" ref-type="bibr">107</xref><xref rid="ref108" ref-type="bibr">108</xref><xref rid="ref109" ref-type="bibr">109</xref>] Phytochemical studies have shown that the medicinal and organoleptic properties are due to the presence of crocetin [<xref ref-type="fig" rid="F1">Figure 1</xref>], crocin, picrocrocin, and safranal.[<xref rid="ref106" ref-type="bibr">106</xref><xref rid="ref107" ref-type="bibr">107</xref><xref rid="ref108" ref-type="bibr">108</xref><xref rid="ref109" ref-type="bibr">109</xref>] Crocetin, an important carotenoid of saffron, has been widely studied in the prevention and as a therapy for cancer.[<xref rid="ref110" ref-type="bibr">110</xref>]</p><p>With respect to saffron's effectiveness in UC, studies have shown that oral administration of crocetin to mice [<xref ref-type="fig" rid="F1">Figure 1</xref>] (25-100 mg/kg b. wt. per day) for 8 days significantly ameliorated TNBS-induced UC.[<xref rid="ref111" ref-type="bibr">111</xref>] The animals administered crocetin had reduced diarrhea and disruption of colonic architecture. Optimal effects were observed at 50 mg/kg/day dosage. Crocetin-treated mice had reduced levels of NO, neutrophil infiltration, and lipid peroxidation in the inflamed colon, favorable expression of TH1 and TH2 cytokines, and down-regulation of NF-&#x003ba;B. These observations indicate that crocetin exerts beneficial effects in experimental UC.[<xref rid="ref111" ref-type="bibr">111</xref>]</p></sec><sec id="sec2-15"><title>Malabar tamarind</title><p><italic>Garcinia cambogia</italic>, also known as Malabar tamarind, is a plant mostly utilized in India and parts of southeast Asia for its culinary uses and therapeutic effects.[<xref rid="ref112" ref-type="bibr">112</xref><xref rid="ref113" ref-type="bibr">113</xref>] The fruits, which are the most important part, resemble a miniature pumpkin and are used as an acidulant in the curries and to prepare sherbat. Its main component is hydroxycitric acid (HCA), which is an inhibitor of adenosine triphosphate (ATP) citrate lyase that reduces the biosynthesis of fatty acid and thus aids in weight reduction.[<xref rid="ref114" ref-type="bibr">114</xref><xref rid="ref115" ref-type="bibr">115</xref>] The decoction of the fruit is an essential preparation to treat ulcers and inflammation. It has been shown that the fruit extract possesses hypolipidemic properties and anti-adipogenic and appetite-suppressor effects.[<xref rid="ref116" ref-type="bibr">116</xref><xref rid="ref117" ref-type="bibr">117</xref>] A recent finding suggests that the extract blocked TNBS-induced colitis in rats by preventing epithelial damage, decreasing the activity of MPO, decreasing the expression of COX-2 and iNOS, reducing colonic PGE2 and IL-1&#x003b2; levels, and reducing epithelial cell DNA damage.[<xref rid="ref117" ref-type="bibr">117</xref>]</p></sec><sec id="sec2-16"><title>Fenugreek</title><p><italic>Trigonella foenum-graecum</italic>, colloquially known as fenugreek and belonging to the Fabaceae family, is an important dietary and medicinal agent.[<xref rid="ref118" ref-type="bibr">118</xref><xref rid="ref119" ref-type="bibr">119</xref>] The plants are native to India and northern Africa and have been used in the various traditional and folk systems of medicine to treat numerous indications, including labor induction, aiding digestion, and as a general tonic to improve metabolism and health.[<xref rid="ref119" ref-type="bibr">119</xref>] Scientific studies have shown that fenugreek possesses pleiotropic actions and is useful in the amelioration of hypertension, cataract, inflammation, thyroid dysfunction, malaria, endothelial dysfunction, hyperlipidemia, and diabetes.[<xref rid="ref119" ref-type="bibr">119</xref>]</p><p>With regard to its protective effects, animal studies in colitis have shown that saponin diosgenin [<xref ref-type="fig" rid="F1">Figure 1</xref>], a compound in fenugreek, suppresses inflammation.[<xref rid="ref120" ref-type="bibr">120</xref>] Diosgenin was also found to suppress ovalbumin-induced intestinal allergic reaction; gut inflammation, which reduced the frequency of diarrhea; infiltration and degranulation of mast cells; and increased the presence of mucin-containing goblet cells in mice's duodenum.[<xref rid="ref121" ref-type="bibr">121</xref>] Furthermore, diosgenin reduced the crypt depth in the intestinal epithelium and inhibited systemic ovalbumin-specific IgE and total IgE.[<xref rid="ref121" ref-type="bibr">121</xref>] Another anti-inflammatory property of diosgenin is that it suppresses TNF-induced NF-&#x003ba;B activation as determined by DNA binding, activation of I&#x003ba;Ba kinase, I&#x003ba;Ba phosphorylation, I&#x003ba;Ba degradation, p65 phosphorylation, and p65 nuclear translocation through Akt inhibition.[<xref rid="ref120" ref-type="bibr">120</xref>] It also down-regulated TNF-induced expression of NF-&#x003ba;B&#x02013;regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc) and anti-apoptosis (IAP1, Bcl-2, Bcl-XL, Bfl-1/A1, TRAF1, and cFLIP).[<xref rid="ref120" ref-type="bibr">120</xref>]</p></sec><sec id="sec2-17"><title>Turmeric (&#x08591;&#x09ec3;Ji&#x00101;ng Hu&#x000e1;ng)</title><p><italic>Curcuma longa</italic> Linn., a perennial shrub belonging to the family Zingiberaceae, is an indigenous plant of India, but is also cultivated in China, Sri Lanka, and other tropical countries nowadays.[<xref rid="ref122" ref-type="bibr">122</xref>] The roots are the most important part of the plant and are used as a religious, culinary, and medicinal agent in India.[<xref rid="ref122" ref-type="bibr">122</xref>] Turmeric is one of the highly investigated plants and studies have shown it to contain curcuminoids like curcumin [<xref ref-type="fig" rid="F1">Figure 1</xref>], Desmethoxycurcumin, bisdemethoxy curcumin, monodememthoxy curcumin, dihydro curcumin, and cyclocurcumin.[<xref rid="ref122" ref-type="bibr">122</xref>] Curcumin has been shown to possess potent antioxidant,[<xref rid="ref123" ref-type="bibr">123</xref>] anti-inflammatory,[<xref rid="ref124" ref-type="bibr">124</xref>] and cytoprotective effects.[<xref rid="ref125" ref-type="bibr">125</xref>]</p><p>With respect to its protective effects in UC, numerous preclinical studies have shown that when administered orally or systemically, either as a prophylactic or curative agent, curcumin improved the survival rate, and decreased the wasting and discomfort induced by various ulcerogens, such as DSS,[<xref rid="ref126" ref-type="bibr">126</xref><xref rid="ref127" ref-type="bibr">127</xref><xref rid="ref128" ref-type="bibr">128</xref><xref rid="ref129" ref-type="bibr">129</xref><xref rid="ref130" ref-type="bibr">130</xref>] dinitrobenzene sulfonic acid (DNB),[<xref rid="ref131" ref-type="bibr">131</xref>] dinitrochlorobenzene (DNCB),[<xref rid="ref132" ref-type="bibr">132</xref>] TNBS,[<xref rid="ref133" ref-type="bibr">133</xref><xref rid="ref134" ref-type="bibr">134</xref>] acetic acid, and also in genetically predisposed IL-10-knockout[<xref rid="ref135" ref-type="bibr">135</xref><xref rid="ref136" ref-type="bibr">136</xref>] and mdr1a-/- mice.[<xref rid="ref137" ref-type="bibr">137</xref><xref rid="ref138" ref-type="bibr">138</xref>] The results from these observations have all been summarized in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>List of articles published on the protective effects afforded by curcumin in IBD in experimental animals with emphasis on the mechanism/s of action</p></caption><graphic xlink:href="JTCM-4-203-g004"/></table-wrap><p>At the tissue level, curcumin decreased the macroscopic scores of mucosal erosions significantly.[<xref rid="ref139" ref-type="bibr">139</xref>]It has the property of scavenging the free radicals, influencing multiple signaling pathways, especially involving the kinases, extracellular signaling kinase (AKT, MAPK,ERK), inhibiting COX-1, COX-2, lipoxygenase,[<xref rid="ref140" ref-type="bibr">140</xref>] TNF-&#x003b1;, IFN-&#x003b3;, iNOS, inhibiting transcription factors such as NF-&#x003ba;&#x003b2; and AP-1, and modulating Nrf2-dependent cytoprotective pathways.[<xref rid="ref137" ref-type="bibr">137</xref>] Cumulatively, these studies strongly indicate that curcumin is a promising medication for improving remission in IBD patients and that randomized controlled clinical investigations in large cohorts of patients are needed to fully evaluate its clinical potential in the treatment of IBD.[<xref rid="ref137" ref-type="bibr">137</xref>]</p><p>The most encouraging observations were accrued from a clinical study where curcumin (360 mg) reduced the relapse episodes in patients with quiescent IBD, when administered three or four times a day for 3 months.[<xref rid="ref141" ref-type="bibr">141</xref>] Additionally, recent studies by Suskind and coworkers have also shown that curcumin was well tolerated at a high dose by children with IBD. In the study, the investigators prescribed 500 mg of curcumin twice per day for 3 weeks along with the standard therapy, and then by using the forced-dose titration design enhanced the curcumin doses to 1 g twice per day at week 3 for a total of 3 weeks and then to 2 g twice per day at week 6 for 3 weeks. At the end of the study period, it was observed that all patients tolerated curcumin well and that the only untoward symptom observed was increase in flatulence. The authors also observed that combining curcumin with the standard therapy resulted in the improvement of Pediatric Crohn's Disease Activity Index (PCDAI) or Pediatric Ulcerative Colitis Activity Index (PUCAI) score and suggested that curcumin may be useful as an adjunctive therapy in IBD.[<xref rid="ref142" ref-type="bibr">142</xref>]</p></sec></sec><sec id="sec1-5"><title>PHYTOCHEMICALS WITH BENEFICIAL EFFECTS</title><sec id="sec2-18"><title>Resveratrol</title><p>Resveratrol (3,5,4&#x02032;-trihydroxy-<italic>trans</italic>-stilbene) is a phytoalexin produced by several plants including grapes, peanuts, mulberries, raspberries, and blueberries.[<xref rid="ref143" ref-type="bibr">143</xref><xref rid="ref144" ref-type="bibr">144</xref>] Its polyphenolic compound possesses multiple pharmacological benefits.[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref146" ref-type="bibr">146</xref>] Preclinical studies provided evidence that resveratrol is effective in preventing DSS- and TNBS-induced colitis in mice and rats, respectively.[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref146" ref-type="bibr">146</xref><xref rid="ref147" ref-type="bibr">147</xref><xref rid="ref148" ref-type="bibr">148</xref>] It was also shown that resveratrol (300 ppm) reduced the tumor burden of AOM + DSS-induced colorectal inflammation and cancer in mice.[<xref rid="ref147" ref-type="bibr">147</xref>]</p><p>In 2010, S&#x000e1;nchez-Fidalgo and coworkers observed that mice fed with a diet enriched with 20 mg/kg of resveratrol for 30 days and administered 3% DSS for 5 days showed lower clinical scores, lower inflammation, and an increase in survival rate, as compared to the DSS-treated mice which were fed control diet. Therefore, resveratrol can be linked to a better disease prognosis in case of acute UC. Subsequent studies have also shown the ability of resveratrol to halt the weight loss and reduce the colonic inflammation in mice treated with DSS[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref147" ref-type="bibr">147</xref>] and rats treated with TNBS.[<xref rid="ref146" ref-type="bibr">146</xref>] It also causes a concentration-dependent reduction in inflammation.</p><p>Mechanistically, resveratrol decreases the percentage of neutrophils in the mesenteric lymph nodes and lamina propria[<xref rid="ref147" ref-type="bibr">147</xref>] and modulates the number of CD3 (+) T cells[<xref rid="ref147" ref-type="bibr">147</xref>] and down-regulates inflammatory and stress markers, namely p53 and p53-phospho-Ser (15) proteins.[<xref rid="ref147" ref-type="bibr">147</xref>] Resveratrol prevented the depletion of glutathione[<xref rid="ref146" ref-type="bibr">146</xref>] and reduced the levels of MPO[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref146" ref-type="bibr">146</xref>] and lipid peroxides in the colon,[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref146" ref-type="bibr">146</xref>] concomitantly increasing the activities of SOD and GSH-Px in the colonic tissue.[<xref rid="ref145" ref-type="bibr">145</xref>]</p><p> <list list-type="bullet"><list-item><p>Furthermore, studies have been successful in showing a decrease in the expression levels of TNF-&#x003b1;,[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref147" ref-type="bibr">147</xref><xref rid="ref148" ref-type="bibr">148</xref>] IL-1&#x003b2;,[<xref rid="ref148" ref-type="bibr">148</xref>] IFN-&#x003b3;,[<xref rid="ref145" ref-type="bibr">145</xref><xref rid="ref147" ref-type="bibr">147</xref>] IL-8,[<xref rid="ref145" ref-type="bibr">145</xref>] p22(phox),[<xref rid="ref145" ref-type="bibr">145</xref>] and gp91(phox),[<xref rid="ref145" ref-type="bibr">145</xref>] and an increase in the levels of the anti-inflammatory cytokine IL-10[<xref rid="ref148" ref-type="bibr">148</xref>] with resveratrol administration. Additionally, resveratrol also caused a reduction in the levels of PGE synthase-1 (PGES-1), COX-2, and iNOS protein expressions by down-regulation of p38-mediated MAPK signaling pathway[<xref rid="ref148" ref-type="bibr">148</xref>] and suppressing intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in the colon and serum.[<xref rid="ref146" ref-type="bibr">146</xref>]</p></list-item></list> </p></sec><sec id="sec2-19"><title>Quercetin</title><p>Quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-t r i hydroxy-4H-chromen-4-one] [<xref ref-type="fig" rid="F1">Figure 1</xref>] is a flavonoid ubiquitously found in fruits, vegetables, leaves, and grains, and is one of the highly investigated phytochemicals.[<xref rid="ref149" ref-type="bibr">149</xref>] It is a glycone form of a number of other flavonoid glycosides, such as rutin and quercitin, found in citrus fruit, buckwheat, and onions. Quercetin possesses many pharmacological benefits, including scavenging the free radicals, and has antioxidant and anti-inflammatory properties.[<xref rid="ref150" ref-type="bibr">150</xref>] Animal studies have shown that quercitin (1 and 5 mg/kg) is effective when administered in the early stages (24 h) of TNBS-induced colitis.[<xref rid="ref151" ref-type="bibr">151</xref>] Biochemical end points showed that treatment with the flavonoids prevented an increase in colonic MDA, inhibited iNOS and alkaline phosphatase activities, but had no significant effects on observable damage. However, histopathologic observations showed no changes in the neutrophil infiltration.[<xref rid="ref151" ref-type="bibr">151</xref>]</p></sec><sec id="sec2-20"><title>Kaempferol</title><p>The flavanoid kaempferol [3, 5, 7 - t r i hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] [<xref ref-type="fig" rid="F1">Figure 1</xref>] is ubiquitously present in many edible plants such as broccoli, cabbage, kale, beans, endive, leek, tomato, strawberries, and grapes, and in plants or botanical products commonly used in traditional medicine (e.g. <italic>Ginkgo biloba, Tilia spp, Equisetum spp, Moringa oleifera, Sophora japonica</italic>, and propolis).[<xref rid="ref152" ref-type="bibr">152</xref>] Myriad preclinical studies have shown that kaempferol and some of its glycosides have a wide range of pharmacological activities, including anticancer, antioxidant, anti-inflammatory, antimicrobial, antidiabetic, anti-osteoporotic, estrogenic/antiestrogenic, anxiolytic, analgesic, and antiallergic activities, and cardioprotective, and neuroprotective activities.[<xref rid="ref153" ref-type="bibr">153</xref>]</p><p>With regard to its role in UC treatment, recent studies by Park <italic>et al</italic>.[<xref rid="ref165" ref-type="bibr">165</xref>] have shown that feeding kaempferol (0.1% or 0.3%) was effective in decreasing the DSS-induced colitis in mice. They found lower levels of plasma leukotriene B4 (LTB4) in all the groups fed kaempferol, while the levels of NO and PGE2 and the activity of MPO in colonic mucosa were significantly decreased in 0.3% kaempferol pre- and post-fed groups. Additionally, the level of Trefoil factor 3 (TFF3) mRNA, a marker for goblet cell function, was up-regulated in kaempferol pre-fed animals indicating its usefulness.[<xref rid="ref154" ref-type="bibr">154</xref>]</p></sec><sec id="sec2-21"><title>Rutoside or rutin</title><p>Rutoside, alsok nown as rutin, quercetin-3-<italic>O</italic>-rutinoside, or sophorin, is a flavonol glycoside between quercetin and the disaccharide rutinose [&#x003b1;-l-rhamnopyranosyl-(1 &#x02192; 6))-&#x003b2;-d-glucopyranose] [<xref ref-type="fig" rid="F1">Figure 1</xref>]. It is found in many plants such as buckwheat, tobacco, forsythia, hydrangea, and viola, and has important pharmacological effects.[<xref rid="ref155" ref-type="bibr">155</xref>] Studies revealed that oral pre- and post-treatment of rutoside (10 or 25 mg/kg) was effective in ameliorating TNBS-induced colitis. It promoted colonic healing in rats[<xref rid="ref156" ref-type="bibr">156</xref>] by increasing the levels of colonic glutathione and reducing the levels of colonic oxidative stress.[<xref rid="ref156" ref-type="bibr">156</xref>] In another study, a diet containing 0.1% rutin, but not quercetin, ameliorated 5% DSS-induced body weight loss and shortening of the colorectum, and dramatically improved colitis histological scores in ICR mice.[<xref rid="ref157" ref-type="bibr">157</xref>] Importantly, pretreatment with rutin for 2 weeks or therapeutic post-treatment for 4 days starting 3 days after DSS administration was shown to produce significant beneficial effects by attenuating proinflammatory gene expression levels, namely IL-1&#x003b2; and IL-6, in colonic mucosa, in a dose-dependent manner.[<xref rid="ref157" ref-type="bibr">157</xref>]</p></sec><sec id="sec2-22"><title>Naringenin</title><p>One of the major polyphenols isolated from the citrus fruit is naringenin. It has been shown to possess nephro- and hepatoprotective,[<xref rid="ref158" ref-type="bibr">158</xref>] antioxidative,[<xref rid="ref159" ref-type="bibr">159</xref>] anti-inflammatory, and anticancer[<xref rid="ref160" ref-type="bibr">160</xref>] properties, as suggested by several cell- and animal model-based studies. Naringenin was found to be effective in reducing the lead-induced oxidative stress in rat models by increasing the activities of SOD, CAT, and GPx.[<xref rid="ref69" ref-type="bibr">69</xref><xref rid="ref159" ref-type="bibr">159</xref>] It reduces inflammation by inhibition of pro-inflammatory cytokines NF-&#x003ba;B and COX-2 and phosphorylation of transcription factor proto-oncogene&#x02013;encoded AP-1 in macrophages in lipopolysa ccharide (LPS)-induced model.[<xref rid="ref161" ref-type="bibr">161</xref>] Naringenin ameliorates the DSS-induced colitis by reducing colonic damage, shortening the colon length, protecting the tight junction barrier, and decreasing pro-inflammatory cytokines&#x02019; expression, especially those of IFN-&#x003b3;, Il-6, MIP-2, and IL-17A.[<xref rid="ref162" ref-type="bibr">162</xref>] These <italic>in vivo</italic> and <italic>in vitro</italic> studies make naringenin a strong contender for clinical studies for colitis and other related disease models.</p></sec></sec><sec sec-type="conclusions" id="sec1-6"><title>CONCLUSIONS</title><p>A number of dietary supplements and phytochemicals are widely used in preventing or alleviating the symptoms of UC in experimental animal models [Graphical summary]. Studies on the efficacy and mechanism of various dietary plant extracts and pure phytochemicals in this field are reviewed and highlighted here [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Such evidence-based updated information is very important for IBD patients and healthcare providers to make informed decisions about the benefits and limitations of the use of dietary bioactive compounds. We also emphasize the need for further clinical studies assessing the long-term efficacy and safety of most commonly used dietary bioactives in UC. Studies providing deeper mechanistic insights using <italic>in vitro</italic> systems and <italic>in vivo</italic> animal models will also be equally important to help develop better compounds or combination therapies ultimately aimed at the development of more optimized, robust, and safer treatment strategies in future based on natural dietary supplements and phytochemicals. There is a need for more clinical studies to declare these bioactive compounds as completely safe and effective.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Molecular targets affected by various dietary agents and their phytochemicals in the prevention/amelioration of ulcerative colitis. &#x02191;, increase; &#x02193;, decrease</p></caption><graphic xlink:href="JTCM-4-203-g005"/></fig></sec>

Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) &#x02013; An Emerging Adjunct in Routine Oral Care

<p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) (&#x02018;acus&#x02019; (needle) + &#x02018;punctura&#x02019; (to puncture)) is the stimulation of specific points along the skin of the body involving various methods such as penetration by thin needles or the application of heat, pressure, or laser light. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) aims to treat a range of medical and dental ailments, though is most commonly used for pain relief. This article reviews about the various possible roles of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in clinical dental practice. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has potential in supplementing conventional treatment procedures by its diverse applicability outreach. Role of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in dental practice has been well supported by clinical trials. Its role in alleviating facial pain, pre-operative and post-operative dental pain has led to its widespread application. Its role as sole analgesic for treatment procedure has to be tested. It's It is a thought that acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) may prove an indispensible supplement to conventional treatment modalities and more of clinical trials and studies are required to prove the efficacy. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is not a miracle cure and is not going to replace the drill. However, the technique can be a supplement to conventional treatments in TMDs, facial pain, pain management Sjoegrens syndrome, and in phobias and anxiety. The application and use of Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) comes with some side effects. Proper training needs to be obtained before commencement of any procedure related to acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;). Various training programs are offered to train clinical practitioners the apt method to use acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;).</p>

<contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Devanand</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Dalai</surname><given-names>Deepak Ranjan</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Swapnadeep</surname><given-names/></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Mehta</surname><given-names>Parul</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Indra</surname><given-names>B Niranjanaprasad</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Rastogi</surname><given-names>Saurabh</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Ankita</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chaturvedi</surname><given-names>Mudita</given-names></name><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Saumya</given-names></name><xref ref-type="aff" rid="aff8">8</xref></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Sanjeev</given-names></name><xref ref-type="aff" rid="aff9">9</xref></contrib><contrib contrib-type="author"><name><surname>Gill</surname><given-names>Shruti</given-names></name><xref ref-type="aff" rid="aff10">10</xref></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Nisha</given-names></name><xref ref-type="aff" rid="aff11">11</xref></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Rajendra Kumar</given-names></name><xref ref-type="aff" rid="aff12">12</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is one of the &#x0201c;complementary and alternative medicine (CAM)&#x0201d; techniques used to treat a variety of diseases and disorders. Up to one third of the public in many countries has been shown to consult a CAM practitioner at least once a year.</p><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is defined as the stimulation of the so-called &#x02018;acupoints&#x02019; for disease prevention, treatment or maintenance of health. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) can utilize various methods including solid needles, lasers, electro-acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) and transcutaneous nerve stimulation.</p><p>Evidence of origin of Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) dates back to 3000 years.[<xref rid="ref1" ref-type="bibr">1</xref>] It was China which came up with the idea of Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;). The earliest account of this is found in the book called Nei Jing which in English means the Yellow Emperor's Classic of Internal Medicine. The contents of this book date somewhere around 200 BC. Back then, they did not use metal needles and instead used those made out of stone, moxibustion and herbs to treat a patient. It is being relied and used practically as an useful adjunct in various medical and allied health sciences. Several studies are suggestive of the pivotal role of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in healing of several diseases and alleviating pain.[<xref rid="ref2" ref-type="bibr">2</xref>]. Literature has proved the role of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in withdrawal of narcotics.[<xref rid="ref3" ref-type="bibr">3</xref>] There are several smoking cessation medications and therapies available presently. However acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is a recommended alternative procedure, especially where conventional therapies have already failed. We feel, however, that serious consideration of this issue is beyond the scope of this paper. It is thought that acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is a technique involving ancient knowledge of Chinese philosophy and is of no use in dentistry as it works on the placebo concept but the tremendous research on acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has proved it wrong.[<xref rid="ref4" ref-type="bibr">4</xref>] Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is effective in numerous conditions like temporomandibular disorders (TMDs), pain management, and clinical conditions like Sjogren's syndrome.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] As because the use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has evolved since last few decades, Skeptics shrug off the positive effects of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) as merely placebo effects. Believers in acupuncture, (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) however, say that the benefits have simply not yet been proven. Believers promote that the treatment is harmless and can be used as a complement to western medicine.</p><p>Published controlled studies on the effect of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in dentistry are still relatively few, but those which fulfill predefined methodological criteria are reviewed to assess if acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is effective in this field. Anxiety related to dental treatment is rife, and there is a clinical impression that acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) can offer an alternative to the sedative drugs commonly used although no controlled Studies are available.</p><p>In recent years interest for acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in dentistry has increased at least partly because of published results of its efficacy. However the literature is not extensive and results vary considerably: from no effect to significant improvement. The main of this article is to discuss and review methodologically the published clinical trials to determine whether clear conclusions can be obtained with the use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;).</p></sec><sec id="sec1-2"><title>DEFINITION AND TYPES</title><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is a medical technique in which needles are inserted into the skin and underlying tissues. One or more small metal needles are inserted at precise points along 12 meridians (pathways) in the body, through which the vital life force (qi) is believed to flow, in order to restore yin-yang balance and treat disease caused by yin-yang imbalance. Practitioners of Chinese traditional medicine believe in energy flows. This energy, called Qi, circulates around the body using pathways called meridians. Meridians run very close to the surface of the skin in certain areas and can be accessed by needles. Much like plumbing, these pipes can get blocked or go the wrong way, causing health problems. The insertion of needles at these points is aimed to help loosen blocks and normalize flow. The Chinese describe acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) by the character &#x0201c;Chen,&#x0201d; which literally means &#x0201c;to prick with a needle,&#x0201d; a graphic description of this therapeutic technique. It is a treatment procedure in which generally steel, silver, or gold needles are inserted into specific acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>]</p><p>Various techniques used in acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) are as follows:[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>]</p><p> <list list-type="bullet"><list-item><p>Traditional body acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) (It uses eight principles of complementary opposites to create harmony in the body. These include yin/yang, internal/external, excess/deficiency, hot/cold.)</p></list-item><list-item><p>Microsystems acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) such as ear acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;)</p></list-item><list-item><p>Electro-acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;)</p></list-item><list-item><p>Trigger point acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;)</p></list-item><list-item><p>Laser treatment</p></list-item><list-item><p>Moxibustion</p></list-item><list-item><p>Acupressure</p></list-item><list-item><p>Okibari &#x02013; Japanese style</p></list-item></list> </p><p>At times, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is more effective when the needles are first heated. This technique is known as &#x0201c;moxibustion.&#x0201d; Here, the acupuncturist lights a small bunch of the dried herb called moxa or mugwort and holds it above the needles. The herb, which burns slowly and gives off a little smoke and a pleasant, incense-like smell, will never directly touch one's body. Another variation is electrical acupuncture. (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) Here, electrical wires are hooked up to the needles and a weak current runs through it which may cause no or little sensation at all. There is also auricular acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) where it is believed that the ear is a microcosm of the body. This means that acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) needles are placed on certain points on the ear so it can treat certain addiction disorders.</p></sec><sec id="sec1-3"><title>HISTORICAL EVIDENCE</title><p>In tradiational Chinese Medicine (TCM), The history of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) can be dated back to 2000 years ago i.e during the Warring states and the Qin and Han dyansties.[<xref rid="ref15" ref-type="bibr">15</xref>] Authors cite about the use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) among the wounded soldiers of a battle.[<xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>] Chinese mythology says, Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) practice began in the Stone Ages, when the sharp edgd tools called as &#x0201c;Bian&#x0201d; were used to puncture and drain abscesses.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>]</p><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is a tried and tested system of traditional medicine, which has been used in China and other eastern cultures for thousands of years to restore promote and maintain good health. Its benefits are now widely acknowledged all over the world and in the past decade traditional acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has begun to feature more prominently in mainstream healthcare in the UK. In conjunction with needling, the practitioner may use techniques such as moxibustion, cupping, massage or electro-acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;). They may also suggest dietary or lifestyle changes.</p><p>Traditional acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) takes a holistic approach to health and regards illness as a sign that the body is out of balance. The exact pattern and degree of imbalance is unique to each individual. The traditional acupuncturist's skill lies in identifying the precise nature of the underlying disharmony and selecting the most effective treatment. The choice of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points will be specific to each patient's needs. Traditional acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) can also be used as a preventive measure to strengthen the constitution and promote general wellbeing.</p></sec><sec id="sec1-4"><title>ACUPUNCTURE POINTS AND FEATURES</title><p>In general, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is believed to stimulate the nervous system and cause the release of neurochemical messenger molecules. The resulting biochemical changes influence the body's homeostatic mechanisms, thus promoting physical and emotional well-being. Stimulation of certain acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points has been shown to affect areas of the brain that are known to reduce sensitivity to pain and stress, as well as promoting relaxation and deactivating the &#x02018;analytical&#x02019; brain, which is responsible for anxiety.</p><p>The Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points are basically areas of low electrical resistance. The insertion of needles is made into the skin and muscle. The needles used in acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) are very small and one probably feels a slight twitch when the needle goes in. They will be inserted carefully to 1/2&#x0201d; or an inch in depth and will stay there from 45 minutes to an hour depending on the condition of the patient. If inserted properly, patient should feel some cramping, distention, electric sensation or tingling which means that the treatment is working. Around 70-80% of these acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points are similar to the trigger points and most of the acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points are also similar to muscular motor points.[<xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] There are various acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points which play their role in dentistry, such as body points, relevant trigger points, and ear points.</p><p>The General points are described as under:</p><p><inline-graphic xlink:href="JTCM-4-218-g001.jpg"/></p></sec><sec id="sec1-5"><title>MECHANISM OF ACTION</title><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) prevents and treats diseases by inserting very fine needles into the skin specifically at the anatomic points of the body. The principle behind this concept is that illnesses occur because there is an imbalance in your life force otherwise known as Qi. It is believed that this flows in 14 channels in the human body known as meridians which branch out to bodily organs and functions. If there is a blockage or obstruction in any of them, this is when you succumb to a disease or an illness. The imbalance in the Qi may go one way or the other because of Yin and Yang. The person can only be normal if there is harmony between the two which is what acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is trying to achieve. This can only be restored by stimulating these acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points so your Qi can be adjusted, balanced and harmonized. Apart from using needles, practitioners also use friction, heat, impulses of electromagnetic energy and pressure to stimulate these points in order to balance the movement of energy in the body to reduce one's health.</p><p>The mechanism used in Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) basically stimulates the myelinated nerve fibres in muscles which activates midbrain and pituitary-hyothalmus via spinal cord. The various nerotransmitters playing role are Enkephalin, b-endorphin, Dynorphin, Serotonin, and Noradrenalin.[<xref rid="ref25" ref-type="bibr">25</xref>] The insertion of a needle in an acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) point creates a small inflammatory process with release of neurotransmitters such as bradykinin, histamine, etc., and subsequently stimulate A&#x01d5f; fibers located in the skin and muscle. The A&#x01d5f; fibers terminating in the second layer of the black horn inhibit the incoming painful sensations by release of enkephalin. This step acts as a Pain relieving step for Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;).[<xref rid="ref26" ref-type="bibr">26</xref>] From the second layer of the back horn, the A&#x01d5f; fiber continues to the fifth layer of the black horn, crosses over to the opposite side, and ascends via spinothalamic tract to the midbrain where the raphe magnus nucleus is stimulated.[<xref rid="ref27" ref-type="bibr">27</xref><xref rid="ref28" ref-type="bibr">28</xref>]</p></sec><sec id="sec1-6"><title>ACCUPUNCTURE (&#x091dd;&#x07078; ZH&#x00112;N JI&#x0016c;) ORAL AND DENTAL HEALTH</title><p>According to the World Health Organization, here is a list of some illnesses where acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is used. These include acute bronchitis, the common cold, cataract, toothaches, hiccups, ulcers, constipation, diarrhea, headache and migraine, Meniere's disease, osteoarthritis and a lot more. In the US, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is often used to treat chronic pain conditions and mind body disorders.</p><sec id="sec2-1"><title>Management of dental pain (pre-op and post-op), TMDs, oro-facial pain and facial palsy</title><p>Like other clinical disciplines dentists sometimes meet problems which do not respond to orthodox treatment. Facial pain may give rise to diagnostic difficulties, sometimes though failure to consider musculoskeletal causes. Orthodox treatment may involve surgery for instance on sinuses, trigeminal nerve ablation, or removal of teeth, occasionally unnecessarily. There are many causes of facial pain and there is often a musculoskeletal component but in general high percentage respond to acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) clinically.</p><p>According to TCM theory, local acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points on facial regions such as ST6 and ST7 and distant points like LI4 can be used to treat dental pain. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in dental considerations might not be involved in treating the cause of dental pain but it acts as an adjunct in achieving anesthesia before dental procedures are carried out. In some countries, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has even been used to replace chemical anaesthesia prior to surgery as there are some patients who are not able to tolerate regular anaesthesia. Studies have shown that the onset time for regional anesthesia after administration of prilocaine hydrochloride is around 2 min. A pilot study was conducted to investigate whether the induction time of local anesthetic can be reduced if acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is given before injection.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref29" ref-type="bibr">29</xref>]In 1995 and 1999 randomized placebo-controlled trials conducted by Lao <italic>et al</italic>. reported that the group that received acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) treatment after the surgical removal of impacted lower third molar had significantly longer pain-free postoperative time compared to the placebo group.[<xref rid="ref30" ref-type="bibr">30</xref><xref rid="ref31" ref-type="bibr">31</xref>] Subjects treated with acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) reported 181 min pain-free time compared with 71 min in the placebo group. More randomized controlled clinical trials may be necessary to verify the role of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) therapy in dental pain management, particularly in postoperative pain.</p><p>Although acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) therapy may not be helpful in treating the cause of TMD but it may aid in relieving the pain caused by TMD and can provide comfort to the patient. It has been documented that acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) can help in muscle relaxation and reduce muscle spasms. A systematic review of randomized controlled trials for assessing the efficacy of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) for symptomatic cure of TMDs was conducted.[<xref rid="ref32" ref-type="bibr">32</xref>] Raustia <italic>et al</italic>. compared the effectiveness of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) and conventional treatment modalities in the management of TMD and found no difference in the two modalities as far as subjective and objective variables are concerned.[<xref rid="ref33" ref-type="bibr">33</xref>]</p><p>There are several case reports and case series in Chinese literature on successful acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) treatment for patients with trigeminal neuralgia. Studies have shown that by the use of filiform needles and using both local and distal points, trigeminal neuralgia can be treated.[<xref rid="ref34" ref-type="bibr">34</xref>] Positive results have also been obtained with combined use of three needling and point injections.[<xref rid="ref35" ref-type="bibr">35</xref>] Researches were also done with the combination of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) and Chinese herbs which also showed positive results.[<xref rid="ref36" ref-type="bibr">36</xref>]</p><p>The use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) to treat Bell's palsy is based on the TCM concept that needle manipulation at both the local and distal sites can regulate the flow of qi in the meridians, harmonize qi&#x02013;blood balance, and strengthen the body's resistance to external wind pathogens. It may also help in increasing the excitability of nerves and in promoting the regeneration of nerve fibers. Some local acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points used include ST6 Jiache, located near the angle of the mandible at the prominence of the masseter muscle, and ST7 Xiaguan, located at the depression between the zygomatic arch and the mandibular notch. These two points are found to be anatomically close to branches of the facial nerve.[<xref rid="ref37" ref-type="bibr">37</xref><xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref>]</p><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) for orofacial pain and facial paralysis works primarily through cranial nerve stimulation of primarily the trigeminal and facial nerves, respectively. In treating xerostomia, needles stimulate parasympathetic function. Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) for craniomandibular pain targets sensitive spots primarily in the muscles of mastication. The value of practicing acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) from a neurophysiologic perspective is that one can more readily and justifiably extrapolate treatments from humans onto animals, based on neuro anatomically relevant transposition of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points across species.</p></sec><sec id="sec2-2"><title>Management of dental anxiety and gag reflex</title><p>Several reports on the use of auricular acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) for treating chronic and acute anxiety have shown promising results.[<xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref>] There was no difference in the efficacy of Midazolam and auricular acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in the management of anxiety related to dental treatment.[<xref rid="ref42" ref-type="bibr">42</xref>]</p><p>The stimulation of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) points like PC6 Neiguan and CV24 Chengjiang has proved to significantly reduce gag reflex.[<xref rid="ref43" ref-type="bibr">43</xref>] Reports suggest auricular acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) to be helpful in treating severe gag reflex.[<xref rid="ref44" ref-type="bibr">44</xref>] The points known to reduce anxiety are: Top inside of ear (relaxation), Lower inside of ear, just above where the lobe attached to the side of the face (tranquilizer), Lower inside of the ear and near the upper part of the lobe (master cerebral). Stimulation of an acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) point on the ear prior to undergoing treatment effectively controls the gag reflex, allowing dentists to perform a variety of procedures without compromising the patient's safety and comfort. More studies should be carried out to verify the effectiveness of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in controlling gag reflex.</p></sec></sec><sec id="sec1-7"><title>EFFICACY AND PROGNOSIS OF TREATMENT</title><p>The cure of Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is not a miraculous. Not everyone is impressed with acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) itself. Most western medical professionals have expressed either doubt or indifference to the oriental method while others have downright driven it down into the earth with criticisms and brutal skepticism. However, recent research shows the efficacy (or lack thereof) of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;), and while more research has to be done, it has been proven to actually positively affect some, but not all, forms of ailments that it claims to cure. The mechanism does not work in all patients, so its efficacy varies from one person to other. A lack of response should always result in re-examination and refinement of diagnosis.[<xref rid="ref45" ref-type="bibr">45</xref>] Adequate positive results have come out in treatment of pain in teeth and bite problems, various types of neuralgia, anxiety, etc.</p></sec><sec id="sec1-8"><title>ADVERSE EFFECTS OF ACUPUNCTURE (&#x091dd;&#x07078; ZH&#x00112;N JI&#x0016c;)</title><p>Like other strategies used in treating health conditions, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) may have some side effects. Medications have side effects and allergic reactions, surgeries have risk of infection and complications. There have been numerous reports of adverse effects following acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;). These are (to mention a few) risk of injury, rare infections, minor bleeding, small bruises, some dizziness, pneumothorax, endocarditis, and hepatitis, some resulting in fatalities.[<xref rid="ref46" ref-type="bibr">46</xref>] However, it must be appreciated that most of these results are due to the lack of basic anatomical knowledge or because of not applying aseptic procedures by non-medical/dental qualified practitioners. When these are factored out of analysis, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) proves to be a very safe technique in the hands of a properly trained practitioner.[<xref rid="ref47" ref-type="bibr">47</xref><xref rid="ref48" ref-type="bibr">48</xref><xref rid="ref49" ref-type="bibr">49</xref><xref rid="ref50" ref-type="bibr">50</xref><xref rid="ref51" ref-type="bibr">51</xref><xref rid="ref52" ref-type="bibr">52</xref><xref rid="ref53" ref-type="bibr">53</xref><xref rid="ref54" ref-type="bibr">54</xref><xref rid="ref55" ref-type="bibr">55</xref>]</p><p>To promote acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;), the NIH or National Institute of Health in 1997 has stated that this technique is very useful in treating various health conditions. These include ear, nose, throat, respiratory, gastrointestinal, eye, nervous system and muscular disorders. In some cases, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is able to prevent some of them from happening. This was further strengthened by the fact that the side effects of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) are much less than that of conventional drugs that are being sold by pharmaceutical companies.</p></sec><sec sec-type="conclusion" id="sec1-9"><title>CONCLUSION</title><p>Although, the application of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has a long history, it still proves to be an effective treatment modality in TCM sector. With the growing acceptance of alternative medicine in Western cultures, acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is quickly becoming a popular practice. More and more people today are choosing acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) over western medicine to treat bodily pains, relieve stress, or to promote overall health. In the control of postoperative pain or in the management of TMD and facial pain, it has come out to be a useful alternative to the conventional therapeutic armamentarium of the general dental practitioner. But acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) is not risk free. Hematoma may develop if the needle punctures a circulatory structure. It is also possible that pneumothorax may occur if the needle is inserted too deep. There is also the risk of HIV and hepatitis if the needle is not properly sterilized. One must not forget that it is merely an alternative and not a proper form of medical treatment. This means one should still be examined by a professional in the medical field who can determine the severity of the illness or disease. Some physicians may wish to expand the scope of their practice by taking additional training to administer acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;).[<xref rid="ref46" ref-type="bibr">46</xref><xref rid="ref55" ref-type="bibr">55</xref><xref rid="ref56" ref-type="bibr">56</xref><xref rid="ref57" ref-type="bibr">57</xref><xref rid="ref58" ref-type="bibr">58</xref><xref rid="ref59" ref-type="bibr">59</xref><xref rid="ref60" ref-type="bibr">60</xref>] Unfortunately, standards of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) have not been fully approved by the FDA due to many unregulated practices that still exist such as the re-use of needles.</p></sec><sec id="sec1-10"><title>RECOMMENDATIONS</title><p> <list list-type="bullet"><list-item><p>Dentists should treat their patients with acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) only after obtaining the relevant knowledge and ability.</p></list-item><list-item><p>Acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) should only be employed after a thorough diagnosis of the patient's condition. Meanwhile, employment of conventional therapies may be considered as appropriate.</p></list-item><list-item><p>A separate informed consent for acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) may be required. Standard infection control procedures must be in place.</p></list-item><list-item><p>Single use of needles must be employed.</p></list-item><list-item><p>As it is the case with all therapeutic measures, the use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) has to be documented appropriately and detailed records must be kept.</p></list-item><list-item><p>Multidisciplinary research into the effective use of acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) in dentistry is encouraged.</p></list-item><list-item><p>Teaching and professional training in acupuncture (&#x091dd;&#x07078; Zh&#x00113;n Ji&#x001d4;) could be considered as an optional part of undergraduate, postgraduate and continuing professional development.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref46" ref-type="bibr">46</xref>]</p></list-item></list> </p></sec>

Revisiting <italic>Terminalia arjuna</italic> &#x02013; An Ancient Cardiovascular Drug

<p><italic>Terminalia arjuna</italic>, commonly known as <italic>arjuna</italic>, belongs to the family of Combretaceae. Its bark decoction is being used in the Indian subcontinent for anginal pain, hypertension, congestive heart failure, and dyslipidemia, based on the observations of ancient physicians for centuries. The utility of <italic>arjuna</italic> in various cardiovascular diseases needs to be studied further. Therefore, the present review is an effort to give a detailed survey of the literature summarizing the experimental and clinical studies pertinent to <italic>arjuna</italic> in cardiovascular disorders, which were particularly performed during the last decade. Systematic reviews, meta-analyses, and clinical studies of <italic>arjuna</italic> were retrieved through the use of PubMed, Google Scholar, and Cochrane databases. Most of the studies, both experimental and clinical, have suggested that the crude drug possesses anti-ischemic, antioxidant, hypolipidemic, and antiatherogenic activities. Its useful phytoconstituents are: Triterpenoids, &#x003b2;-sitosterol, flavonoids, and glycosides. Triterpenoids and flavonoids are considered to be responsible for its beneficial antioxidant cardiovascular properties. The drug has shown promising effect on ischemic cardiomyopathy. So far, no serious side effects have been reported with <italic>arjuna</italic> therapy. However, its long-term safety still remains to be elucidated. Though it has been found quite useful in angina pectoris, mild hypertension, and dyslipidemia, its exact role in primary/secondary coronary prevention is yet to be explored.</p>

<contrib contrib-type="author"><name><surname>Dwivedi</surname><given-names>Shridhar</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Chopra</surname><given-names>Deepti</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p><italic>Arjuna</italic> is a potential cardioprotective agent belonging to the Combretaceae family. It is an ayurvedic remedy that has been mentioned since vedic period in many ancient Indian medicinal texts including Charaka Samhita, Sushruta Samhita, and Astang Hridayam. It was Vagabhatta who, for the first time, advocated the use of stem bark powder in heart ailments.[<xref rid="ref1" ref-type="bibr">1</xref>]</p></sec><sec id="sec1-2"><title>ETHNOMEDICAL USES</title><p>The bark has been described as an astringent, demulcent, expectorant, cardiotonic, styptic, antidysenteric, urinary astringent, and has shown to be useful in fracture, ulcers, leukorrhea, diabetes, anemia, cardiopathy, and cirrhosis.[<xref rid="ref2" ref-type="bibr">2</xref>] Chakradatta, the great ancient physician, recommended it to be given as a decoction of bark with milk or as a ghrita (a preparation with ghee or butter).[<xref rid="ref3" ref-type="bibr">3</xref>] Decoction of the bark has been used as ulcer wash, while bark ashes have been prescribed for snakebite and scorpion sting.[<xref rid="ref4" ref-type="bibr">4</xref>] Traditional healers from Kancheepuram district, Tamil Nadu boil the bark powder with water, and inhale it to cure headache and to kill worms in teeth. They also use fruit paste topically on wounds.[<xref rid="ref5" ref-type="bibr">5</xref>] Fresh leaf juice is used for the treatment of earache and bark powder for treating heart ailments by Malabar tribe, Kerala.[<xref rid="ref6" ref-type="bibr">6</xref>] Tribals living in Sundargarh District, Orissa use dried bark powder along with rice washed water to treat blood in urine, and tribes living in Malkangiri district chew the fresh bark and swallow the juice as an antacid.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>]</p></sec><sec id="sec1-3"><title>HABITAT</title><p><italic>Arjuna</italic> tree is about 60-80 ft in height, and is seen along rivers, streams, and dry water bodies throughout the Indo-sub-Himalayan tracts of Uttar Pradesh, southern Bihar, Chota Nagpur, Burma, Madhya Pradesh, Delhi, and Deccan region [<xref ref-type="fig" rid="F1">Figure 1</xref>]. It is also found in the forests of Sri Lanka and Mauritius.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref9" ref-type="bibr">9</xref>] It grows almost in all types of soils, but prefers humid, fertile loam and red lateritic soils. It can tolerate half submergence for a few weeks. <italic>Arjuna</italic> is propagated by seeds; Germination takes 50-70 days with 50-60% germination.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><italic>Terminalia arjuna</italic> tree</p></caption><graphic xlink:href="JTCM-4-224-g001"/></fig></sec><sec id="sec1-4"><title>PHARMACOGNOSTIC FEATURES</title><p>The outer surface of the bark is smooth, while the inner surface has longitudinal striation and is pinkish in color.[<xref rid="ref2" ref-type="bibr">2</xref>] The bark gets flaked off itself in the month of April&#x02013;May [<xref ref-type="fig" rid="F2">Figure 2</xref>].[<xref rid="ref11" ref-type="bibr">11</xref>]</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Bark stem of <italic>Terminalia arjuna</italic></p></caption><graphic xlink:href="JTCM-4-224-g002"/></fig><p>On microscopic examination of the mature bark, a cork consisting of 9-10 layers of tangentially elongated cells, 2-4 cells thick phellogen, and phelloderm consisting of tangentially elongated cells are seen. The phloem is broad, consisting of ceratenchyma, phloem parenchyma, phloem fibers, and crystal fibers with rosette crystals of calcium oxalate. Periderm and secondary phloem are present in the old bark.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Leaves are sub-opposite, coriaceous, oblong/elliptic, dull green from the upper side and pale brown on the lower side, often unequal sided with 10-15 pairs of nerves [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Flowers are white in color and bisexual, arranged in spikes with linear bracteoles [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Fruits are ovoid/oblong with 5-7 hard angles or wings. The lines on wings are oblique and curving upward [<xref ref-type="fig" rid="F5">Figure 5</xref>].[<xref rid="ref2" ref-type="bibr">2</xref>]</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Leaves of <italic>Terminalia arjuna</italic></p></caption><graphic xlink:href="JTCM-4-224-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Flower of <italic>Terminalia arjuna</italic></p></caption><graphic xlink:href="JTCM-4-224-g004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Fruits of <italic>Terminalia arjuna</italic> (ripe, fresh)</p></caption><graphic xlink:href="JTCM-4-224-g005"/></fig><p>Major chemical constituents of <italic>arjuna</italic> have been shown in <xref ref-type="table" rid="T1">Table 1</xref>.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>]</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Major chemical constituents of arjuna</p></caption><graphic xlink:href="JTCM-4-224-g006"/></table-wrap><p>Various extracts of the stem bark of <italic>arjuna</italic> have shown to possess many pharmacological properties including inotropic, anti-ischemic, antioxidant, blood pressure lowering, antiplatelet, hypolipidemic, antiatherogenic, and antihypertrophic.[<xref rid="ref18" ref-type="bibr">18</xref>] Thus, in the present article, we have made an attempt to review and give up-to-date information pertinent to the usage of <italic>arjuna</italic> as a potential cardioprotective agent.</p></sec><sec id="sec1-5"><title>EXPERIMENTAL STUDIES</title><sec id="sec2-1"><title>Effects on cardiac hemodynamics, coronary flow, and blood pressure</title><p>Bark stem of <italic>arjuna</italic> possesses diuretic, inotropic, and chronotropic properties.[<xref rid="ref9" ref-type="bibr">9</xref>] In the Langendorff's rabbit heart preparation, the aqueous extract has demonstrated to cause an increase in the coronary flow.[<xref rid="ref19" ref-type="bibr">19</xref>] Substantiating the earlier findings recently, an experimental study showed that the aqueous extract of <italic>arjuna</italic> increased the force of contraction of cardiac muscle in frog's heart <italic>in situ</italic>, hypodynamic frog's heart <italic>in situ</italic>, and isolated perfused rabbit heart. It increased the coronary flow in isolated perfused rabbit heart and produced bradycardia.[<xref rid="ref20" ref-type="bibr">20</xref>] The inotropic effect is considered to be mediated through the high concentration of Ca⁺⁺ present in the plant.[<xref rid="ref21" ref-type="bibr">21</xref>]</p><p>Aqueous and alcoholic bark extract, when given intravenously, intracerebrally, and intravertebrally in dog, resulted in a dose-dependent decrease in blood pressure.[<xref rid="ref9" ref-type="bibr">9</xref>] Singh <italic>et al</italic>. reported that an aqueous solution of 70% alcoholic bark extract produced dose-dependent decrease in heart rate and blood pressure in dogs, though the mechanism was not determined.[<xref rid="ref22" ref-type="bibr">22</xref>]</p><p>Takahashi <italic>et al</italic>. demonstrated that the hypotensive effect of <italic>arjuna</italic> was observed with a fraction containing tannin-related compounds separated from the aqueous extract, which was not affected by pretreatment of rats with propranolol, but was attenuated by pretreatment with atropine. This suggested that the hypotensive effect may be mediated by cholinergic mechanisms.[<xref rid="ref23" ref-type="bibr">23</xref>] Later on, it was documented that the 70% alcoholic extract produced dose-dependent hypotension of peripheral origin which might be due to adrenergic &#x003b2;2-receptor agonistic and/or direct action on the heart muscle. It was also suggested that muscarinic or histaminergic mechanisms are not likely to be involved in the hypotension produced.[<xref rid="ref24" ref-type="bibr">24</xref>]</p><p>In a recent study, it has been established that the method of administration and/or selective omission of the hydrophobic components from the bark powder could be crucial to the efficacy and safety of <italic>arjuna</italic> bark in cardiac therapy.[<xref rid="ref25" ref-type="bibr">25</xref>]</p></sec><sec id="sec2-2"><title>Antioxidant and cardioprotective effect</title><p>Dried, pulverized bark has been shown to augment endogenous antioxidant compounds of rat heart and prevent oxidative stress associated with ischemic&#x02013;reperfusion injury of the heart.[<xref rid="ref26" ref-type="bibr">26</xref>]</p><p>It was suggested that the alcoholic extract of <italic>arjuna</italic> in rabbit induces myocardial heat shock protein 72 and augments myocardial endogenous antioxidants which offer cardioprotection against oxidative stress associated with myocardial ischemic&#x02013;reperfusion injury.[<xref rid="ref27" ref-type="bibr">27</xref>] The cardioprotective effect of the active phytoconstituents of <italic>arjuna</italic> bark against carbon tetrachloride and sodium fluoride induced oxidative stress, probably via its antioxidant properties, has also been documented. In the above models, ferric reducing/antioxidant power assay revealed that ethanol extract enhanced the cardiac intracellular antioxidant activity.[<xref rid="ref28" ref-type="bibr">28</xref><xref rid="ref29" ref-type="bibr">29</xref>] In a recent study, the methanol extract yielded the highest phenolic and flavonoid content and was found to possess the highest total antioxidant capacity. Thus, it can be inferred that there exists a linear correlation between the antioxidant capacity and the total phenolic content of the extracts.[<xref rid="ref30" ref-type="bibr">30</xref>] In another study, both alcoholic and aqueous extracts of the bark attenuated H₂O₂-mediated reactive oxygen species generation in human monocytic cells by promoting catalase and glutathione peroxidase (GPO) activities and by sustaining cellular reducing power. Moreover, the extracts inhibited lipid peroxidation (LPO) and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, but had no effect on lipoprotein lipase.[<xref rid="ref31" ref-type="bibr">31</xref>]</p><p>In isoprenaline-induced myocardial ischemia (MI), <italic>arjuna</italic> has been found to possess prostaglandin E2-like activity with coronary vasodilatation and hypotension.[<xref rid="ref1" ref-type="bibr">1</xref>] The bark extract has shown to significantly prevent isoprenaline-induced increase in oxidative stress and decline in endogenous antioxidant level.</p><p>Arjunolic acid has been found to prevent the decrease in the levels of superoxide dismutase, catalase, GPO, ceruloplasmin, &#x003b1;-tocopherol, reduced glutathione, ascorbic acid, lipid peroxide, and myeloperoxidase.[<xref rid="ref32" ref-type="bibr">32</xref>]</p><p>Further, the bark extract has also shown protective effects against doxorubicin-induced DNA damage and cardiotoxicity.[<xref rid="ref33" ref-type="bibr">33</xref><xref rid="ref34" ref-type="bibr">34</xref>] Kumar <italic>et al</italic>. demonstrated that <italic>arjuna</italic> protects the heart against myocardial changes induced by chronic &#x003b2;-adrenoceptor stimulation.[<xref rid="ref35" ref-type="bibr">35</xref>] Substantiating this, in a recent experiment, the bark extract significantly attenuated cardiac dysfunction and myocardial injury in rats with congestive heart failure (CHF). Cardioprotective action of <italic>arjuna</italic> was comparable to fluvastatin. <italic>Arjuna</italic> bark extract has a significant prophylactic and therapeutic beneficial effect in protecting heart against catecholamine-induced CHF, possibly through maintaining endogenous antioxidant enzyme activities and inhibiting LPO and cytokine levels.[<xref rid="ref36" ref-type="bibr">36</xref>]</p><p>Recently, Mythili <italic>et al</italic>. confirmed the earlier findings that triterpenoids derived from <italic>arjuna</italic> extract containing arjunolic acid show cardioprotective activity by boosting endogenous antioxidant defense system.[<xref rid="ref37" ref-type="bibr">37</xref>]</p></sec><sec id="sec2-3"><title>Hypolipidemic and antiatherogenic activity</title><p>Earlier animal experiments have demonstrated that <italic>arjuna</italic> bark powder/extract reduces the total cholesterol (TC) and triglyceride (TG) levels.[<xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref><xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref>] On comparing the hypolipidemic property of the bark in different solvent fractions (petroleum ether, solvent ether, ethanol, and water) in hyperlipidemic rats, it was observed that only the ethanolic fraction exerted significant lipid-lowering effect. Solvent ether and ethanolic fractions caused a decrease in the plasma levels of lipids in triton as well as in high fat diet (HFD) fed models of hyperlipidemia in hamsters. In an <italic>in vitro</italic> experiment with <italic>arjuna</italic> fractions at concentrations of 50-500 &#x003bc;g/ml, they were found to inhibit the oxidative degradation of lipids induced by metal ions in human low density lipoprotein (LDL) and rat liver microsomes. When these fractions were tested against the generation of oxygen free radicals, they counteracted the formation of superoxide anions and hydroxyl radicals in nonenzymic test systems. The efficacy of <italic>arjuna</italic> fractions was found to be in the order: Ethanol fraction &#x0003e; solvent ether fraction &#x0003e; petroleum ether fraction.[<xref rid="ref42" ref-type="bibr">42</xref>]</p><p>The ethanolic fraction possesses potent antioxidant and hypolipidemic properties compared to other fractions, and this has been substantiated by other studies also.[<xref rid="ref43" ref-type="bibr">43</xref><xref rid="ref44" ref-type="bibr">44</xref>] Subsequent work done by Sharma <italic>et al</italic>. also substantiated the hypolipidemic and antioxidant effect of <italic>arjuna</italic>. In addition to this, they also found that recipes (<italic>Arjuna</italic> Omelette and <italic>Arjuna</italic> En Upma) incorporating <italic>arjuna</italic> bark showed good acceptability, meriting their inclusion in the daily diet of the people needing long-term intervention for elevated lipids and oxidative stress levels.[<xref rid="ref45" ref-type="bibr">45</xref>]</p><p>The hypolipidemic action is thought to be mediated through increased hepatic clearance of cholesterol, down-regulation of lipogenic enzymes, and inhibition of HMG-CoA reductase.[<xref rid="ref46" ref-type="bibr">46</xref>] Further, Parmar <italic>et al</italic>. showed that there is a possibility of involvement of thyroid hormones (suppression of thyroid function) in the amelioration of cardiac and hepatic LPO by the bark extract in albino rats.[<xref rid="ref47" ref-type="bibr">47</xref>]</p></sec></sec><sec id="sec1-6"><title>CLINICAL USES</title><sec id="sec2-4"><title>Angina/myocardial infarction</title><p>The anti-ischemic effect of bark powder was evaluated in 30 patients of stable angina/post-infarct angina (500 mg tds). The authors observed that the mean anginal frequency decreased significantly, along with a significant decrease in systolic blood pressure (SBP), improvement in ECG changes, and reduction in plasma cortisol and serum cholesterol levels.[<xref rid="ref48" ref-type="bibr">48</xref>]</p><p>Later, in a study, 500 mg of bark powder was administered twice daily to 25 coronary artery disease (CAD) patients for 3 months. A reduction in the grade of positivity of treadmill test (TMT) response was observed in six patients, in addition to improvement in exercise tolerance and a reduction in the frequency of anginal attacks and use of sublingual nitrates.[<xref rid="ref49" ref-type="bibr">49</xref>]</p><p>Subsequently, in an open-label trial, it was demonstrated that there was a 50% reduction in angina episodes along with a significant delay in the time to the onset of angina on TMT and appearance of ST&#x02013;T changes in ECG after <italic>arjuna</italic> therapy was administered in stable angina patients. Significant lowering of SBP and body mass index, with a marginal improvement in left ventricular ejection fraction (LVEF) and a slight increase in high density lipoprotein (HDL) levels were also observed. In unstable angina patients, there was an insignificant reduction in anginal frequency. These results suggest that monotherapy with <italic>arjuna</italic> is fairly effective in patients with stable angina, but has a limited role in unstable angina.[<xref rid="ref50" ref-type="bibr">50</xref>]</p><p>In yet another study, 500 mg of bark powder was administered 8 hourly to 10 patients of post-myocardial infarction angina and 2 patients of ischemic cardiomyopathy for a period of 3 months. These patients were compared with matched patients of post-myocardial infarction angina receiving only conventional treatment. Significant reduction in anginal frequency, improvement in LVEF (from 42.25 &#x000b1; 9.96% to 52.57 &#x000b1; 12.32%), and reduction in left ventricular mass (LVM; from 159.18 &#x000b1; 51.11 g/m² to 140.62 &#x000b1; 55.65 g/m²) was noted.[<xref rid="ref51" ref-type="bibr">51</xref>]</p><p>The efficacy of Hartone (an herbal product containing <italic>arjuna</italic>) was studied in 10 stable angina patients. The results were compared with those of 10 patients of stable angina on 20 mg of isosorbide mononitrate (ISMN) administered twice daily. It was observed that Hartone gave symptomatic relief in 80% of patients as compared to 70% in ISMN alone group. In addition, <italic>arjuna</italic> was better tolerated than ISMN.[<xref rid="ref52" ref-type="bibr">52</xref>]</p><p>In a randomized, double-blind, cross-over study, 58 male patients with chronic stable angina (class II-III) with evidence of provocable ische mia on TMT received 500 mg of 90% alcohol extract 8 hourly, ISMN (40 mg/day), or a matching placebo for 1 week each after a washout period of at least 3 days. It was found that <italic>arjuna</italic> therapy was associated with a significant decrease in the frequency of angina and the need for isosorbide dinitrate. Improvements in clinical and TMT parameters were observed with both <italic>arjuna</italic> and ISMN as compared to placebo. No significant differences were observed in the above parameters when <italic>arjuna</italic> and ISMN therapies were compared.[<xref rid="ref53" ref-type="bibr">53</xref>]</p></sec><sec id="sec2-5"><title>CHF/hypertension</title><p>In one of the earliest studies, 10 patients with CHF received 4 g of <italic>arjuna</italic> bark powder twice daily for 1 month. The researchers observed improvement in the functional class, breathlessness, and overall well-being with significant diuresis, and a fall in both systolic and diastolic blood pressure.[<xref rid="ref54" ref-type="bibr">54</xref>]</p><p>Subsequently, the effect of bark extract (500 mg 8 hourly) was studied in a double-blind placebo-controlled two-phase trial comprising 12 patients with refractory CHF. In the first phase, <italic>arjuna</italic> was administered for a period of 2 weeks. A decrease in echo-left ventricular end-diastolic and end-systolic volume indices, an increase in left ventricular stroke volume index, and an increase in LVEF were recorded suggesting improvement. On long-term evaluation (20-28 months), in addition to continued improvement in symptoms and signs, they also reported an improvement in quality of life.[<xref rid="ref55" ref-type="bibr">55</xref>]</p><p>A study done with abana (herbal formulation containing <italic>arjuna</italic>) in hypertensive individuals revealed an improvement in cardiac function as indicated by an increase in ejection fraction and a significant reduction of the SBP, echocardiographic left ventricular internal diameter, posterior wall thickness, and interventricular septal thickness.[<xref rid="ref56" ref-type="bibr">56</xref>]</p><p>Recently, <italic>arjuna</italic> has also been shown useful in improving cardiovascular endurance and in lowering SBP in normal healthy subjects.[<xref rid="ref57" ref-type="bibr">57</xref>]</p></sec><sec id="sec2-6"><title>Rheumatic heart disease</title><p>Efficacy of <italic>arjuna</italic> in decompensated rheumatic heart disease was studied in a double-blind study in which 30 patients of rheumatic valvular heart disease with CHF were administered 200 mg <italic>arjuna</italic> thrice daily. The results revealed a significant improvement in LVEF, exercise duration, and significant reduction in heart size.[<xref rid="ref58" ref-type="bibr">58</xref>]</p></sec><sec id="sec2-7"><title>Ischemic mitral regurgitation</title><p>In a randomized, double-blind, placebo-controlled study done in patients with ischemic mitral regurgitation (IMR) following acute myocardial infarction, <italic>arjuna</italic> was found to significantly decrease IMR and anginal frequency. In addition, there was also significant improvement in diastolic dysfunction (E/A ratio; from 0.93 &#x000b1; 0.31 to 1.38 &#x000b1; 0.40 at 12 weeks).[<xref rid="ref59" ref-type="bibr">59</xref>]</p></sec><sec id="sec2-8"><title>Cardiomyopathy</title><p>In addition to its anti-ischemic property, <italic>arjuna</italic> was found to reduce LVM and improve LVEF.[<xref rid="ref59" ref-type="bibr">59</xref>] A recent observational study revealed that when patients of dilated cardiomyopathy with reduced LVEF received <italic>arjuna</italic> in addition to their standard therapy, there was a significant improvement in left ventricular parameters as well as functional capacity.[<xref rid="ref60" ref-type="bibr">60</xref>]</p></sec><sec id="sec2-9"><title>Platelet aggregation</title><p>The bark extract has been found to decrease platelet activation and possess antithrombotic properties <italic>in vitro</italic> in 20 patients of angiographically proven CAD and 20 age- and sex-matched controls. The possible mechanism could be by desensitizing platelets by competing with platelet receptor or by interfering with signal transduction.[<xref rid="ref61" ref-type="bibr">61</xref>]</p><p>In another recent randomized, double-blind, parallel-group, placebo-controlled study in patients with type 2 diabetes mellitus, 500 mg of <italic>arjuna</italic> administered thrice daily resulted in a significant increase in mean cardiac output from 4.34 &#x000b1; 0.38 to 4.86 &#x000b1; 0.20 (l/min). In addition to this, there was a reduction in mean systemic vascular resistance from 1729 &#x000b1; 93.52 to 1484 &#x000b1; 115.5 (dyne sec/cm⁵). <italic>Arjuna</italic> also caused significant inhibition of platelet aggregation.[<xref rid="ref62" ref-type="bibr">62</xref>]</p></sec><sec id="sec2-10"><title>Oxidative stress/dyslipidemia</title><p>In a study on 21 patients with coronary heart disease administered 1 g of bark powder twice daily with milk for 4 months, the patients showed improvement in lipid profile. In addition to this, patients got symptomatic relief after 1 month of treatment.[<xref rid="ref63" ref-type="bibr">63</xref>]</p><p>Antioxidant effect of bark powder (500 mg) has been demonstrated to be comparable to vitamin E (400 IU) in a randomized, controlled, open trial done in 105 patients with coronary heart disease. The authors also observed a significant decrease in TC, LDL, and lipid peroxide levels. The hypocholesterolemic effect was attributed to the soluble fibers and sitostanol content, while the antioxidant effect was attributed to the flavonoids.[<xref rid="ref64" ref-type="bibr">64</xref>] Further, it was observed in a study that when the bark powder was given along with statin for 3 months, it resulted in 15% reduction in TC, 11% reduction in TG, and 16% reduction in LDL, while there was minimal decline in lipoprotein (a) and nitrite levels.[<xref rid="ref65" ref-type="bibr">65</xref>]</p><p>In a prospective cohort study, dyslipidemic patients received <italic>arjuna</italic> powder (5 g, BD) for 3 weeks followed by Arogyavardhini Vati (500 mg, BD) for 4 weeks. A significant reduction in TC, LDL, TG, serum C-reactive protein, blood glucose, and an increase in HDL level were found, which supported the role of <italic>arjuna</italic> in dyslipidemic patients.[<xref rid="ref66" ref-type="bibr">66</xref>]</p></sec><sec id="sec2-11"><title>Lipoprotein(a)</title><p>A significant reduction in lipoprotein(a) levels amounting to 24.71% following the administration of <italic>arjuna</italic> in a patient of &#x003b2;-thalassemia associated with hyperlipoproteinemia and metabolic syndrome has been reported.[<xref rid="ref67" ref-type="bibr">67</xref>]</p></sec><sec id="sec2-12"><title>Endothelial dysfunction</title><p>In a double-blind, placebo-controlled, cross-over study involving 18 healthy male smokers and an equal number of age-matched non-smoker controls, it was observed that the hydroalcoholic extract of bark when given for 2 weeks led to significant regression of the endothelial abnormality amongst smokers.[<xref rid="ref68" ref-type="bibr">68</xref>]</p></sec><sec id="sec2-13"><title>Thrombotic condition</title><p>In a recent study done to investigate the <italic>in vitro</italic> thrombolytic and membrane-stabilizing action of four Bangladeshi medicinal plants including <italic>arjuna</italic>, the methanol extract was found to possess significant thrombolytic activity (30.57%). It also significantly inhibited the hemolysis of RBCs in both hypotonic solution and heat-induced conditions. This showed that it has moderate thrombolytic activity; however, more research is needed to isolate the secondary metabolites responsible for the activity.[<xref rid="ref69" ref-type="bibr">69</xref>]</p><p>Not much data is available to comment on the effect of <italic>arjuna</italic> on cytochrome P450 (CYP450) enzyme. Results from a recent <italic>in vitro</italic> study indicate that <italic>arjuna</italic> extracts contain constituents that can potently inhibit the activity of CYP1A.[<xref rid="ref70" ref-type="bibr">70</xref>]</p></sec></sec><sec id="sec1-7"><title>TOXICITY AND SIDE EFFECTS</title><p>Mild side effects like nausea, gastritis, headache, bodyache, constipation, and insomnia have been reported. No hematological, renal, or metabolic toxicity has been reported even after more than 24 months of its administration.[<xref rid="ref48" ref-type="bibr">48</xref><xref rid="ref50" ref-type="bibr">50</xref><xref rid="ref53" ref-type="bibr">53</xref><xref rid="ref55" ref-type="bibr">55</xref>] However, Parmar <italic>et al</italic>. noticed that administration of <italic>arjuna</italic> resulted in reduction of thyroid hormone concentration in euthyroid animals, whereas the hepatic LPO was increased. Thus, high amounts of the plant extract should not be consumed, as it may induce hepatotoxicity as well as hypothyroidism.[<xref rid="ref47" ref-type="bibr">47</xref>] The results from a recent acute and oral toxicological study done in animals showed that administration of ethanolic extract at a limit dose of 2000 mg/kg orally did not produce any kind of toxicity and death in animals.[<xref rid="ref46" ref-type="bibr">46</xref>]</p></sec><sec sec-type="conclusion" id="sec1-8"><title>CONCLUSION</title><p>The eternal interest in medicinal plants has led to the discovery of new chemical constituents and pharmacological actions of <italic>arjuna</italic>. Its efficacy as an anti-ischemic agent, a potent antioxidant, and an antiatherogenic agent has been amply demonstrated in various experimental and clinical studies. However, major lacunae of these studies include the lack of phytochemical standardization of the extract, bioavailability studies, and well-designed studies to evaluate its long-term toxicity effects. Its exact role in primary/secondary coronary prevention needs to be investigated. In addition to this, studies to look for the effect of <italic>arjuna</italic> on CYP450 enzymes and its interactions with other drugs like statin, aspirin, angiotensin-converting enzyme (ACE) inhibitors, and &#x003b2;-blocker need to be designed. Increasing the awareness regarding its medicinal usage can give a direction to the physicians to respond to the challenges in treating cardiovascular diseases.</p></sec>

Pomegranate from Oman Alleviates the Brain Oxidative Damage in Transgenic Mouse Model of Alzheimer's disease

<p>Oxidative stress may play a key role in Alzheimer's disease (AD) neuropathology. Pomegranates (&#x077f3;&#x069b4; Sh&#x000ed; Li&#x000fa;) contain very high levels of antioxidant polyphenolic substances, as compared to other fruits and vegetables. Polyphenols have been shown to be neuroprotective in different model systems. Here, the effects of the antioxidant-rich pomegranate fruit grown in Oman on brain oxidative stress status were tested in the AD transgenic mouse. The 4-month-old mice with double Swedish APP mutation (APPsw/Tg2576) were purchased from Taconic Farm, NY, USA. Four-month-old Tg2576 mice were fed with 4% pomegranate or control diet for 15 months and then assessed for the influence of diet on oxidative stress. Significant increase in oxidative stress was found in terms of enhanced levels of lipid peroxidation (LPO) and protein carbonyls. Concomitantly, decrease in the activities of antioxidant enzymes was observed in Tg2576 mice treated with control diet. Supplementation with 4% pomegranate attenuated oxidative damage, as evidenced by decreased LPO and protein carbonyl levels and restoration in the activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione (GSH), and Glutathione S transferase (GST)]. The activities of membrane-bound enzymes [Na⁺ K⁺-ATPase and acetylcholinesterase (AChE)] were altered in the brain regions of Tg2576 mouse treated with control diet, and 4% pomegranate supplementation was able to restore the activities of enzymes to comparable values observed in controls. The results suggest that the therapeutic potential of 4% pomegranate in the treatment of AD might be associated with counteracting the oxidative stress by the presence of active phytochemicals in it.</p>

<contrib contrib-type="author"><name><surname>Subash</surname><given-names>Selvaraju</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Essa</surname><given-names>Musthafa Mohamed</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Al-Asmi</surname><given-names>Abdullah</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Al-Adawi</surname><given-names>Samir</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Vaishnav</surname><given-names>Ragini</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Braidy</surname><given-names>Nady</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Manivasagam</surname><given-names>Thamilarasan</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Guillemin</surname><given-names>Gilles J.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most prevalent form of dementia characterized by a progressive decline in memory, behavior, and cognitive functions in the elderly population.[<xref rid="ref1" ref-type="bibr">1</xref>] It affects millions of people and has become a major medical and social burden in developed and developing countries.[<xref rid="ref2" ref-type="bibr">2</xref>] The disease has been the sixth leading cause of death across all ages and the fifth leading cause of death in those aged 65 and above. The neuropathology of AD is characterized at first by the deposition of senile plaques mainly composed of amyloid beta protein (A&#x003b2;) and neurofibrillary tangles containing hyperphosphorylated tau protein in the brain and later by the loss of neurons and their processes.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Cognitive impairment appears to be most closely correlated in time with the loss of neurons and neuronal processes.[<xref rid="ref5" ref-type="bibr">5</xref>] At present, the etiology of AD is still not well understood. Accumulation of A&#x003b2; peptide causes an increase in intracellular reactive free radicals and reactive oxygen species (ROS). The generation of free radicals (ROS) due to the A&#x003b2; peptide can induce functional and structural damage to cell membranes through lipid peroxidation (LPO) and protein carbonyl formation which may be involved in the pathogenesis of AD.[<xref rid="ref6" ref-type="bibr">6</xref>] These abnormal events in cells lead to oxidative neuronal cell death and cognitive decline in patients with AD.[<xref rid="ref7" ref-type="bibr">7</xref>] However, the relationship between plaque and tangle deposition and the neuronal degeneration that follows it is not clearly understood.</p><p>More recently, the interest in the role of dietary antioxidants in human health has prompted research in the field of AD. Fruits are good sources of these bioactives, and there are a number of commercial polyphenol-rich beverages which base their marketing strategies on antioxidant potency. Naturally occurring compounds from plants have been shown to have therapeutic potential f or AD.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] Curcumin and <italic>Ginkgo biloba</italic> extract are such natural compounds that have been shown to be protective against the progression of AD pathology in AD murine models.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] Mediterranean and Middle East countries are the main regions where pomegranate (&#x077f3;&#x069b4; Sh&#x000ed; Li&#x000fa;) is cultivated and produced.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>] Pomegranates (<italic>Punica granatum</italic> Linn.) contain very high levels of polyphenols, as compared to other fruits and vegetables. They have been extensively used in Unani, Ayurvedic, and Chinese systems of medicine.[<xref rid="ref15" ref-type="bibr">15</xref>] Different parts of the fruit have been successfully evaluated for various diseases, including peptic ulcer, hepatic damage, and snakebite. The ripe fruit is a tonic, astringent to the bowels, aphrodisiac, and alleged panacea for a myriad of conditions, including biliousness, fever, heart diseases, sore throat, and stomatitis. The rind of the fruit is antihelminthic and useful in diarrhea, dysentery, and ulcer (Ayurveda).[<xref rid="ref16" ref-type="bibr">16</xref>] Recently, we have reported that the four different pomegranate varieties grown in Oman offer protection to Parkinson's disease like neurotoxicity in human primary neurons.[<xref rid="ref17" ref-type="bibr">17</xref>] Dietary supplementation of pregnant mice with pomegranate juice was shown to protect against neurodegeneration in neonatal mice subjected to hypoxic&#x02013;ischemic brain injury.[<xref rid="ref18" ref-type="bibr">18</xref>] A recent study suggested that 3 months of supplementation with pomegranate may attenuate AD progression by offering the brain anti-inflammatory effects in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice.[<xref rid="ref19" ref-type="bibr">19</xref>] But till now, there are no studies conducted to find out the effect of long-term dietary supplementation of pomegranate on oxidative stress status in APPSw/Tg2576 transgenic mouse model. To fill the information gap, we designed this study to find out whether long-term dietary supplementation (15 months) with pomegranate would influence AD-like oxidative stress in an APPsw/Tg2576 mouse model of AD.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Collection and preparation</title><p>Fresh pomegranate (&#x077f3;&#x069b4; Sh&#x000ed; Li&#x000fa;) &#x0201c;Helow&#x0201d; (literally, sweet) variety of Oman fruits were purchased from a local farm in Al-Jabal Al-Akhdar, Oman. The fruits were transported to our laboratory in an electric cooler box maintained at 9&#x000b0;C. Then the edible parts were separated and freeze dried at &#x02212;40&#x000b0;C for 5 days. The samples were then grinded into fine powder by using a KMF grinder (KIKA Werke, Wilmington, Delaware USA) at 6000 rpm. Powders were kept in air-tight plastic containers and stored at &#x02212;40&#x000b0;C until they were sent for the diet preparation. Before sending, we analyzed the samples for the qualitative presence of polyphenols. Phytochemicals such as anthocycanin, hydroxycinnamic acid derivatives (e.g. caffeic acid, etc.), hydrolyzable tannins (e.g. ellagic acid, quercetin-3-<italic>O</italic>-glucoside, punicalin, etc.), hydroxybenzoic acids (gallic acid, protocatechuic acid, etc.), hydroxycyclohexane carboxylic acids (quinic acid), and hydroxyphenyls (kaempferol, catechin, etc.) have been reported to be present in pomegranate. This was confirmed by qualitative analysis by high performance liquid chromatography (HPLC; data not included).</p></sec><sec id="sec2-2"><title>Diet preparation for the animals</title><p>The ground pomegranate samples were sent to USA to prepare the diet for the mice. Based on the our primary dose-dependent study (2, 4, 6, 8 and 10% on the amyloid beta 1-42 induced AD like status in mice) results, we have chosen the 4% (data not shown). The diet was prepared by mixing the pomegranate (4%) with regular diet as per National Institute Health, USA protocol by Research Diet, Inc, NJ, New Brunswick USA. The constituents of the diet are given in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Consituents of rodent diet with 60 kcal% fat and 4% pomegranate (prepared by Research Diet, Inc, USA)</p></caption><graphic xlink:href="JTCM-4-232-g001"/></table-wrap></sec><sec id="sec2-3"><title>Animals and treatment</title><p>Twelve transgenic females (APPsw/Tg2576) and six wild control (non-transgenic) mice (Taconic Farm, Hudson St, Manhattan NY, USA) were used. Animals were quarantined for 7 days after shipping and individually housed in plastic cages in an animal room which was maintained at a temperature of 22 &#x000b1; 2&#x000b0;C, a relative humidity of 50 &#x000b1; 10%, and a 12-h light/dark automatic light cycle (0800-2000 h). Tap water was offered <italic>ad libitum</italic> throughout the study. The study was approved by the Animal Care and Use Committee of the Sultan Qaboos University, Oman (SQU/AEC/2010-11/3).</p><p>All these animals were free from pathogens and viruses. Experimental period commenced from the age of 4 months. The animals were divided into three groups as follows: Group 1: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Group 2: AD transgenic mice also fed with regular diet; and Group 3: AD mice fed with 4% pomegranate fruit diet. Dose of pomegranate was designed based on the preliminary study done with different percentages of fruits and 4% showed better effects. These experimental and control mice were examined at the age of 15 months, and oxidative stress, antioxidants and membrane bound enzymes were investigated. All animal experiments in the present study were conducted in compliance with the Animal Care and Use Committee of the Sultan Qaboos University, Oman.</p></sec><sec id="sec2-4"><title>Tissue sample collection</title><p>Following the behavioral assessments, the animals were decapitated with the head transferred onto dry ice, followed by rapid dissection of the hippocampus and the cerebral cortex, and homogenized in 9 volumes (1:9 w/v) of cold saline for preparation of a 10% cerebral homogenate in an ice bath, and centrifuged for supernatant collection. Whole brains were rapidly removed simultaneously and chilled in an ice-cold saline solution. The tissue samples were stored at &#x02212;80&#x000b0;C until assay.</p></sec><sec id="sec2-5"><title>Assay of oxidative parameters in the brain</title><p>The hippocampus and the cerebral cortex were obtained for measurements of oxidative parameters.</p></sec><sec id="sec2-6"><title>Superoxide dismutase activity</title><p>Measurements of the superoxide dismutase (SOD) activity were performed as described by Sun <italic>et al</italic>.[<xref rid="ref20" ref-type="bibr">20</xref>] Estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, both of which react with nitroblue tetrazolium (NTB) to form formazan dye, followed by measurement of SOD activity at 550 nm by the degree of inhibition of this reaction. One unit of enzyme was defined as the amount of enzyme required at the inhibition rate of 50%. The activity of SOD was expressed as units/mg protein.</p></sec><sec id="sec2-7"><title>Determination of catalase</title><p>Catalase (CAT) activity was measured according to the method of Aebi.[<xref rid="ref21" ref-type="bibr">21</xref>] Briefly, 0.1 ml of supernatant was added to a cuvette containing 1.9 ml of 50 mmol/L phosphate buffer (pH 7.0). The reaction was started with the addition of 1 ml of freshly prepared 30 mmol/L H₂O₂. The variations of decomposition rate of H₂O₂ were determined by spectrophotometry at 240 nm. The activity of catalase was expressed as units/mg protein.</p></sec><sec id="sec2-8"><title>Determination of glutathione</title><p>Glutathione (GSH) concentration was determined by the procedure of Elmann.[<xref rid="ref22" ref-type="bibr">22</xref>] Briefly, 0.5 ml homogenate was mixed with 1.5 ml 0.15 M KCl and 3 ml deproteinized solution. Each sample was centrifuged at 3000 rpm for 10 min and the resultant supernatant was obtained, followed by the addition of 2 ml phosphate solution and 0.5 ml 5,5&#x02019;-Dithiobis-2-Nitrobenzoic Acid (DTNB) into 0.5 ml supernatant, with the absorbance read at 412 nm and compared with glutathione standards.</p></sec><sec id="sec2-9"><title>Determination of glutathione peroxidase</title><p>Hippocampal and cerebrocortical glutathione peroxidase (GPx) activity was determined based on the protocol developed by Wendel[<xref rid="ref23" ref-type="bibr">23</xref>] by indirectly measuring the consumption of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. The GPx uses GSH to reduce the <italic>tert</italic>-butyl hydroperoxide, producing glutathione disulfide (GSSG), which is readily reduced to GSH by glutathione reductase (GR) using NADPH as a reducing equivalent donor. SOD activity was assayed spectrophotometrically as described previously.[<xref rid="ref24" ref-type="bibr">24</xref>] This method is based on the capacity of SOD to inhibit autoxidation of adrenaline to adrenochrome. The color reaction was measured at 480 nm. One unit of enzyme was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%. The enzymatic activity was expressed as units (U) per milligram protein.</p></sec><sec id="sec2-10"><title>Determination of GR</title><p>Hippocampal and cerebrocortical GR activity was determined based on the protocol developed by Carlberg and Mannervik.[<xref rid="ref25" ref-type="bibr">25</xref>] Briefly, GR reduces glutathione disulfide (GSSG) to GSH at the expense of NADPH, whose disappearance was followed at 340 nm.</p></sec><sec id="sec2-11"><title>Determination of malondialdehyde</title><p>Malondialdehye (MDA) was measured by the method previously described.[<xref rid="ref26" ref-type="bibr">26</xref>] The reagents acetic acid 1.5 ml (20%) pH 3.5, 1.5 ml thiobarbituric acid (0.8%), and 0.2 ml sodium lauryl sulfate (8.1%) were added to 0.1 ml supernatant samples and heated at 95&#x000b0;C for 60 min. The mixture was cooled with tap water, followed by centrifugation at 4000 rpm for 10 min, separation of the organic layer, and absorbance measurement at 532 nm by microplate spectrophotometry. Protein estimation was conducted according to Lowry's method and the data were expressed as nmol/mg protein.</p></sec><sec id="sec2-12"><title>Measurement of the protein carbonyl content</title><p>The total protein carbonyl content was determined by a previously described method for brain tissue.[<xref rid="ref27" ref-type="bibr">27</xref>] Briefly, 1 ml aliquots of the homogenates containing 759-800 mg protein/ml in each sample were mixed with 0.2 ml of 2,4-dinitrophenylhydrazine (DNPH, 10 mM) or 0.2 ml HCl (2 M). After incubation at room temperature for 1 h in a dark room, 0.6 ml of denaturing buffer [150 mM sodium phosphate buffer, pH 6.8, containing 3% sodium dodecyl sulfate (SDS)], 1.8 ml of heptane (99.5%), and 1.8 ml of ethanol (99.8%) were added sequentially and mixed by vortex agitation for 40 s; the mixture was then centrifuged for 15 min. Next, the protein that was isolated from the interface was washed two times with 1 ml of ethyl acetate/ethanol 1:1 (v/v) and suspended in 1 ml of denaturing buffer. Each DNPH sample was read at 370 nm in a Hitachi U-2001 spectrophotometer against the corresponding HCl sample (blank); the level of total carbonylation was calculated using a molar extinction coefficient of 22,000 M¹ cm¹.</p></sec><sec id="sec2-13"><title>Assay of acetylcholinesterase activity</title><p>The acetylcholinesterase (AChE) activity in the hippocampus and the cerebral cortex was determined according to the method of Ellman.[<xref rid="ref28" ref-type="bibr">28</xref>] 0.4-ml aliquots of homogenates were added to a cuvette containing 2.6 ml of phosphate buffer (pH 8.0, 0.1 M). Then, 100 &#x003bc;1 of DTNB was added to the photocell. The absorbance was measured at 412 nm. When the reading had stopped increasing, the photometer slit was opened to set the absorbance to zero. Then, 20 &#x003bc;l of the substrate was added. Changes in absorbance were recorded and calculated as changes in absorbance per minute.</p></sec><sec id="sec2-14"><title>Spectrophotometric analysis of Na⁺ K⁺-ATPase activity</title><p>The reaction mixture for the Na⁺ K⁺-ATPase assay contained 5 mM MgCl₂, 80 mM NaCl, 20 mM KCl, 40 mM Tris&#x02013;HCl buffer, pH 7.4, and purified synaptic membranes (approximately 3 &#x003bc;g of protein) in a final volume of 200 &#x003bc;l. The enzymatic assay was performed at 37&#x000b0;C for 5 min and started by the addition of ATP (disodium salt, vanadium free) to a final concentration of 3 mM. The reaction was stopped by the addition of 200 &#x003bc;l of 10% trichloroacetic acid. Ouabain-insensitive Mg2⁺-ATPase was assayed under the same conditions with the addition of 1 mM ouabain. Na⁺ K⁺-ATPase activity was calculated by determining the difference between the two assays and the inorganic phosphate (Pi) released was measured. Enzyme-specific activities were calculated as nmol of Pi released/min/mg protein and expressed as percentage of controls.[<xref rid="ref29" ref-type="bibr">29</xref>]</p></sec><sec id="sec2-15"><title>Statistical analysis</title><p>Statistical analysis was performed using the software statistical package SPSS 12 (SPSS, Chicago, IL, USA). A univariate analysis of variance was performed using genotype (wild-type and transgenic), treatment (transgenic + pomegranate), and their interactions as between-individuals fixed factors. According to this, differences between treatments and genotype, or differences between transgenic and treatment, were analyzed. For pairwise comparison, Scheffe test (as a <italic>post-hoc</italic> test) was applied to determine the level of significance between groups. Results are given as mean values &#x000b1; standard deviation (SD). For all tests, the level of statistical significance was set at <italic>P</italic> &#x0003c; 0.05.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-16"><title>The effect of 4% pomegranate (&#x077f3;&#x069b4; Sh&#x000ed; Li&#x000fa;) on LPO and protein carbonyls in APPsw (Tg2576) mice</title><p>Disease control APPsw (Tg2576) mice showed significant increase in LPO levels in both brain regions studied (cortex and hippocampus), as compared to wild control mice [<xref ref-type="table" rid="T2">Table 2</xref>]. However, treatment with 4% pomegranate dietary supplementation in APPsw (Tg2576) mice for15 months attenuated LPO levels to values comparable to wild control mice. <xref ref-type="table" rid="T2">Table 2</xref> depicts significantly elevated levels of protein carbonyls in control APPsw (Tg2576) mice as compared to wild control mice (cortex and hippocampus). Dietary supplementation with 4% pomegranate significantly attenuated the protein carbonyl levels in APPsw (Tg2576) mice.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Effect of pomegranate on lipid peroxidation and protein carbonyl content in the cortex and hippocampus</p></caption><graphic xlink:href="JTCM-4-232-g002"/></table-wrap></sec><sec id="sec2-17"><title>Effect of 4% pomegranate on changes in the antioxidant enzymes in APPsw (Tg2576) mice</title><p>It is evident from the data that there was a significant decrease in the activity of SOD in cerebral cortex and hippocampus in AD mice when compared to wild control mice [<xref ref-type="table" rid="T3">Table 3</xref>]. However, the SOD activity was significantly elevated by 4% pomegranate dietary supplementation in the cerebral cortex and hippocampus of APPSw (Tg2576) AD mice. Disease control transgenic mice showed significant reduction in CAT activity in the cortex and hippocampus. Dietary supplementation with 4% pomegranate subsequently enhanced the CAT activity both in the cortex and hippocampus [<xref ref-type="table" rid="T3">Table 3</xref>] in AD mice.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Effect of pomegranate on superoxide dismutase and catalase activities in the brain of mice</p></caption><graphic xlink:href="JTCM-4-232-g003"/></table-wrap><p>GSH activity of disease control APPsw (Tg2576) mice was significantly lower in the cortex and hippocampus as compared to wild control mice [<xref ref-type="table" rid="T4">Table 4</xref>]. However, treatment with 4% pomegranate dietary supplementation restored the GSH activity to near-normal levels in the cortex and hippocampus. The data for GPx activity are depicted in <xref ref-type="table" rid="T4">Table 4</xref>. A significant decrease in GPx activity in the cortex and hippocampus was observed in disease control AD mice. Dietary supplementation with 4% pomegranate was found to protect the GPx activity; the activity was increased in the cortex and hippocampus, respectively. GR activity was significantly reduced in the brain regions, cerebral cortex and hippocampus, in AD mice. The activity was restored with long-term supplementation of 4% pomegranate [<xref ref-type="table" rid="T4">Table 4</xref>].</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Protective effect of pomegranate dietary supplementation on glutathione-dependent antioxidant enzymes</p></caption><graphic xlink:href="JTCM-4-232-g004"/></table-wrap></sec><sec id="sec2-18"><title>The effect of 4% pomegranate on alterations in membrane-bound enzymes in AD transgenic mice</title><p>AChE activity significantly increased in the cortex and hippocampus of AD mice and 4% pomegranate dietary supplementation for 15 months attenuated AChE activity in the cerebral cortex and hippocampus. Disease control APPsw (Tg2576) mice showed significantly inhibited activity of Na⁺ K⁺-ATPase in the cortex and hippocampus. In 4% pomegranate dietary supplemented mice, there was a significant improvement in the activities of membrane-bound enzymes [<xref ref-type="table" rid="T5">Table 5</xref>].</p><table-wrap id="T5" position="float"><label>Table 5</label><caption><p>Influence of pomegranate on acetylcholinesterase and Na⁺ K⁺-ATPase activities in brain</p></caption><graphic xlink:href="JTCM-4-232-g005"/></table-wrap></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Studies demonstrate that the beneficial action of pomegranate (&#x077f3;&#x069b4; Sh&#x000ed; Li&#x000fa;) is mediated through attenuation of oxidative stress in APPSw (Tg2576) mice. The present study suggests that the anti-AD&#x02013;like effects of pomegranate grown in Oman might be related to its high antioxidant activities.[<xref rid="ref30" ref-type="bibr">30</xref><xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>] ROS can damage essential cellular constituents such as lipids and proteins, which can be measured by identification of their byproducts malondialdehyde and protein carbonyl, respectively.[<xref rid="ref33" ref-type="bibr">33</xref>] We observed increase in MDA production and protein carbonylation in the cerebral cortex and hippocampus of AD mice, indicating that oxidative stress occurs as a consequence of AD, thereby contributing to brain damage. Dietary supplementation of pomegranate notably inhibited the accumulation of MDA and protein carbonyl levels in the cortex and hippocampus of Tg2576 mice, which is an oxidized byproduct of LPO. In this context, pomegranate has been demonstrated to have direct antioxidant effects,[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>] and decreased MDA and protein carbonyl content were demonstrated after daily administration of pomegranate juice.[<xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref>]</p><p>GSH is the primary defense in neurons against oxidative stress and maintains cellular redox homeostasis.[<xref rid="ref37" ref-type="bibr">37</xref>] In our experiment, we observed a significant decrease in the GSH levels in the brain of Tg2576 disease control mice as compared to wild controls. It is known that GSH depletion is the first indicator of oxidative stress during neurodegenerative diseases.[<xref rid="ref38" ref-type="bibr">38</xref>] Pomegranate supplementation to AD mice was able to reverse the decreased GSH levels. Ajaikumar <italic>et al</italic>.[<xref rid="ref39" ref-type="bibr">39</xref>] reported the reversal in GSH levels in aspirin- and ethanol-induced gastric ulceration following pomegranate administration, suggesting the efficacy of pomegranate in preventing the oxidative damage and associated changes.</p><p>SOD is responsible for catalyzing the conversion of superoxide anions into hydrogen peroxide[<xref rid="ref40" ref-type="bibr">40</xref>] which is further decomposed into water and oxygen by CAT.[<xref rid="ref41" ref-type="bibr">41</xref>] The activities of SOD and CAT were found to be significantly diminished in the cortex and hippocampus of Tg2576 disease control mice. Dietary supplementation of pomegranate to Tg2576 AD mice prevented the reduction in the activities of SOD and CAT. Studies have shown that pomegranate could directly inhibit the superoxide anion formation which could activation of SOD and CAT. This result suggests that the neuroprotective effects of pomegranate might be due to its antioxidant activity.[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref><xref rid="ref39" ref-type="bibr">39</xref><xref rid="ref42" ref-type="bibr">42</xref>]</p><p>GPx and GR represent a crucial defensive system to protect cells against ROS.[<xref rid="ref43" ref-type="bibr">43</xref>] The activities of GPx and GR were significantly decreased in the brain regions of AD mice, and a significant decrease in the activities of these enzymes during experimental dementia was reported.[<xref rid="ref44" ref-type="bibr">44</xref>] On the other hand, Fan <italic>et al</italic>.[<xref rid="ref45" ref-type="bibr">45</xref>] have reported decreased GPx and GR activities as a result of oxidative stress in scopolamine-induced amnesia in the hippocampus and cerebral cortex. Pomegranate supplementation significantly attenuated the elevated levels of GPx and GR in the brain regions. Pomegranate has been shown to be successful in increasing the activities of GPx and GR enzymes in animals with aspirin- and ethanol-induced gastric ulceration[<xref rid="ref39" ref-type="bibr">39</xref>] and improving the antioxidant function in elderly subjects.[<xref rid="ref36" ref-type="bibr">36</xref>] The mechanism involved in restoration of the activities of these enzymes by pomegranate might include prevention of critical &#x02013;SH group of these enzymes that is involved in catalysis.</p><p>AChE is an acetylcholine hydrolyzing enzyme that is responsible for the termination of cholinergic response.[<xref rid="ref46" ref-type="bibr">46</xref>] The AChE activity was found to be markedly elevated in the brain regions of Tg2576 AD mice. This observation collaborates with previous reports whereby Intracerebrobentricular (i.c.v.) administration of streptozotocin at sub-diabetogenic dose has been shown to induce memory deficits, along with an increase in oxidative stress and AChE activity.[<xref rid="ref47" ref-type="bibr">47</xref>] Koladiya <italic>et al</italic>.[<xref rid="ref48" ref-type="bibr">48</xref>] have reported that AChE activity was significantly increased in the hippocampus in L-methionine&#x02013;induced model of vascular dementia. The activity of AChE depends largely on the membrane characteristics, since the enzyme is membrane bound. Amyloid beta peptides can induce Ca²⁺ influx that leads to increased activity of AChE which is attributed to Ca²⁺-mediated oxidative stress.[<xref rid="ref49" ref-type="bibr">49</xref>] In our study, long-term pomegranate supplementation thorugh diet was found to inhibit AChE activity. Previous studies supporting the leaf extracts of pomegranate were obtained for the AChE inhibitory activity and antioxidant effects.[<xref rid="ref50" ref-type="bibr">50</xref>] The mechanism of anticholinesterase activity of pomegranate appears to be complicated and needs further investigation.</p><p>Modification in Na⁺ K⁺-ATPase activity may induce neuronal death with features of both apoptosis and necrosis.[<xref rid="ref51" ref-type="bibr">51</xref>] In the current study, the activity of Na⁺ K⁺-ATPase was found to be decreased in Tg2576 AD mice, which is in line with the other studies reporting reduced enzyme activity during aging.[<xref rid="ref52" ref-type="bibr">52</xref><xref rid="ref53" ref-type="bibr">53</xref>] Na⁺ K⁺-ATPase is known to be highly susceptible to changes in the membrane lipids, which may be further attributed to the progressive increase in the LPO.[<xref rid="ref54" ref-type="bibr">54</xref><xref rid="ref55" ref-type="bibr">55</xref>] ROS overproduction inhibits the activity of ATPase via thiol- and lipid-dependent mechanisms.[<xref rid="ref56" ref-type="bibr">56</xref>] It has been demonstrated that the reduced activity of Na⁺ K⁺-ATPase caused by oxidative stress cannot drive the ion pumps to maintain depolarization of neurons and, thus, may become lethal to neurons.[<xref rid="ref57" ref-type="bibr">57</xref>] The mechanism of action of pomegranate in improving Na⁺ K⁺-ATPase activity is uncertain, as its multiple active compounds such as anthocyanins, ascorbic acid, ellagic acid, gallic acid, fumaric acid, caffeic acid, catechin, Epigallocatechingallate (EGCG), quercetin, rutin, tannins, alkaloids, and flavanoids have multiple functions, making it pharmacologically complex. However, the antioxidant properties of pomegranate have been well documented, which include free radical scavenging and inhibition of LPO as well as enhancement of antioxidant status[<xref rid="ref30" ref-type="bibr">30</xref><xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>] and neuroprotection.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref58" ref-type="bibr">58</xref>] The results of our present study are supported by the previous studies on short-term oral juice supplementation in AD mice.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref58" ref-type="bibr">58</xref>]</p><p>We recently reported that chronic dietary term supplementation of fruits could be beneficial for behavior and oxidative stress related changes in AD transgenic mice[<xref rid="ref59" ref-type="bibr">59</xref><xref rid="ref60" ref-type="bibr">60</xref><xref rid="ref61" ref-type="bibr">61</xref><xref rid="ref62" ref-type="bibr">62</xref>] which also support our present findings.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Pomegranates grown in Oman provide possible protection against the oxidative stress and have antioxidant effects in Tg2576 AD mice brain, and the mechanism of protection may be related to their antioxidant activity and phenolic constituents. These results warrant further exploration of how the anti-ROS property of pomegranate affords such beneficial effects on the AD mice brain. This study also supports an important concept that the onset of neurodegenerative disease may be delayed or mitigated by the use of dietary preventive agents that protect against AD by reducing the oxidative stress.</p></sec>

Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of <italic>Astragalus membranaceus</italic> using Bioassay-guided Fractionation

<p>The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, ³H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 &#x000b1; 0.27) at 0.125 mg/ml (<italic>P</italic> &#x0003c; 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), ³H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells <italic>in vitro</italic>.</p>

<contrib contrib-type="author"><name><surname>Lai</surname><given-names>Patrick Kwok-Kin</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Judy Yuet-Wa</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kwok</surname><given-names>Hin-Fai</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Ling</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Hua</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lau</surname><given-names>Ching-Po</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Leung</surname><given-names>Ping-Chung</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Fung</surname><given-names>Kwok-Pui</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Lau</surname><given-names>Clara Bik-San</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1"/></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Angiogenesis, defined as the formation of new blood vessels from pre-existing vessels, plays crucial roles in many physiological processes.[<xref rid="ref1" ref-type="bibr">1</xref>] During the wound healing process, angiogenesis is involved in proliferation, migration, and differentiation of endothelial cells with a new basement membrane (Hoeben <italic>et al</italic>., 2004). These promote wound healing process through the delivery of oxygen and nutrients to the wound site. However, in impaired wound, due to poor circulation and reduced oxygenation, angiogenesis will also be affected.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] The resulting chronic wound formed may eventually lead to ulceration. One of the examples is diabetic foot ulcer.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] If the ulcer is not properly treated, the patient may need amputation which may further lead to morbidity and mortality.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Astragali Radix (AR), or the root of <italic>Astragalus</italic> (&#x09ec3;&#x08006; Hu&#x000e1;ng Q&#x000ed;) <italic>membranaceus</italic> (Fisch.) Bunge (Fabaceae), is a common Chinese herb that has been traditionally used in treating various diseases such as anemia, fever, wounds, chronic fatigue, uterine bleeding, diabetes, and immune-related diseases.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] It is also one of the herbs commonly found in Chinese herbal formulae used for ulcer healing.[<xref rid="ref10" ref-type="bibr">10</xref>] In our previous clinical study, two herbal formulae comprising AR as one of the component herbs were shown to rescue 85% of the legs condemned to amputation due to non-healing chronic diabetic ulcer.[<xref rid="ref11" ref-type="bibr">11</xref>] In addition, AR was shown to be the major component in our simplified herbal formula (NF3) which was demonstrated to enhance diabetic wound healing in rats through tissue regeneration, pro-angiogenesis, and anti-inflammation and also exhibited pro-angiogenic effect in zebrafish embryo <italic>in vivo</italic> and rat aortic ring <italic>in vitro</italic>.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>] As a single herb, the research focused on its constituents including polysaccharides, triterpene saponins, isoflavonoids, and trace elements.[<xref rid="ref14" ref-type="bibr">14</xref>] In our recent study, two major anti-inflammatory AR active fractions, which may enhance wound healing, were identified using bioassay-guided fractionation, and formononetin (an isoflavone) was one of the active ingredients in the active fractions(Lai <italic>et al</italic>., 2013a). Their anti-inflammatory properties were further confirmed by reducing the release of inflammatory mediators and inactivation of nuclear factor kappa B (NF&#x003ba;B) through mitogen-activated protein kinase (MAPK) signaling pathway(Lai <italic>et al</italic>., 2013b). In another study, AR extract that is enriched in saponin and isoflavone constituents has been demonstrated to exhibit pro-angiogenic effect <italic>in vitro</italic>.[<xref rid="ref15" ref-type="bibr">15</xref>] Calycosin, one of the flavonoids found in AR, induces angiogenesis in human umbilical vein endothelial cell (HUVEC) and zebrafish embryos.[<xref rid="ref16" ref-type="bibr">16</xref>] However, other AR angiogenic active components might be present, but are not yet identified.</p><p>Hence, in this study, we aim to systematically identify active fraction(s) from AR which is/are responsible for the pro-angiogenic effect by bioassay-guided isolation method in <italic>in vivo</italic> zebrafish model. The isolated fraction(s) will be chemically characterized and its angiogenic effects will also be investigated in zebrafish model as well as in human microvascular endothelial cell (HMEC-1) model.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Materials</title><p>The raw herb of AR was purchased from mainland China and its voucher specimen was deposited in the museum of the Institute of Chinese Medicine, The Chinese University of Hong Kong, with voucher specimen number 2008-3201. The procedures of authentication and preparation of aqueous crude extract were described in our previous publication(Lai <italic>et al</italic>., 2013a). Extraction yield of the crude extract was about 30% (w/w). All chemicals and solvents were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified. The transgenic zebrafish line TG (flil: EGFP) with endothelial cells expressing enhanced Green Fluorescent Protein (eGFP) was purchased from Zebrafish International Resource Center (University of Oregon, USA) and was maintained as described in the previous report.[<xref rid="ref17" ref-type="bibr">17</xref>] The handling of the zebrafish was under the animal licence issued and endorsed by Department of Health, the Government of the Hong Kong Special Administrative Region and the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong, respectively [reference no.: (09-530) in DH/HA and P/8/2/1 Pt. 10]. HMEC-1 was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in MCDB 131 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, CA, USA), 10 ng/ml epidermal growth factor, 1 &#x003bc;g/ml hydrocortisone, penicillin (100 IU/ml), and streptomycin (100 &#x003bc;g/ml). They were grown in 37&#x000b0;C humidified incubator supplied with 5% CO₂.</p></sec><sec id="sec2-2"><title><italic>in vivo</italic> zebrafish model</title><p>Collection of zebrafish embryos and herbal treatment were performed as described in our previous report.[<xref rid="ref18" ref-type="bibr">18</xref>] Briefly, embryos at 1-4 cell stage were placed into six-well plates with 20-30 embryos per well depending on the assay. AR crude extract and its isolated fractions were dissolved into embryo medium [19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO₄&#x02027;7H₂O, 0.2 mM Ca (NO₃)₂, 1.67 mM HEPES, pH 7.2] and then filtered. The medium of the wells was replaced with the filtered AR extract and fractions in various concentrations. After incubation at 28&#x000b0;C for 96 h, sprout formation in sub-intestinal vessel (SIV) region of the treated embryos was examined under fluorescence microscope.[<xref rid="ref16" ref-type="bibr">16</xref>] The number of sprouts formed in each embryo was counted.</p></sec><sec id="sec2-3"><title>Bioassay-guided isolation of active fractions from AR</title><p>AR aqueous crude extract was used for the isolation of active fractions by bioassay-guided fractionation method. For each fraction, zebrafish model was applied to evaluate the angiogenic activities. The active fractions were selected for further sub-fractionation until the most potent fraction(s) or component(s) were identified and isolated. Finally, an active fraction P1-1-1 was identified. A simplified diagram [<xref ref-type="fig" rid="F1">Figure 1</xref>] shows the isolation of P1-1-1. Firstly, AR aqueous crude extract was re-dissolved in water, and solvent precipitation method using 95% ethanol was employed. The resulting precipitate (P1) and supernatant (P2) were concentrated under reduced pressure and lyophilized to dryness. P1 was re-dissolved in water and further fractionated into two sub-fractions, P1-1 and P1-2, by solvent partition method using chloroform: <italic>n</italic>-butanol (4:1). Among these sub-fractions, P1-1, which showed the most promising activity [<xref ref-type="table" rid="T1">Table 1</xref>], was selected for further fractionation into five sub-fractions (P1-1-1, P1-1-2, P1-1-3, P1-1-4, and P1-1-5) by Sephadex G-100 column. According to the result shown in <xref ref-type="table" rid="T1">Table 1</xref>, P1-1-1 showed the highest angiogenic activity among these five fractions. Thus, in the following experiments, P1-1-1 was used to characterize its biological activities.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Schematic diagram showing the method for bioassay-guided fractionation of AR aqueous crude extract and representative images of zebrafish study. (a) Active fractions at each level (shown in gray boxes) were selected for further fractionation. P1-1-1 was the fraction finally selected for mechanistic study. (b) Zebrafish embryos treated with (i) embryo medium only and zebrafish embryos treated with P1-1-1 at (ii) 0.03125 mg/ml, (iii) 0.0625 mg/ml, and (iv) 0.125 mg/ml. Red arrows indicate the smooth basket-like structure of sub-intestinal vessel (SIV) appearing at the bottom of each embryo. Yellow arrows indicate the new blood vessels (sprouts) formed on the SIV of embryos treated with P1-1-1 which were observed in (iii) and (iv)</p></caption><graphic xlink:href="JTCM-4-239-g001"/></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Summary of the angiogenic effects of AR crude extract and isolated fractions on <italic>in vivo</italic> zebrafish model</p></caption><graphic xlink:href="JTCM-4-239-g002"/></table-wrap></sec><sec id="sec2-4"><title>Chemical characterization of active fractions</title><p>In order to characterize the chemical property of P1-1-1, this fraction was further processed to divide into two sub-fractions, P1-1-1-A and P1-1-1-B, as shown in <xref ref-type="fig" rid="F1">Figure 1</xref>. Molecular weight distribution of the three isolated active fractions (P1-1-1, P1-1-1-A, and P1-1-1-B) was determined by gel filtration chromatography (GFC) using Waters (Milford, MA, USA) ACQUITY ultra performance liquid chromatograph (UPLC) equipped with a calibrated 7.8 mm &#x000d7; 30 cm, 7 &#x003bc;m TSKgel G3000 column (Tosoh Bioscience, Tokyo, Japan) and evaporative light scattering detector (Alltech, Columbia, USA). The column was equilibrated with water at a flow rate of 0.5 ml/min. The molecular mass range of dextran standards (Fluka, Buches, Switzerland) used for calibration was 5-2000 kDa. The retention times plotted as a function of the logarithm of molecular mass and the respective linear regression obtained were used for molecular mass determination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with the fractions (10 mg/ml) using 10% (w/v) acrylamide in gels. The reference markers used were the high-range rainbow markers (GE Healthcare, Buckinghamshire, UK). Glycoprotein was detected by the periodic acid-Schiff (PAS) staining of the gel.[<xref rid="ref14" ref-type="bibr">14</xref>] The carbohydrate and protein content in the fractions were determined by anthrone reagent[<xref rid="ref19" ref-type="bibr">19</xref>] and bicinchoninic acid (BCA) protein assay, respectively.</p></sec><sec id="sec2-5"><title>Quantitation of mRNA expression level of angiogenic markers by real-time polymerase chain reaction</title><p>Total RNA was isolated from 20 zebrafish embryos treated with or without various concentrations of active fraction (P1-1-1) for 72 h using NucleoSpin RNA kit (Macherey-Nagel, Duren, Germany). Reverse transcription and polymerase chain reaction (PCR) amplification were carried out using iScript One-Step real-time polymerase chain reaction (RT-PCR) kit supplied by Bio-Rad (CA, USA). The reaction mixture contained 15 &#x003bc;l of RT-PCR reagent mix, 3 &#x003bc;l of 10 mM primer mix of target gene (VEGFA, Flt-1, or KDR) or house-keeping gene (&#x003b2;-actin), 0.2 &#x003bc;l of reverse transcriptase, 5 &#x003bc;l of RNA template, and 11.8 &#x003bc;l of nuclease-free water. Their primer sequences are as follows: VEGFA, 5&#x02032;-TCCAGGAGTATCCCGATGAG-3&#x02032; and 5&#x02032;-GCTTTGACTTCTGCCTTTGG-3&#x02032;; Flt-1, 5&#x02032;-ATGGGAACAGCAGCACTCTT-3&#x02032; and 5&#x02032;-TTGAAGACGGAGGGACAATC-3&#x02032;; KDR, 5&#x02032;-TGTGGTCAGCTATGCTGGAG-3&#x02032; and 5&#x02032;-AGCCTCTCATGCTGTGGACT-3&#x02032;; &#x003b2;-actin, 5&#x02032;-CTCTTCCAGCCTTCCTTCCT-3&#x02032; and 5&#x02032;-CTTCTGCATACGGTCAGCAA-3&#x02019;. RT-PCR was then performed by CFX96 Real-Time System (Bio-Rad, CA, USA). Analysis of expression of VEGFA, Flt-1, and KDR was preformed using &#x003b2;-actin as the control gene for normalization.</p></sec><sec id="sec2-6"><title>Measurement of cell viability by MTT assay</title><p>Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HMEC-1 cells (5 &#x000d7; 10³ cells/well) were seeded in 96-well culture plates and incubated overnight for attachment. Then, the cells were arrested with 0.5% (v/v) FBS in MCDB 131 medium for another 24 h. After further treating them with various concentrations of P1-1-1 for 24 or 48 h, the culture medium was removed and 40 &#x003bc;l of MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well. After incubation for 3 h at 37&#x000b0;C, MTT solution was removed and 100 &#x003bc;l of dimethyl sulfoxide was added to dissolve the crystals formed. Then, absorbance at 540 nm was measured using a microplate reader. The percentage cell viability was calculated as [Absorbance (_treatment)/Absorbance (_{negative control})] &#x000d7;100%.</p></sec><sec id="sec2-7"><title>Measurement of DNA synthesis by ³H-thymidine uptake assay</title><p>HMEC-1 cells (5 &#x000d7; 10³ cells/well) were seeded in 96-well culture plates and incubated overnight for attachment. Then, the cells were arrested with 0.5% (v/v) FBS in MCDB 131 medium for another 24 h. After further treating them with various concentrations of P1-1-1 for 24 or 48 h, 0.5 &#x003bc;Ci of ³H-thymidine in PBS (Invitrogen, USA) was added to each well and the cells were incubated at 37&#x000b0;C for 6 h. Then, DNA was harvested on microfilters with a cell harvester (Beckman Coulter, Brea, CA, USA). The amount of DNA synthesized was determined by measuring the radioactivity of the filter using a microplate scintillation counter (Beckman Coulter).</p></sec><sec id="sec2-8"><title>Cell cycle analysis</title><p>HMEC-1 cells (3 &#x000d7; 10⁵ cells/well) were seeded into six-well culture plates and incubated overnight for attachment. After synchronization with 0.5% FBS in MCDB 131 medium for 24 h, the cells were treated with various concentrations of P1-1-1 or vehicle for a further 24 or 48 h. Then, the cells were harvested and fixed in 70% ethanol. Before performing flow cytometry, ethanol was removed and the cells were incubated with RNase (8 &#x003bc;g/ml) and propidium iodide (10 &#x003bc;g/ml) for 30 min. Cell cycle distribution was then detected using a flow cytometer (BD FACSCanto, BD BioSciences, CA, USA), and the results were analyzed using ModfitLT version 3.0 software.</p></sec><sec id="sec2-9"><title>Statistical analysis</title><p>All experiments were performed in not less than three replicates and the results were presented as mean &#x000b1; standard deviation (SD) for <italic>in vitro</italic> studies or mean &#x000b1; standard error of the mean (SEM) for <italic>in vivo</italic> studies. One-way analysis of variance (ANOVA) with Dunnett's multiple comparison test was used for comparisons among various treatment groups and the control group. Results were considered statistically significant when <italic>P</italic> &#x0003c; 0.05.</p></sec></sec><sec id="sec1-3"><title>RESULTS AND DISCUSSION</title><sec id="sec2-10"><title>Angiogenic effects of AR crude extract and its fractions</title><p>In the present study, we identified the major angiogenic active fraction that possibly contributed to the wound healing property of AR by bioassay-guided fractionation [<xref ref-type="fig" rid="F1">Figure 1a</xref>] using zebrafish embryo <italic>in vivo</italic> model. As shown in <xref ref-type="table" rid="T1">Table 1</xref>, AR crude extract at 0.25 mg/ml was demonstrated to enhance angiogenesis significantly by the increased number of sprouts (1.78 &#x000b1; 0.21) formed in treated embryos when compared with control (0.10 &#x000b1; 0.01, <italic>P</italic> &#x0003c; 0.001). Two downstream fractions, P1 (macromolecules) and P2 (small molecules) separated by ethanol precipitation method, were also found to have significant activities at 0.25 mg/ml and, thus, fractionation was conducted on both fractions. Among seven resulting sub-fractions tested, P1-1 showed the most promising angiogenic effect (2.46 &#x000b1; 0.24) and, therefore, was further fractionated into five sub-fractions (P1-1-1 to P1-1-5) by Sephadex G-100 column. P1-1-1 was selected as it showed the highest enhancing effect (2.03 &#x000b1; 0.27) among the five fractions tested. When comparing to the mother fraction (P1-1), the effective concentration of P1-1-1 was lower (0.125 mg/ml). When P1-1-1 was further fractionated into P1-1-1-A and P1-1-1-B, it was found that the activities of these sub-fractions were decreased. Therefore, P1-1-1 was identified as the major active angiogenic fraction of AR. Zebrafish (<italic>Danio rerio</italic>) embryos were chosen as <italic>in vivo</italic> model for discovery of bioactive drugs from natural sources due to their high genetic similarity to human, high reproducibility, and short generation time, ease of drug administration, as well as their optical transparency for allowing visualization of drug effects on internal organs and tissues.[<xref rid="ref20" ref-type="bibr">20</xref>] Additionally, transgenic zebrafish with florescent blood vessels have been extensively used for study of embryonic blood vessel formation in the area of angiogenesis since they offer a less labor-intensive method of visualizing blood vessels in the zebrafish embryo.[<xref rid="ref21" ref-type="bibr">21</xref>]</p></sec><sec id="sec2-11"><title>Chemical characterization of major active fraction P1-1-1</title><p>P1-1-1 was isolated from P1 which was composed of macromolecules. The chemical property of P1-1-1 has been characterized in terms of P1-1-1-A and P1-1-1-B. The results of GFC [<xref ref-type="fig" rid="F2">Figure 2</xref>] showed that three groups of macromolecules (P1-1-1-A1, P1-1-1-B1, and P1-1-1-B2) were present in P1-1-1, and their molecular weights were estimated to be 229-2396 kDa, 5.3-143 kDa, and 1.6 kDa, respectively, with P1-1-1-A1 originating from the sub-fraction P1-1-1-A and the others from P1-1-1-B [<xref ref-type="table" rid="T2">Table 2</xref>]. Additionally, when P1-1-1 was analyzed by SDS-PAGE, a broad band of large molecular size (&#x0003e;76 kDa) and a band of small molecular size (about 38 kDa) were observed [<xref ref-type="fig" rid="F3">Figure S1</xref>, Supplementary Information]. When compared with the results of GFC, these two bands were identified as P1-1-1-A1 and P1-1-1-B1, respectively [<xref ref-type="table" rid="T2">Table 2</xref>]. It was suggested by PAS staining that the fractions were glycoproteins [<xref ref-type="fig" rid="F3">Figure S1</xref>, Supplementary Information]. The carbohydrate content of the fractions was analyzed and the results are summarized in <xref ref-type="table" rid="T2">Table 2</xref>. They were estimated to contain 45.1-70.6% (w/w) of carbohydrate and 11.7-15.9% (w/w) of protein.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Gel filtration chromatogram (GFC) of the major active fraction P1-1-1 and its sub-fractions (P1-1-1-A and P1-1-1-B). P1-1-1-A1, P1-1- 1-B1, and P1-1-1-B2 were three groups of macromolecules identified in P1-1-1 by GFC</p></caption><graphic xlink:href="JTCM-4-239-g003"/></fig><fig id="F3" position="float"><label>Figure S1</label><caption><p>SDS-PAGE analysis of angiogenic active fraction (P1-1-1) and its sub-fractions (P1-1-1-A and P1-1-1-B). The resulting gel was further stained with PAS. The bands (P1-1-1-A1 and P1-1-1-B1) in the lanes of P1-1-1, P1-1-1-A and P1-1-1-B were stained positively with PAS</p></caption><graphic xlink:href="JTCM-4-239-g004"/></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Chemical characterization of P1-1-1, P1-1-1-A, and P1-1-1-B</p></caption><graphic xlink:href="JTCM-4-239-g005"/></table-wrap></sec><sec id="sec2-12"><title>P1-1-1 stimulated sprout formation in zebrafish embryos by up-regulating expression of angiogenic markers</title><p>The angiogenic activity and underlying mechanism of P1-1-1 was further evaluated using zebrafish model. Results indicated the average number of sprouts formed per embryo was significantly increased from 0.76 to 2.03 (<italic>P</italic> &#x0003c; 0.001) after treating the embryos with 0.0625 mg/ml and 0.125 mg/ml of P1-1-1 and the stimulation was in a concentration-dependent manner [<xref ref-type="fig" rid="F4">Figure 3a</xref>]. The representative pictures of P1-1-1 treated zebrafish embryo and control embryo are shown in [<xref ref-type="fig" rid="F1">Figure 1b</xref>]. In order to identify the molecular targets of the angiogenic effects of P1-1-1 in zebrafish embryos, RT-PCR was used to quantify the mRNA expression levels of selected genes involved in vascular endothelial growth factor (VEGF) signaling pathway. VEGF and its tyrosine kinase receptors (VEGFRs) are the key regulators in angiogenesis and are highly specific to endothelial cells.[<xref rid="ref22" ref-type="bibr">22</xref>] VEGFA, the most important member of VEGF, binds and activates the two VEGFRs, VEGFR-1 (Flt-1) and VEGFR-2 (KDR), subsequently initiates the main signaling pathway.[<xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] As shown in [Figure <xref ref-type="fig" rid="F4">3b</xref>-<xref ref-type="fig" rid="F4">d</xref>], 0.125 mg/ml of P1-1-1 was demonstrated to significantly up-regulate mRNA expression of these regulators, 5.7-fold for Flt-1, 2.6-fold for KDR, and 8.2-fold for VEGFA (<italic>P</italic> &#x0003c; 0.05). Therefore, our results suggested that the angiogenic effect of P1-1-1 observed in zebrafish embryos was at least partly mediated via VEGF signaling pathways.</p><fig id="F4" position="float"><label>Figure 3</label><caption><p>Effects of P1-1-1 on sprout formation and mRNA expression of three angiogenic markers (Flt-1, KDR, VEGFA) in zebrafish embryos. (a) Sprout formation was expressed as number of sprouts per embryo (mean &#x000b1; SEM) of not less than three independent experiments. For (b) Flt-1, (c) KDR, and (d) VEGFA, the expression levels were normalized and expressed as mean &#x000b1; SEM of not less than three independent experiments. *<italic>P</italic> &#x0003c; 0.05, **<italic>P</italic> &#x0003c; 0.01, and ***<italic>P</italic> &#x0003c; 0.001 indicate significant difference when compared to control using one-way ANOVA with Dunnett's multiple comparison test</p></caption><graphic xlink:href="JTCM-4-239-g006"/></fig></sec><sec id="sec2-13"><title>P1-1-1 stimulated HMEC-1 cell viability, DNA synthesis and cell population in S phase</title><p>Proliferation of endothelial cells is the first and the primary event in angiogenesis. Therefore, HMEC-1 were used to examine the angiogenic effects of P1-1-1 by measuring cell proliferation using MTT assay, ³H-thymidine uptake assay, and cell cycle analysis. After the cells were incubated with P1-1-1 at a concentration range of 0.03125-0.125 mg/ml for 24 or 48 h, increased cell viability was observed in MTT assay when compared to the control which was set to 100% (<italic>P</italic> &#x0003c; 0.001) [Figure <xref ref-type="fig" rid="F5">4a</xref> and <xref ref-type="fig" rid="F4">b</xref>]. In a similar manner, P1-1-1 also significantly accelerated the uptake of ³H-thymidine under the same concentration range (<italic>P</italic> &#x0003c; 0.01) [<xref ref-type="fig" rid="F5">Figure 4c</xref> and <xref ref-type="fig" rid="F4">d</xref>]. The results indicated that cellular DNA synthesis was enhanced by P1-1-1. This was further verified by the increased S (synthesis) phase population in cell cycle analysis. As shown in <xref ref-type="table" rid="T3">Table 3</xref>, P1-1-1 at concentrations of 0.0625 mg/ml and 0.125 mg/ml significantly increased the cell population in S phase after 24 h (from 27.4 to 32.7%) and 48 h (from 20.7 to 26.0%) incubation (<italic>P</italic> &#x0003c; 0.05).</p><fig id="F5" position="float"><label>Figure 4</label><caption><p>Effect of P1-1-1 on HMEC-1 cell viability and DNA synthesis. The cells were treated with P1-1-1 (0.03125-0.125 mg/ml). (a, b) MTT assay and (c, d) 3H-thymidine uptake assay were performed after 24 and 48 h. Data were expressed as mean &#x000b1; SD of not less than three independent experiments. **<italic>P</italic> &#x0003c; 0.01 and **<italic>P</italic> &#x0003c; 0.001 indicate significant difference when compared to control using one-way ANOVA with Dunnett's multiple comparison test</p></caption><graphic xlink:href="JTCM-4-239-g007"/></fig><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Effects of P1-1-1 on cell cycle distribution of HMEC-1 cells</p></caption><graphic xlink:href="JTCM-4-239-g008"/></table-wrap><p>Previous <italic>in vitro</italic> and <italic>in vivo</italic> studies of AR extract and its constituents (calycosin, astragaloside IV, and polysaccharides) have confirmed their pro-angiogenic activities.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>] However, very few biological studies of its glycoprotein were found, except a pathogenesis-related class 10 protein (PR-10), a glycoprotein isolated from AR, which was shown to exhibit ribonuclease activity.[<xref rid="ref14" ref-type="bibr">14</xref>] It was found that the isolated AR active fraction P1-1-1, which consisted of glycoproteins, stimulated angiogenesis in zebrafish embryos via up-regulated expression of VEGF and its tyrosine kinase receptors, and proliferation of HMEC-1 cells by increasing cell viability, DNA synthesis and the shift of cell population to S phase of cell cycle in which DNA replication was stimulated. Taken together, it was suggested that P1-1-1 was a glycoprotein-containing active fraction which partially contributed to the angiogenic activity of AR.</p><p>The analysis of gene expression of VEGFA, KDR, and Flt in HMECs, as well as cell migration and differentiation will be included in our future study.</p></sec></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>In the present work is presented the isolation of an angiogenic active fraction of AR by bioassay-guided fractionation using <italic>in vivo</italic> model. The fraction comprising glycoproteins exhibited angiogenic activity in zebrafish embryos via VEGF signaling pathway, and stimulated the proliferation of HMEC-1 cells by increasing cell viability and DNA synthesis.</p></sec>

Wound-healing Activity of the Aqueous Leaf Extract and Fractions of <italic>Ficus exasperata</italic> (Moraceae) and its Safety Evaluation on Albino Rats

<p><italic>Ficus exasperata</italic> have been reported to have wide applications in the treatment of many human diseases. However, its traditional use in the treatment of wounds has not been validated by any scientific study. Also, its safety in the management of chronic disease conditions requires attention. We evaluated the wound-healing activity of the aqueous extract and fractions of <italic>F. exasperata</italic>, as well as its safety after subchronic oral administration. Similar percentage of wound contraction was observed with 5% w/w extract ointment application and administration of cicatrin powder (standard) on the 4^th day, while better contraction than the standard was recorded with higher concentrations of the extract ointment. Of all the fractions tested, significant (<italic>P</italic> &#x0003c; 0.05) contraction was only noticed in chloroform fraction, though lower than that of the aqueous extract. The extract also showed concentration-dependent inhibition of all the tested microbial isolates. Extract administered up to 5000 mg/kg (single dose administration) did not cause any mortality after 24 h. Mortality was, however, recorded at 4000 mg/kg within the first 20 days of subchronic administration of the extract. Significant (<italic>P</italic> &#x0003c; 0.05) increases in alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and in particular, alkaline phosphatase (ALP) were observed at different doses and time periods. Pathological and histological changes were noticed in the liver and kidney on the 91^st day of the study with 4000 mg/kg of the extract. Except for the significant (<italic>P</italic> &#x0003c; 0.05) reduction in WBC on the 91^st day, no other significant (<italic>P</italic> &#x0003c; 0.05) changes were observed in other hematological parameters. The aqueous extract demonstrated better wound-healing activity than its fractions; however, the extract may not be safe at higher doses for subchronic oral administration, as may be the case in the management of chronic disease conditions.</p>

<contrib contrib-type="author"><name><surname>Umeh</surname><given-names>Victoria Nonyelum</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ilodigwe</surname><given-names>Emmanuel Emeka</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ajaghaku</surname><given-names>Daniel Lotanna</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Erhirhie</surname><given-names>Earnest Oghenesuvwe</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Moke</surname><given-names>Goodies Emuesiri</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Akah</surname><given-names>Peter Achunike</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Population rise, inadequate supply of drugs, high cost of treatment, side effects, and drug resistance have led to increased use of plant materials as a source of medicine for various human ailments.[<xref rid="ref1" ref-type="bibr">1</xref>] The valuable medicinal properties inherent in plants have long been acknowledged, as biologically active molecules and lead structures for the development of modified derivatives with enhanced activity have been obtained through various natural product researches.[<xref rid="ref2" ref-type="bibr">2</xref>] It is not therefore surprising that over three-quarters of the world population rely mainly on plants and plant extracts for health care.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p><italic>Ficus exasperata</italic>, popularly referred to as &#x0201c;sandpaper leaf tree,&#x0201d; finds wide application in ethnomedicine.[<xref rid="ref4" ref-type="bibr">4</xref>] In African traditional medicine, all the plant parts are considered medicinally important, but the leaves are much valued in the treatment of diseases.[<xref rid="ref5" ref-type="bibr">5</xref>] The leaf extract has been shown to cause a significant decrease in hyperglycemia, polyurea, and hyperlipidemia and enhanced serum insulin levels in streptozotocin-induced diabetic rats.[<xref rid="ref6" ref-type="bibr">6</xref>] Reduced blood pressure and restoration of microanatomy of the blood vessels to almost normal levels have also been achieved using the leave extract of <italic>F. exasperata</italic> in spontaneously hypertensive and obese Zucker rats.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Hydro-ethanol extract of the leaves of this plant has also been shown to exhibit radical scavenging activity comparable to <italic>n</italic>-propyl gallate and also inhibits lipid peroxidation in rat brain.[<xref rid="ref8" ref-type="bibr">8</xref>] Anti-inflammatory,[<xref rid="ref9" ref-type="bibr">9</xref>] antiarthritic,[<xref rid="ref8" ref-type="bibr">8</xref>] antinociceptive,[<xref rid="ref9" ref-type="bibr">9</xref>] anticonvulsant,[<xref rid="ref10" ref-type="bibr">10</xref>] anxiolytic,[<xref rid="ref11" ref-type="bibr">11</xref>] antiulcer,[<xref rid="ref12" ref-type="bibr">12</xref>] antipyretic,[<xref rid="ref13" ref-type="bibr">13</xref>] and antimicrobial[<xref rid="ref14" ref-type="bibr">14</xref>] activities of the leaf extract have all been validated in numerous scientific studies.</p><p>In south-eastern Nigeria, the leaves are mainly used for the treatment of topical wounds and for the management of diabetes and hypertension. Since no studies have been done to validate its wound-healing activity and its safety in managing chronic ailments, we evaluated the wound-healing activity of the aqueous extract and fractions, as well as its safety after subchronic administration in albino rats.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Plant materials</title><p>Leaves of <italic>F. exasperata</italic> were collected from Nibo town in Awka South Local Government Area, Nigeria and were authenticated by Dr. J. E. Amadi of the Department of Botany, Nnamdi Azikiwe University, Awka, Anambra state, Nigeria where a voucher specimen (PCG/423A/019) was kept.</p></sec><sec id="sec2-2"><title>Animals</title><p>Adult albino rats (150-180 g) were obtained from the animal house, Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University. They were maintained in standard animal house conditions and were fed with growers feed (by Top feeds Nigeria Limited) and water <italic>ad libitum</italic>. All animal experiments were conducted in compliance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals (Pub. No. 85-23 Revised 1985) and approved by the Nnamdi Azikiwe University, Awka Ethical Committee for the use of laboratory animals.</p></sec><sec id="sec2-3"><title>Methods</title><sec id="sec3-1"><title>Extraction and fractionation</title><p>The shade-dried leaves were pulverized, cold macerated for 24 h, and filtered, and the filtrate was concentrated using freeze-drier. Part of the freeze-dried extract (100 g) was adsorbed on silica gel and eluted in succession with <italic>n</italic>-hexane, chloroform, ethyl acetate, and methanol. The filtrate so obtained was evaporated to a constant weight using rotary evaporator at 40&#x000b0;C.</p></sec><sec id="sec3-2"><title>Qualitative phytochemical analysis</title><p>Screening for the presence of secondary metabolites was performed following standard phytochemical tests as described by Harborne.[<xref rid="ref15" ref-type="bibr">15</xref>]</p></sec><sec id="sec3-3"><title>Antimicrobial evaluation</title><p>Agar well diffusion technique, as described by Adeniyi <italic>et al</italic>.,[<xref rid="ref16" ref-type="bibr">16</xref>] was used. The bacterial isolates of <italic>Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, and Proteus vulgaris</italic> were used. The extract concentrations used were 10, 50, 100, 150, 200, and 250 mg/ml at an equal volume of 1000 &#x003bc;l. Each concentration was tested in triplicate and the zones of inhibition were measured in millimeters.</p></sec></sec><sec id="sec2-4"><title>Wound healing evaluation</title><sec id="sec3-4"><title>Formulation of herbal ointment</title><p>The medicated ointment was prepared by incorporating different quantities of the extract or fractions into the ointment base (composed of cetosteryl alcohol, hard paraffin, wool fat, and white soft paraffin) to get 5, 15, 20, and 25% w/w of the extract in ointment. Also 10, and 20% w/w of each fraction in ointment was prepared. The fusion method was adopted in the formulation of the herbal ointment. The required quantity of the ointment base was weighed and melted at a temperature of about 70&#x000b0;C in a water bath.</p><p>The designated quantity of the extract (or fraction) was added to the melted base at 40&#x000b0;C in a water bath, stirred gently and continuously until a homogenous dispersion was obtained. The above exercise was repeated using different weights of the crude extract (or fractions) in order to obtain the above-mentioned concentrations.</p></sec><sec id="sec3-5"><title>Wound creation</title><p>Thirty adult albino rats divided into six groups of five rats each were used for the study. Excision wounds were created after shaving the left dorsal thoracic region 1 cm away from the vertebral column and 5 cm away from the ear. The animals were anesthetized prior to and during creation of wounds with chloroform using open mask method. Excision wounds of about 3 cm in diameter were created. All wounds were of full thickness, extending down to adipose tissue.[<xref rid="ref17" ref-type="bibr">17</xref>] The wound areas were measured every 4 days by retracing the wound on a millimeter scale graph paper,[<xref rid="ref18" ref-type="bibr">18</xref>] cleaned with methylated spirit, and the appropriate ointment concentration applied once a day until complete healing was observed. Wound contraction was calculated as percentage reduction in the wound area with respect to the initial wound area, while epithelialization time was noted as the number of days required after wound infliction for the scab to fall off leaving no raw wounds behind:</p><p><inline-graphic xlink:href="JTCM-4-246-g001.jpg"/></p><p>where</p><p>W_A0= wound area on day 0</p><p>W_AT= wound area on day T (after induction).</p></sec><sec id="sec3-6"><title>Acute toxicity test</title><p>Acute oral toxicity test was conducted as per the Organisation for Economic Co-operation and Development (OECD) guidelines 423.[<xref rid="ref19" ref-type="bibr">19</xref>] A total of 18 albino mice were divided into three groups of six animals each. The control group received 10 ml/kg of normal saline, while the second and third groups received a single dose of 2000 and 5000 mg/kg of the extract, respectively. The animals were observed for obvious signs of toxicity and mortality at hourly intervals for the next 24 h and thereafter for a total of 14 days.</p></sec><sec id="sec3-7"><title>Subchronic toxicity test</title><p>Sixty albino rats were divided into four groups as A, B, C, and D, with each group consisting of 15 rats. After pre-treatment and determination of basal biochemical and hematological parameters, groups B, C, and D received the extract orally at doses of 1000, 2000, and 4000 mg/kg, respectively, daily for 90 days. Group A served as negative control and received 10 ml/kg normal saline. Physical observation of the animals was done on a daily basis.</p><p>Five rats from each group were anesthetized using chloroform on the 31^st, 61^st, and 91^st days of the study. Blood samples were collected through retro-orbital plexus and used for the estimation of hemoglobin (Hb), packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) count.[<xref rid="ref20" ref-type="bibr">20</xref>] Serum samples were used for analysis of biochemical markers, i.e. alanine aminotransaminase (ALT),[<xref rid="ref21" ref-type="bibr">21</xref>] aspartate aminotransaminase (AST),[<xref rid="ref21" ref-type="bibr">21</xref>] and alkaline phosphatase (ALP).[<xref rid="ref22" ref-type="bibr">22</xref>] Histological studies were done on liver and kidney isolates from animals of different dose groups on the 91^st day.</p></sec></sec><sec id="sec2-5"><title>Statistical analysis</title><p>The data were expressed as mean &#x000b1; SEM. The results were analyzed by SPSS version 19 using one-way analysis of variance. Graphical representation was done using Microsoft Excel 2010. The differences between mean values were considered significant at <italic>P</italic> &#x0003c; 0.05.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-6"><title>Qualitative phytochemistry</title><p>Qualitative phytochemical study of the extract [<xref ref-type="table" rid="T1">Table 1</xref>] revealed the presence of many phytocompounds. Phlobatannins and terpenoids were found at a high concentration in the chloroform fraction, while alkaloids and saponins were more in the methanol fraction. Ethyl acetate fraction contained saponins, terpenoids, and flavonoids. The <italic>n</italic>-hexene fraction was more of fats and oil. Due to the low percentage yield of <italic>n</italic>-hexane fraction, no further study was done with this fraction.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Phytochemical constituents of aqueous extract and fractions of <italic>Ficus exasperata</italic></p></caption><graphic xlink:href="JTCM-4-246-g002"/></table-wrap></sec><sec id="sec2-7"><title>Antimicrobial activities of the extract</title><p><xref ref-type="table" rid="T2">Table 2</xref> shows the results of the antimicrobial activities of different concentrations of the aqueous crude extract on some human pathogens. There was a concentration-dependent inhibition of all the tested micro-organisms. The minimum inhibitory concentration of the leaf extract ranged from 10 to 250 mg/ml.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Inhibition zone diameters (mm) of <italic>F. exasperate</italic> against test microorganisms</p></caption><graphic xlink:href="JTCM-4-246-g003"/></table-wrap></sec><sec id="sec2-8"><title>Wound-healing activity</title><p>Progressive wound contraction was observed in all the extract-treated groups [<xref ref-type="fig" rid="F1">Figure 1</xref>]. At the lowest concentration of 5% w/w extract ointment, significant (<italic>P</italic> &#x0003c; 0.05) contraction similar to that of cicatrin powder (standard) was observed on the 4^th day. Better contraction than the standard was recorded at higher concentrations of the extract ointment. Epithelialization was observed on the 11^th day in the 20% and 25% w/w extract ointment treated groups, while it occurred on the 14^th day in the cicatrin powder treated group just like the 15% w/w extract ointment treated group [<xref ref-type="table" rid="T3">Table 3</xref>]. The group treated with blank ointment took a longer time (24 days) to achieve epithelialization. Less contraction activity was recorded for the fractions [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Only the chloroform fraction (20% w/w) showed significant contraction from the 12^th day. Epithelialization time (14 days) for this fraction was, however, the same as that of cicatrin powder.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Wound-healing activity of the aqueous leaf extract of <italic>F. exasperata</italic>.*<italic>p</italic> &#x0003c; 0.05 compared with control</p></caption><graphic xlink:href="JTCM-4-246-g004"/></fig><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Epithelialization time of the different formulated extract and fractions ointments</p></caption><graphic xlink:href="JTCM-4-246-g005"/></table-wrap><fig id="F2" position="float"><label>Figure 2</label><caption><p>Wound-healing activity of different fractions of aqueous leaf extract of <italic>F. exasperata</italic>. *<italic>P</italic> &#x0003c; 0.05 compared with control</p></caption><graphic xlink:href="JTCM-4-246-g006"/></fig></sec><sec id="sec2-9"><title>Acute toxicity test</title><p>No obvious physical and behavioral side effect was observed. Up to 5000 mg/kg, the extract did not cause any mortality after 24 h and subsequently for 14 days. The LD₅₀ was, therefore, estimated to be greater than 5000 mg/kg.</p></sec><sec id="sec2-10"><title>Subchronic toxicity tests</title><p>By day 17 of daily oral administration of the extract, obvious signs of weakness were observed in some of the rats that received 4000 mg/kg of the extract and, subsequently, eight rats died in this group within the first 20 days. Thereafter, no death was recorded in all the groups. Significant (<italic>P</italic> &#x002c2; 0.05) increase in ALT was observed only for the highest dosed group on the 61^st and 91^st days [<xref ref-type="fig" rid="F3">Figure 3a</xref>]. Groups that received 2000 and 4000 mg/kg of the extract exhibited significant (<italic>P</italic> &#x0003c; 0.05) increase in AST on the 61^st day, while significant (<italic>P</italic> &#x0003c; 0.05) increase was observed with all doses on the 91^st day [<xref ref-type="fig" rid="F3">Figure 3b</xref>]. Also, significant (<italic>P</italic> &#x0003c; 0.05) increase in ALP was observed in all the extract-treated groups on the 61^st and 91^st days. It was also observed that increase in ALP in the group treated with 4000 mg/kg became significant (<italic>P</italic> &#x0003c; 0.05) even on the 31^st day [<xref ref-type="fig" rid="F3">Figure 3c</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Effects of extract on liver enzymes: (a) Alanine aminotransaminase, (b) Aspartate aminotransaminase and (c) Alkaline phosphatase. *<italic>p</italic> &#x0003c; 0.05 compared with control</p></caption><graphic xlink:href="JTCM-4-246-g007"/></fig><p>Histological changes in the liver of the highest dosed group correlated with the biochemical changes observed. Histology of the liver in this group showed proliferation of the tissue stroma, edema, dilatation, hemorrhage, and increased size of the hepatocytes [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Histology of the kidney of negative control revealed intact glomeruli. As the dose of the extract was increased (1000, 2000, and 4000 mg/kg), there was a progressive decrease in the number of glomeruli [<xref ref-type="fig" rid="F5">Figure 5</xref>]. Changes in the kidney histology became obvious in the group that received 4000 mg/kg b.wt. of the extract. Most glomeruli were either shrunken or lost, in addition to tubular hemorrhage occurring in this group. Hematological analysis revealed no significant (<italic>P</italic> &#x002c3; 0.05) changes in PCV [<xref ref-type="fig" rid="F6">Figure 6a</xref>], Hb [<xref ref-type="fig" rid="F6">Figure 6b</xref>], and RBC [<xref ref-type="fig" rid="F6">Figure 6c</xref>] values in all the groups. Significant (<italic>P</italic> &#x002c2; 0.05) decrease in the WBC was observed in all the extract-treated groups on the 91^st day [<xref ref-type="fig" rid="F6">Figure 6d</xref>].</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Liver histology. (a) Negative control, with normal liver histology, (b) no significant changes observed with 100 mg/kg, (c) proliferation of the tissue observed with 2000 mg/kg, (d) proliferation of the tissue stroma, oedema (p), dilatation and haemorrhage within the central vein (v), increase in size of the hepatocytes (h) observed with 4000 mg/kg</p></caption><graphic xlink:href="JTCM-4-246-g008"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Kidney histology. (a) Negative control with normal features, (b) 1000 mg/kg of extract with no significant change, (c) 2000/kg of extract with no significant change, (d), 4000 mg/kg of extract showing significant damages: oedema (H), shrunken /loss of glomeruli(S), haemorrhage (H), tissue stromal distortion(S)</p></caption><graphic xlink:href="JTCM-4-246-g009"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p>Effects of extract on haematological parameters: (a) Packed cell volume (PCV), (b) Hemoglobin (HB), (c) Red blood cell (RBC) and (d) White blood cell (WBC). *<italic>p</italic> &#x0003c; 0.05 compared with control</p></caption><graphic xlink:href="JTCM-4-246-g010"/></fig></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Wounds provide a conducive environment for the growth of micro-organisms. Usually, microbial contaminants of wounds involve a variety of organisms such as <italic>P. aeruginosa, Sta. aureus, Streptococcus faecalis, E. coli, Clostridium perfringens, Clostridium tetani</italic>, coliform bacilli, and enterococcus.[<xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] Evaluation of the extract in clinically isolated microbial contaminants of wounds showed dose-dependent inhibitory activity against some pathogenic micro-organisms including <italic>P. aeruginosa</italic> and <italic>Sta. aureus</italic>, two organisms mostly implicated in chronic and non-healing wounds.[<xref rid="ref25" ref-type="bibr">25</xref>] Microbial infection in wound delays healing and causes more pronounced acute inflammatory reaction which can lead to further tissue injury and damage.[<xref rid="ref24" ref-type="bibr">24</xref>] Thus, the antimicrobial activity of the extract on these wound isolates may partly contribute to the wound-healing effect by eliminating infection and thus allowing initiation of natural tissue repair processes. It also suggests the leaf extract may play useful role in accelerating the healing of old wounds by eradicating already established infection.</p><p>Phytochemical analysis of the extract and fractions revealed the presence of alkaloids, saponins, sterols, terpenoids, glycosides, tannins, and flavonoids. These metabolites are usually responsible for the pharmacological activities of medicinal plants.[<xref rid="ref26" ref-type="bibr">26</xref>] Saponins and flavonoids have been reported to possess wound-healing activity.[<xref rid="ref27" ref-type="bibr">27</xref>] Terpenoids are known to promote wound-healing process, mainly due to their astringent and antimicrobial activities which seem to be responsible for wound contraction and increased rate of epithelialization.[<xref rid="ref28" ref-type="bibr">28</xref>] Flavonoids and their derivatives are known to decrease lipid peroxidation by improving vascularity leading to slowing down of cell necrosis.[<xref rid="ref29" ref-type="bibr">29</xref>] Phlobatannins have also been demonstrated to have wound-healing activity.[<xref rid="ref30" ref-type="bibr">30</xref>] Therefore, the wound-healing potential of <italic>F. exasperata</italic> can be attributed to the contributions of individual phytoconstituents. Synergistic or complementary effects of the phytoconstituents were further demonstrated by higher activity recorded by the extract compared with the fractions. Significant activity recorded by the chloroform fraction indicates that phlobatannins may have played a significant role in the overall wound-healing activity of the extract.</p><p>Conventional wound treatment requires, more or less, the combined effects of antibiotics, anti-inflammatory agents, astringents, and antipyretics; <italic>F. exasperata</italic> has been reported to possess antibacterial,[<xref rid="ref14" ref-type="bibr">14</xref>] anti-inflammatory,[<xref rid="ref9" ref-type="bibr">9</xref>] antioxidant,[<xref rid="ref8" ref-type="bibr">8</xref>] and antipyretic effects.[<xref rid="ref13" ref-type="bibr">13</xref>] The extract can, therefore, be effectively used in the treatment of wounds.</p><p>Single administration of the extract up to 5000 mg/kg did not produce any sign of toxicity or mortality, an indication that may have led to the assumption that the extract is safe. However, this was not the case observed on subchronic administration, as obvious signs of toxicity and mortality were recorded within the first 20 days in the highest dosed group. These signs alerted us of the inherent toxicity that may be associated with this extract.</p><p>ALT, AST, and ALP are the enzymes associated with liver function and are an indirect measure of liver homeostasis.[<xref rid="ref31" ref-type="bibr">31</xref>] Elevation of these marker enzymes is an indication of hepatotoxicity associated with the extract. It was observed that elevation of ALP preceded that of other marker enzymes. ALP elevation is usually associated with cholestasis due to biliary obstruction or hepatic infiltration,[<xref rid="ref32" ref-type="bibr">32</xref>] which may be a key to hepatotoxicity associated with this extract. Biochemical alterations observed were complemented by histological changes in the liver and kidney. This is in line with the observed increases in liver and kidney weights as well as serum liver enzymes reported following acute administration of aqueous extract of <italic>F. exasperata</italic>.[<xref rid="ref33" ref-type="bibr">33</xref>] Also, Bwititi <italic>et al</italic>.[<xref rid="ref34" ref-type="bibr">34</xref>] and Ijeh and Ukweni[<xref rid="ref35" ref-type="bibr">35</xref>] have indicated toxic injury to the kidney following acute exposure to <italic>F. exasperata</italic>. The toxicity of <italic>F. exasperata</italic> leaf has been attributed to its high content of cyanogenic glycosides[<xref rid="ref36" ref-type="bibr">36</xref>] and may have contributed to the observed toxicity recorded in this study.</p><p>Hematological system is highly susceptible to drug-induced toxicity due to its vital position and functions.[<xref rid="ref37" ref-type="bibr">37</xref>] The reduction in WBC on the 91^st day in all the extract-treated groups may be due to the direct destructive effect of the extract on these cells or their impaired production in the hematopoietic tissues (bone marrow). Moreover, the reduction of WBC following extract treatment might be because of the mobilization of the leukocytes to the tissues surrounding the blood.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>The present study justified the use of the leaf extract of <italic>F. exasperata</italic> in the treatment of cutaneous wounds more than any of its fractions. However, <italic>F. exasperata</italic> may not be safe at higher doses, especially for the management of chronic disease conditions like hypertension and diabetes, as could be observed in the subchronic toxicity and histopathology findings.</p></sec>

Evaluation of Anticancer, Antioxidant, and Possible Anti-inflammatory Properties of Selected Medicinal Plants Used in Indian Traditional Medication

<p>The present study was carried out to evaluate the anticancer, antioxidant, and possible anti-inflammatory properties of diverse medicinal plants frequently used in Indian traditional medication. The selected botanicals such as <italic>Soymida fembrifuga</italic> (Roxb.) A. Juss. (Miliaceae), <italic>Tinospora cordifolia</italic> (Willd.) Miers. (Menispermaceae), <italic>Lavandula bipinnata</italic> (L.) O. Ktze. (Lamiaceae), and <italic>Helicteres isora</italic> L. (Sterculiaceae) extracted in different solvents were evaluated for their <italic>in vitro</italic> anticancer and antioxidant activities. The results obtained indicate that H. isora has potent cytotoxic activity toward the selected cancer cells such as HeLa-B75 (34.21 &#x000b1; 0.24%), HL-60 (30.25 &#x000b1; 1.36%), HEP-3B (25.36 &#x000b1; 1.78%), and PN-15 (29.21 &#x000b1; 0.52%). Interestingly, the selected botanicals selectively inhibited cyclooxygenase-2 (COX-2) more than (COX-1), which are the key enzymes implicated in inflammation. COX-2 inhibition was observed to be in the range of 19.66-49.52% as compared to COX-1 inhibition (3.93-19.61%). The results of the antioxidant study revealed that the selected plants were found to be effective 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH), and superoxide radical (SOR) scavenging agents. High-performance thin layer chromatography (HPTLC) fingerprint of flavonoids was used as a measure of quality control of the selected plant samples. The results of the present findings strengthen the potential of the selected plants as a resource for the discovery of novel anticancer, anti-inflammatory, and antioxidant agents.</p>

<contrib contrib-type="author"><name><surname>Shaikh</surname><given-names>Rafik</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Pund</surname><given-names>Mahesh</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Dawane</surname><given-names>Ashwini</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Iliyas</surname><given-names>Sayyed</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Cancer is one of the most life-threatening diseases, with more than 100 different types occurring due to some molecular changes within the cell. It is the third leading cause of death worldwide following cardiovascular and infectious diseases.[<xref rid="ref1" ref-type="bibr">1</xref>] It is estimated that 12.5% of the population dies due to cancer (WHO, 2004). The disease is widely prevalent, and in the West, almost a third of the population develops cancer at some point of time during their life. Although the mortality due to cancer is high, many advances have been made both in terms of treatment and understanding the biology of the disease at the molecular level.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Breast cancer is the most common form of cancer in women. The incidence of breast cancer is the highest in Pakistan among the South-Central Asian countries. It is the most frequent malignancy in women and accounts for 38.5% of all female cancers. About half (43.7%) of all breast cancers are detected in an advanced stage.[<xref rid="ref3" ref-type="bibr">3</xref>] Colon cancer is the second most common cause of cancer deaths in the US. Prostate cancer is the most frequently diagnosed cancer among men in the US, and ranks second to skin cancer, with an estimated 180,000 new cases and 37,000 deaths expected to occur by the American Cancer Society each year.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Moreover, it is increasingly being realized that many of today's diseases are due to the &#x0201c;oxidative stress&#x0201d; that results from an imbalance between the formation and neutralization of prooxidants. Oxidative stress is initiated by free radicals, which seek stability through electron pairing with biological macromolecules such as proteins, lipids, and DNA in healthy human cells and cause protein and DNA damage along with lipid peroxidation. These changes contribute to cancer, atherosclerosis, cardiovascular diseases, aging, and inflammatory diseases.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] All cells are exposed to oxidative stress, and thus, oxidation and free radicals may be important in carcinogenesis at multiple tumor sites.</p><p>The enzymes cyclooxygenase-1 and -2 (COX-1 and -2) are the key enzymes involved in recruiting inflammation. Nevertheless, the proinflammatory cytokines play a crucial role in the initiation and progression of various cancers.[<xref rid="ref7" ref-type="bibr">7</xref>] Besides the key role of COX in the initiation and progression of inflammation, overexpression of COX has been considered as one of the culprits in the formation of carcinogenic state in the body.[<xref rid="ref8" ref-type="bibr">8</xref>] It is this molecular attribute of the COX upregulation that has made it an attractive target for the design and development of anticancer agents also. Free radical induced oxidative stress and its relevance with inflammation and carcinogenesis is well established.[<xref rid="ref9" ref-type="bibr">9</xref>] Therefore, inflammation, free radicals, and carcinogenesis are closely related with one another. The drug candidates having anti-inflammatory and free radical scavenging activities are more appreciated as anticancer agents.</p><p>Due to lack of effective drugs, cost of chemotherapeutic agents, and the side effects of anticancer drugs, cancer can be a cause of death. Therefore, efforts are still being made to search for effective naturally occurring anticarcinogens that would prevent, slow, or reverse cancer development. Medicinal plants have a special place in the management of cancer. It is estimated that plant-derived compounds in one or the other way constitute more than 50% of anticancer agents.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>] Numerous cancer research studies have been conducted using traditional medicinal plants in an effort to discover new therapeutic agents that lack the toxic side effects associated with the present chemotherapeutic agents. Taking into consideration the above facts, an attempt has been made to evaluate the anticancer, anti-inflammatory, and antioxidant activities of selective medicinal plants used in Indian traditional medicine system.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Materials</title><p>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The COX assay was performed by using colorimetric COX (ovine) inhibitor screening assay kit (catalog no. 760111; Cayman Chemical Company, USA). 1,10-Phenanthroline, phenazine methosulfate (PMS), and nitroblue tetrazolium (NBT) were obtained from SD Fine Chem. (Mumbai, India). Nicotinamide adenine dinucleotide (NADH) was purchased from Spectrochem, Pvt Ltd (Mumbai, India). All other chemicals and reagents used were of AR grade and were obtained from commercial sources.</p></sec><sec id="sec2-2"><title>Collection, identification, and authentication of the selected medicinal plants</title><p>The selected plants, <italic>Soymida fembrifuga</italic> (Roxb.) A. Juss. (SRTH-08), <italic>Tinospora cordifolia</italic> (Willd.) Miers. (SRTH-54), <italic>Lavandula bipinnata</italic> (L.) O. Ktze. (SRTH-24), and <italic>Helicteres isora</italic> L. (SRTH-61), were collected from the nearby regions of Nanded district (Maharashtra) during September 2009. The plants were identified and authenticated by RNG, Head, Department of Botany, School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.[<xref rid="ref12" ref-type="bibr">12</xref>] Voucher specimens of the collected plants were deposited in the herbarium center of the host institute. The shade-dried and powdered plant samples were preserved for further experiments.</p></sec><sec id="sec2-3"><title>Sequential extraction of the plant samples</title><p>The shade-dried, powdered plant samples (10 g) were sequentially extracted in hexane, ethanol, and water as per their boiling points which are (69&#x000b0;C), (79&#x000b0;C), and (100&#x000b0;C), respectively, up to 8 h using Soxhlet's apparatus. The extracted samples were evaporated under reduced pressure at room temperature. The yield of the individual plant extract was measured and the dried extracts were preserved at 4&#x000b0;C in a refrigerator for further analysis.</p></sec><sec id="sec2-4"><title>HPTLC analysis</title><p>High-performance thin layer chromatography (HPTLC) analysis was performed using the instrument from CAMAG (Germany). Thin layer chromatography (TLC) plates (silica gel &#x0003e;60 F254, 20 cm &#x000d7; 10 cm; Merck) were prewashed with methanol. The plates were activated in an oven at 100&#x000b0;C for 10 min. Ten microliters of individual plant extracts (1 mg/ml) was spotted onto the precoated plates using Linomat 5 application system. Rutin hydrate (50, 100, 200 &#x003bc;g/ml) was used as the marker flavonoid. The flavonoids were separated using ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:27) as the mobile phase. Natural product (NP) reagent was used as the flavonoid derivatizing agent, and the spots developed were visualized under CAMAG UV cabinet (366 nm) and digitized using CAMAG photodocumentation system.</p></sec><sec id="sec2-5"><title>Cell lines and culturing</title><p>Human cancer cell lines HeLa-B75, HL-60, HEP-3B, PN-15, and normal liver cell lines were obtained from National Center for Cell Sciences, Pune, Maharashtra. &#x0003e;All cell lines were propagated in Minimum Essential Medium (Eagle) with 2 mM l-glutamine and Earle's BSS (Balanced Salt Solution) adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate 90%, and 10% fetal calf serum. All cell lines were grown in a humidified incubator at 37&#x000b0;C.</p></sec><sec id="sec2-6"><title>MTT cytotoxicity assay for <italic>in vitro</italic> anticancer study</title><p>The cytotoxicity assay was performed according to the microculture MTT method with slight modifications.[<xref rid="ref13" ref-type="bibr">13</xref>] The cells were harvested (1.5 &#x000d7; 10⁴ cells/well) and inoculated in 96-well microtiter plates. They were washed with phosphate-buffered saline (PBS) and the cultured cells were then inoculated with and without the extract (1 mg/ml). After 72 h of incubation, the medium was aspirated. Ten microliters of MTT solution (5 mg/ml in PBS, pH 7.2) was added to each well and the plates were incubated for 4 h at 37&#x000b0;C. After incubation, 100 &#x003bc;l of dimethyl sulfoxide (DMSO) was added to the wells followed by gentle shaking to solubilize the formazan dye for 15 min. Absorbance was read at 540 nm and the surviving cell fraction was calculated. Suramin (100 &#x003bc;M) was used as the reference standard for anticancer activity, and H₂O₂ (1 mM) was used as the cytotoxic agent against normal liver cell lines. The inhibition of cell viability and COX was calculated using the formula:</p><p><inline-graphic xlink:href="JTCM-4-253-g001.jpg"/></p><p>Where T= Absorbance of the test sample</p><p>C= Absorbance of the control sample</p></sec><sec id="sec2-7"><title>COX inhibition assay</title><p>The assay was performed using colorimetric COX (ovine) inhibitor screening assay kit.[<xref rid="ref14" ref-type="bibr">14</xref>] Briefly, the reaction mixture contained 150 &#x003bc;l of assay buffer, 10 &#x003bc;l of heme, 10 &#x003bc;l of enzyme (either COX-1 or COX-2), and 10 &#x003bc;l of the plant sample (1 mg/ml). The assay utilizes the peroxidase component of COX. The peroxidase activity was assayed colorimetrically by monitoring the appearance of oxidized <italic>N, N, N&#x02032;, N&#x02032;</italic>-tetramethyl-<italic>p</italic>-phenylenediamine (TMPD) at 590 nm. Aspirin (acetylsalicylic acid, 1 mM) was used as the reference anti-inflammatory compound.</p></sec><sec id="sec2-8"><title>Antioxidant study</title><sec id="sec3-1"><title>DPPH radical scavenging assay</title><p>DPPH radical scavenging assay was carried out as per the method reported earlier, with slight modifications.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>] Briefly, 1 ml of the test solution (individual plant extracts) was added to an equal quantity of 0.1 mM solution of DPPH in ethanol. After 20 min of incubation at room temperature, the DPPH reduction was measured by reading the absorbance at 517 nm. Ascorbic acid (1 mM) was used as the reference compound.</p></sec><sec id="sec3-2"><title>Hydroxyl (OH) radical scavenging assay</title><p>The OH radical scavenging activity was determined using Fenton reaction.[<xref rid="ref17" ref-type="bibr">17</xref>] The reaction mixture contained 60 &#x003bc;l of FeCl₂ (1 mM), 90 &#x003bc;l of 1,10-phenanthroline (1 mM), 2.4 ml of phosphate buffer (0.2 M, pH 7.8), 150 &#x003bc;l of H₂O₂ (0.17 M), and 1.5 ml of individual plant extracts (1 mg/ml). The reaction was started by adding H₂O₂. After 5 min incubation at room temperature, the absorbance was recorded at 560 nm. Ascorbic acid (1 mM) was used as the reference compound.</p></sec><sec id="sec3-3"><title>Superoxide radical scavenging assay</title><p>The superoxide anion scavenging assay was performed by the method reported earlier.[<xref rid="ref18" ref-type="bibr">18</xref>] Superoxide anion radicals were generated in a non-enzymatic PMS&#x02013;NADH system through the reaction of PMS, NADH, and oxygen. It was assayed by the reduction of NBT. In this experiment, superoxide anion was generated in 3 ml of Tris HCl buffer (100 mM, pH 7.4) containing 0.75 ml of NBT (300 &#x003bc;M), 0.75 ml of NADH (936 &#x003bc;M), and 0.3 ml of the plant sample (1 mg/ml). The reaction was initiated by adding 0.75 ml of PMS (120 &#x003bc;M) to the mixture. After 5 min of incubation at room temperature, the absorbance at 560 nm was measured in a spectrophotometer. Ascorbic acid (1 mM) was used as the reference compound.</p></sec></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-9"><title>Sequential extraction and HPTLC profiling</title><p>The powdered plant samples were extracted sequentially in hexane, ethanol, and water. HPTLC fingerprint of the derivatized flavonoids is shown in <xref ref-type="fig" rid="F1">Figure 1</xref>. The plants were found to contain diverse flavonoids. However, none of the plants showed the presence of rutin (used as marker flavonoid).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>HPTLC profile of the selected medicinal plant extracts. Track No. 1, rutin (50 &#x003bc;g); 2, rutin (100 &#x003bc;g); 3, rutin (200 &#x003bc;g); 4, <italic>Tinospora cordifolia</italic>; 5, <italic>Lavandula bipinnata</italic>, 6, <italic>Helicteres isora</italic>; 7, <italic>Soymida fembrifuga</italic></p></caption><graphic xlink:href="JTCM-4-253-g002"/></fig></sec><sec id="sec2-10"><title>Anticancer activity of the selected medicinal plants</title><p>The results of cytotoxicity of the selected plant samples (ethanol extract) against the selected cancer cells are summarized in <xref ref-type="table" rid="T1">Table 1</xref>. It was observed that the ethanol phytofraction (1 mg/ml) of all the tested plants showed promising anticancer activity toward the selected cancer cell lines. <italic>L. bipinnata</italic> possessed significant anticancer activity by inhibiting PN-15 (35.21 &#x000b1; 1.48%), while <italic>S. fembrifuga</italic> showed moderate inhibition toward PN-15 (06.35 &#x000b1; 0.35%). All the plants under investigation were found to possess moderate cytotoxic activity toward HeLa-B75 and HL-60 cell lines, showing the activity in the range of 12.14-34.21%. The cytotoxic activity was compared with suramin (0.01 mM), which was used as the standard anticancer drug (HeLa-B75, 91.55%; HL-60, 72.51%; HEP-3B, 92.43%; and PN-15, 77.38%). None of the plant samples showed cytotoxicity toward normal &#x0201c;Chang liver&#x0201d; cells, except the standard H₂O₂ (3.13%).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Cytotoxic effect of selected medicinal plant extracts (1 mg/ml) on different cancer cell lines and normal chang liver cells</p></caption><graphic xlink:href="JTCM-4-253-g003"/></table-wrap></sec><sec id="sec2-11"><title>COX inhibitory potential of the selected medicinal plants</title><p>The results of COX inhibitory activity of the ethanolic fraction of the selected medicinal plants are shown in <xref ref-type="fig" rid="F2">Figure 2</xref>. It is interesting to note that all the plant samples preferentially inhibited COX-2 rather than COX-1. <italic>L. bipinnata</italic> showed the maximum activity by inhibiting COX-2 (50.43 &#x000b1; 0.39%) more than COX-1 (18.63 &#x000b1; 0.31%), while all other plant samples inhibited COX-1 and COX-2 in the range of 03.93&#x02013;23.53% and 19.66-50.43%, respectively. Aspirin (COX-1, 08.83 &#x000b1; 0.37% and COX-2, 08.83 &#x000b1; 0.37%) was used as the standard anti-inflammatory agent.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Inhibition of COX-1 and COX-2 by the selected medicinal plants. Data are shown as mean &#x000b1; SD of three similar experiments</p></caption><graphic xlink:href="JTCM-4-253-g004"/></fig></sec><sec id="sec2-12"><title>Antioxidant study</title><sec id="sec3-4"><title>DPPH radical scavenging activity</title><p>The DPPH radical scavenging assay is used for preliminary screening of the plant extracts for their antioxidant activity. The proton radical scavenging action is known to be an important mechanism of antioxidants. The results of this assay are summarized in <xref ref-type="table" rid="T2">Table 2</xref>. Overall, it was observed that the ethanolic fraction of all the selected plants was more potent in stabilizing DPPH radicals. Moreover, the ethanol extract of <italic>S. fembrifuga</italic> (71.43 &#x000b1; 0.42%) possessed the highest DPPH radical scavenging ability, while for the rest of the plants, it was found to be in the range of 06.25-66.67%, as compared to ascorbic acid (82.54 &#x000b1; 0.02%).</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Antioxidant activity of the selected medicinal plants</p></caption><graphic xlink:href="JTCM-4-253-g005"/></table-wrap></sec><sec id="sec3-5"><title>OH radical scavenging activity</title><p>The OH radicals are the most hyperreactive amongst the relative oxygen species and affect every type of molecule found in the living system. Physiologically important biomolecules such as sugar, amino acids, phospholipids, DNA bases, and organic acids may undergo reaction with OH radicals and may change the normal physiological function of cells. The OH radical scavenging activity of the selected medicinal plants is shown in <xref ref-type="table" rid="T2">Table 2</xref>. The water and ethanol extracts showed moderate OH radical scavenging activity in the range of 03.85-17.31%, but not the hexane extract of the individual plant samples. Ascorbic acid (02.82 &#x000b1; 0.02%) was used as the reference compound.</p></sec><sec id="sec3-6"><title>SO radical scavenging activity</title><p>The results of the SO radical scavenging activity of the selected medicinal plants are shown in <xref ref-type="table" rid="T2">Table 2</xref>. Water extract of all the selected plants was found to be an excellent SO radical scavenger, showing the activity in the range of 60.00-71.22%. However, <italic>S. fembrifuga</italic> extracts showed promising SO radical scavenging activity in the order hexane, ethanol, and water (18.46, 59.03, and 71.22%, respectively). A poor activity was observed in the hexane extract of <italic>T. cordifolia</italic> (08.34%).</p></sec></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Over the past decade, herbal medicines have been appreciated and accepted all over the world and they have made an impact on both global health and international trade. Hence, medicinal plants continue to play an important role in the healthcare system of a majority of the world's population.[<xref rid="ref19" ref-type="bibr">19</xref>] Traditional medicine is widely used in India. Even in the US, the use of plants and phytomedicines has increased dramatically in the last two decades, and as a result, a National Centre for Complementary and Alternative Medicine has been established there. The herbal products have been classified under &#x0201c;dietary supplements&#x0201d; and are included with vitamins, minerals, amino acids and other products intended to supplement the diet.[<xref rid="ref20" ref-type="bibr">20</xref>] In fact, there are several medicinal plants all over the world, including India, which are being used traditionally for the prevention and treatment of cancer. However, only a few medicinal plants have attracted the interest of scientists to investigate the remedy for neoplasm (tumor or cancer).</p><p>The plants consist of various phytochemicals which dissolve in specific solvents. According to this, the polar (ethanol, water) and nonpolar (hexane) solvents were selected for the extraction purpose. While describing the mechanism of anticancer, anti-inflammatory, and antioxidant activities of the medicinal plants, various phytochemicals seem to be associated with these activities. In particular, the phytochemicals such as vitamins (A, C, E, K), carotenoids, terpenoids, flavonoids, polyphenols, alkaloids, tannins, saponins, pigments, enzymes, and minerals have been found to elicit antioxidant activities.[<xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref>] With regard to the management of cancers, ellagic acid and a whole range of flavonoids, carotenoids, and terpenoids present in <italic>Fragaria vesca</italic> (strawberries) and <italic>Rubus idaeus</italic> (raspberries) have been reported to be responsible for the antioxidant activity. These chemicals block various hormonal actions and metabolic pathways that are associated with the development of cancer.[<xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] A whole variety of phenolic compounds, in addition to flavonoids, are widely distributed in grains, fruits, vegetables, and herbs. Phenolic compounds such as caffeic and ferulic acids, sesamol, and vanillin have been reported to exhibit antioxidant and anticarcinogenic activities and inhibit atherosclerosis.[<xref rid="ref25" ref-type="bibr">25</xref><xref rid="ref26" ref-type="bibr">26</xref>]</p><p>The severe side effects of the presently used nonsteroidal anti-inflammatory drugs (NSAIDs) have resulted in either withdrawal or replacement of these drugs (nimesulide, bromfenac, ibufenac, and benoxaprofen) from the pharmaceutical market. Selective COX-2 inhibitors were described to be very effective in the management of inflammatory disorders. However, nonselective COX-1 inhibition by these agents imposed restrictions on the usage of selective COX-2 inhibitors on health grounds. In an attempt to discover novel anti-inflammatory agents targeting COX, the drug candidates selectively inhibiting COX-2 and skipping COX-1 are more appreciated as safe anti-inflammatory drugs.[<xref rid="ref27" ref-type="bibr">27</xref>] It is interesting to note that the selected plants preferentially inhibited COX-2 activity more than that of COX-1. This finding indicates the significance of the selected plants as a potential resource for the discovery of novel leads for maneuvering them as effective and safe anti-inflammatory/anticancer agents.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>It can be summarized that the plants selected in the present study having importance in traditional medicine can be considered as a source for the isolation, identification, and development of novel and effective anticancer, anti-inflammatory, and antioxidant agents. Nevertheless, the research data of the present findings may serve as a guideline for the standardization and validation of natural drugs containing the selected medicinal plants as ingredients.</p></sec>

Antimicrobial Potential of <italic>Helicanthus elastica</italic> (Desr.) Danser - A less explored Indian mistletoe Growing on Mango Trees

<p><italic>Helicanthus elastica</italic> (Desr.) Danser (Loranthaceae) is a less-known medicinally important mistletoe species occurring in India. It is used to check abortion, and also in vesical calculi and kidney affections. There are no detailed studies reporting the antimicrobial potential of this plant. Based on the traditional use and the rich phenolic composition of the whole plant, the antimicrobial property of the alcohol extract was analyzed and the results are outlined in the present paper. For the analysis, zone of inhibition, and minimum inhibitory concentration were used, and the total activity was assayed by standard methodologies. The antimicrobial activity was studied against bacteria like <italic>Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Vibrio fischeri, and a fungus Candida albicans</italic>. Of the eight tested bacteria, the alcoholic extract of <italic>H. elastica</italic> was found to be active against <italic>K. pneumoniae</italic>, A. hydrophila, <italic>E. coli</italic>, and <italic>V. fischeri</italic> at concentration ranging from 250 to 500 &#x003bc;g/ml. <italic>C. albicans</italic> showed inhibition only at a concentration of 2000 &#x003bc;g/ml.</p>

<contrib contrib-type="author"><name><surname>Sunil Kumar</surname><given-names>Koppala Narayana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Saraswathy</surname><given-names>Ariyamuthu</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Amerjothy</surname><given-names>Swaminathan</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Ravishankar</surname><given-names>Basaviah</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Screening of medicinal plants for antimicrobial activities is important for finding new compounds for therapeutic use. In particular, the antimicrobial activity of plant oils and extracts has formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine, and natural therapy.[<xref rid="ref1" ref-type="bibr">1</xref>] The development and spread of resistance to the existing antibiotics by microorganisms call for increased efforts in the development of new antibiotics.[<xref rid="ref2" ref-type="bibr">2</xref>] Although a number of plants with antimicrobial activities have been identified, great number still remains unidentified. Many plants are considered as antimicrobial agents for bactericidal or fungicidal applications.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Natural products like quinones, terpenes, alkaloids, and tannins have been reported to curtail the growth of vegetative cells and spores of bacteria and fungi.[<xref rid="ref4" ref-type="bibr">4</xref>] The word mistletoe is applied for hemiparasitic plants, with the exception of the genera <italic>Nuytsia, Atkinsonia</italic>, and <italic>Gaiadendron</italic>, producing parasitic roots on the aerial shoots of other higher plants.[<xref rid="ref5" ref-type="bibr">5</xref>] <italic>Helicanthus elastica</italic> (Desr.) Danser (Loranthaceae) is a common mistletoe species growing on mango trees in India. Its leaves are used to check abortion, and also in vesical calculi and kidney affections.[<xref rid="ref6" ref-type="bibr">6</xref>] The hemiparasite is found to be a rich source of phenolic compounds.[<xref rid="ref7" ref-type="bibr">7</xref>] Hence, a thought has been given to assess the antimicrobial properties of the plant. The prime objective of this work is to evaluate the action of the alcoholic extract of <italic>H. elastica</italic> on different microbial strains to create direct comparable, quantitative, antimicrobial data on these less-known Indian mistletoes.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Laboratory materials</title><p>Chemicals and solvents used in the study were of laboratory and analytical grade and were obtained from Merck chemicals, Mumbai, India. Growth media and their components were obtained from HiMedia, Mumbai, India. Glassware of Borosil were used throughout the study. They were cleaned properly prior to the experiment by soaking them in Lysol for 24 h followed by detergent and were washed thoroughly in running water, rinsed with distilled water, and air dried. Glassware, growth media, and other accessories used in this study were sterilized in a vertical autoclave at 121&#x000b0;C and 15 lbs/sq. inch pressure for 15 min.</p></sec><sec id="sec2-2"><title>Preparation of extract</title><p>Fresh plants of the mistletoe growing on <italic>Mangifera indica</italic> were collected during flowering in August 2009 from Kasaragod District of Kerala. Voucher specimen of the plant collected was deposited (voucher specimen number 00637) at the Pharmacognosy Department of Captain Srinivasa Murti Drug Research Institute for Ayurveda, Chennai. Fifty grams of the coarsely powdered air-dried plant material of <italic>H. elastica</italic> was soaked overnight in absolute ethanol (250 ml). Next day the flask was slightly warmed over a water bath and filtered. The marc was again treated with more of alcohol and filtered. The filtrates were combined and concentrated on a water bath till it became a semi-solid mass (9.5 g) at a temperature not more than 105&#x000b0;C. Further drying was done in a vacuum dryer. The dried extracts were stored in an air-tight amber glass container and kept in a refrigerator until the studies were completed.</p></sec><sec id="sec2-3"><title>Preparation of bacterial inoculum</title><p>The quality and quantity of inoculums have only second-order effects upon the response of an assay system, which is the inhibition of the growth (antibiotics) or stimulation of the growth (vitamins). The suitability of the test organism for the purpose is more important than the quality and quantity of inoculums. The inoculums usually are grown in a medium similar to or identical to the assay medium.</p><p>A single colony was picked from the plate, suspended in 100 ml sterile nutrient broth, and incubated overnight at 37&#x000b0;C. The turbidity was measured in a calibrated photometer, and a suitable quantity was used for inoculating the assay broth to give 5 million cells/ml.</p></sec><sec id="sec2-4"><title>Microbial cultures</title><sec id="sec3-1"><title>Bacterial strains</title><p>The strains used for screening antibacterial and antifungal activity were purchased from National Chemical Laboratory (NCL), Pune. The bacterial strains were methicillin-resistant (MR) <italic>Staphylococcus aureus, Bacillus subtilis</italic> (MTCC 441), <italic>Pseudomonas aeruginosa</italic> (ATCC 27853), <italic>Aeromonas hydrophila</italic> (ATCC 7966), <italic>Streptococcus pyogenes</italic> (ATCC 19615), <italic>Vibrio fischeri</italic> (ATCC 7744), <italic>Klebsiella pneumoniae</italic> (ATCC 15380), and <italic>Escherichia coli</italic> (ATCC 25922).</p></sec><sec id="sec3-2"><title>Fungal strains</title><p>A fungal strain <italic>Candida albicans</italic> (MTCC 227) was obtained from Madras Medical College, Chennai.</p></sec></sec><sec id="sec2-5"><title>Preparation of media</title><sec id="sec3-3"><title>Preparation of nutrient broth</title><p>Nutrient broth (NB) was used for the preparation of suspensions of bacterial cultures which were used as inoculums. NB was composed of peptic digest of animal tissue 5 g/l, sodium chloride 5 g/l, beef extract 1.5 g/l, and yeast extract 1.5 g/l. Exactly 1.3 g of accurately weighed NB was suspended in 100 ml of distilled water and sterilized in an autoclave at 121&#x000b0;C and 15 lbs pressure for 15 min.</p></sec><sec id="sec3-4"><title>Preparation of medium for antibacterial activity</title><p>Mueller Hinton Agar (MHA) from HiMedia Laboratories was used for antibacterial sensitivity test. MHA is composed of beef infusion 300 g/l, casein acid hydrolysate 17.50 mg/l, starch 1.50 g/l, and agar 17 g/l. Exactly 3.8 g of accurately weighed MHA medium was suspended in 100 ml of distilled water and sterilized in an autoclave at 121&#x000b0;C, 15 lbs pressure for 15 min.</p></sec><sec id="sec3-5"><title>Preparation of medium for antifungal activity</title><p>Sabouraud's Dextrose Agar (SDA) from Himedia Laboratories was used for the yeast sensitivity test. SDA is composed of mycological peptone 0.2 g/l, dextrose 4.0 g/l, and agar 1.5 g/l. Exactly 6.8 g of accurately weighed SDA medium was suspended in 100 ml of distilled water and sterilized in an autoclave at 121&#x000b0;C, 15 lbs pressure for 15 min.</p></sec><sec id="sec3-6"><title>Preparation of fungal inoculums</title><p>The fungal cultures were grown on SDA (Himedia Laboratories) by incubating at 25&#x000b0;C for 4 days.</p></sec></sec><sec id="sec2-6"><title>Sterilization</title><p>The microbiology laboratory was fumigated with potassium permanganate and formaldehyde once in a month. Inoculation hood was cleaned with 95% ethyl alcohol and sterilized using UV lamp for 15 min each time before use.</p></sec><sec id="sec2-7"><title>Preparation of sample</title><p>Fifty milligrams of the extract was weighed in a sterile Eppendorf tube and 500 &#x003bc;l of dimethyl sulfoxide (DMSO) was added and mixed well.</p></sec><sec id="sec2-8"><title>Agar well diffusion method[<xref rid="ref8" ref-type="bibr">8</xref>]</title><p>The Petri plates and the medium were sterilized in an autoclave at 121&#x000b0;C and 15 lbs pressure for 15 min. About 25-30 ml of the sterile medium was transferred aseptically to the sterile Petri plates and labeled. The plates were allowed to solidify inside the laminar air flow chamber. Ten microliters of the bacterial inoculums (5 million cells/ml) was transferred using micropipette onto sterile media and a lawn culture was made using sterile &#x0201c;z&#x0201d; rod. Similarly, the SDA plates were inoculated with 72-h-old fungal culture maintained in SDA broth. The plates were labeled. Using separate sterile plungers, wells of 6 mm diameter were made on the solidified loaded media. Then, 20 &#x003bc;l, 10 &#x003bc;l, 5 &#x003bc;l, and 2.5 &#x003bc;l of the extract were aseptically transferred into separate wells. The different wells had the extract concentration of 2000 &#x003bc;g/ml, 1000 &#x003bc;g/ml, 500 &#x003bc;g/ml, and 250 &#x003bc;g/ml, respectively. Standard solution (Ciprofloxacin 5 &#x003bc;g) was used as the positive control. Care was taken to avoid tilting of the plates. The MHA plates were incubated at 37&#x000b0;C for 24 h and the SDA plates at 25&#x000b0;C for 48 h.</p></sec><sec id="sec2-9"><title>Minimum inhibitory concentration</title><p>Minimum inhibitory concentration (MIC) was determined for the plant extract showing antimicrobial activity against the tested pathogens. Broth microdilution method[<xref rid="ref9" ref-type="bibr">9</xref>] was followed for determination of MIC values. Plant extracts were re-suspended in DMSO to a concentration of 2000 &#x003bc;g/ml, and then subjected to twofold serial dilutions and added to broth media present in 96 wells of microtiter plates. Thereafter, 100 &#x003bc;l inoculum [1 &#x000d7; 10⁸ colony forming units (CFU)/ml for bacteria and 1 &#x000d7; 10⁷ cells/ml for fungus] was added to each well. The microtiter plates were incubated at 37&#x000b0;C for 24 h for bacteria and at 28&#x000b0;C for 48 h for fungi. Each extract was assayed in duplicate. A loop full of the broth from a different dilution was inoculated onto the surface of sterile nutrient agar and the plates were incubated at 37&#x000b0;C for 24 h for bacteria and the same was carried out on SDA plates for <italic>C. albicans</italic>. The MIC values were taken as the lowest concentration of the extracts in the well of the microtiter plate that showed no growth after incubation.</p></sec><sec id="sec2-10"><title>Total activity determination</title><p>Total activity (TA) is the volume at which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated, and is expressed in milliliters per gram.[<xref rid="ref10" ref-type="bibr">10</xref>]</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The antibacterial activity of the alcohol extract of <italic>H. elastica</italic> whole plant was studied against <italic>A. hydrophila, B. subtilis</italic>, <italic>E. coli</italic>, <italic>K. pneumoniae</italic>, MR <italic>Sta. aureus</italic>, <italic>P. aeruginosa</italic>, <italic>Str. pyogenes</italic>, and <italic>V. fischeri</italic>. Antifungal activity of the above-mentioned extract was tested against <italic>C. albicans</italic>.</p><sec id="sec2-11"><title>Antimicrobial activity</title><p>The zones of inhibition of the test extract against the selected strains are presented in <xref ref-type="fig" rid="F1">Figure 1</xref> and <xref ref-type="table" rid="T1">Table 1</xref>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Antimicrobial activity of the alcohol extract of Helicanthus elastica Graphic Summary</p></caption><graphic xlink:href="JTCM-4-258-g001"/></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Zone of inhibition of alcoholic extract of <italic>H. elastica</italic> on tested pathogens</p></caption><graphic xlink:href="JTCM-4-258-g002"/></table-wrap><p>The ethanol extract of <italic>H. elastica</italic> at a concentration of 250-2000 &#x003bc;g was screened against the selected pathogenic species. <italic>K. pneumoniae</italic> is the only organism that was susceptible to the extract at the lowest concentration tested (250 &#x003bc;g).</p><p>The observed zone of inhibition against the organisms tested was concentration dependent. <italic>H. elastica</italic> alcohol extract was found to have antifungal activity against <italic>C. albicans</italic> only at a higher concentration of 2000 &#x003bc;g/ml.</p></sec><sec id="sec2-12"><title>Minimum inhibitory concentration</title><p>The MIC values of the alcoholic extract of <italic>H. elastica</italic> are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Minimum inhibitory concentration of the alcoholic extract of <italic>H. elastica</italic></p></caption><graphic xlink:href="JTCM-4-258-g003"/></table-wrap></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>The goal of this study was to ascertain the antimicrobial activity of the alcohol extract of the whole plant. In a previous study,[<xref rid="ref11" ref-type="bibr">11</xref>] the plant extracts were found to possess cytotoxic activity. The test extract was evaluated against several selected bacterial and fungal pathogens which are considered to be the cause of many common infections. The results obtained indicate that the test extract was quite active against <italic>K. pneumoniae</italic>, <italic>A. hydrophila</italic>, <italic>E. coli</italic>, and <italic>V. fischeri</italic> in decreasing order (MIC 62.5, 500, 500, and 500 &#x003bc;g/ml). It produced only a weak effect against other strains tested.</p><p><italic>A. hydrophila</italic> is a heterotrophic, gram-negative, rod-shaped bacterium, mainly found in areas with a warm climate. It can survive in aerobic and anaerobic environments. It is also highly resistant to multiple medications, chlorine, and cold temperature. Gastroenteritis is the common disease caused by these bacteria, especially in children. It is also associated with cellulitis and can cause myonecrosis and eczema in people with compromised immune system. The pathogenic mechanisms of <italic>Aeromonas</italic> spp. still remain unknown. The recently proposed type III secretion system (TTSS) mediated pathogenic mechanism has been proven to play a pivotal role in the pathogenesis of <italic>Aeromonas</italic>. It is a specialized protein secretion machinery that exports virulence factors delivered directly to host cells. These factors subvert normal host cell functions in some ways that are beneficial to the invading bacteria.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p><italic>V. fischeri</italic> is a gram-negative rod frequently found in symbiotic relationships with marine animals like the bobtail squid. Infections associated with non-cholera <italic>Vibrio</italic> species are gastroenteritis, wound infection, and septicemia.[<xref rid="ref13" ref-type="bibr">13</xref>]</p><p><italic>K. pneumoniae</italic> is a non-motile Gram-negative rod. This organism is commonly isolated from water and human and animal feces. It causes infection of the surgical wounds and the urinary tract, and is also commonly found in the respiratory tract.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p><italic>E. coli</italic> is a motile Gram-negative rod. It is one of the most important members of Enterobacteriaceae. It causes frequent opportunistic infections. It is often present in the appendix abscesses, peritonitis, cholecystitis, and septic wounds, and causes bacteremia and endotoxic shock, and occasionally meningitis in neonates. It is the commonest cause of uncomplicated infections of the lower urinary tract (cystitis). An increasing number of strains are recognized as primary gastrointestinal pathogens.[<xref rid="ref8" ref-type="bibr">8</xref>]</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>From the above account, it can be suggested that the tested extract is likely to contain important phytoconstituents possessing antibacterial activity against four important Gram-negative bacteria. This can be considered as a lead for further purification and activity-guided fractionation. Phytochemical study of the extract has shown the presence of numerous constituents. A detailed chemical investigation may result in isolation of therapeutically important antimicrobial agents. Ethyl gallate and gallic acid, isolated from this plant,[<xref rid="ref7" ref-type="bibr">7</xref>] are reported to possess antimicrobial activity.[<xref rid="ref14" ref-type="bibr">14</xref>] It is possible that they may be responsible at least in part to the observed antimicrobial activity of the tested extract. It remains to be elucidated if the other constituents found to be present contribute to the observed activity. Of the eight tested bacteria, the alcoholic extract of <italic>H. elastica</italic> was found to be active against <italic>K. pneumoniae</italic>, <italic>A. hydrophila</italic>, <italic>E. coli</italic>, and <italic>V. fischeri</italic> at concentrations ranging from 250 to 500 &#x003bc;g. When tested on <italic>C. albicans</italic>, the alcoholic extract showed inhibition only at a concentration of 2000 &#x003bc;g.</p></sec>

Comparative Evaluation of the Complementary and Alternative Medicine Therapy and Conventional Therapy Use for Musculoskeletal Disorders Management and Its Association with Job Satisfaction among Dentists of West India

<p>Musculoskeletal problems have become a significant issue in the profession of dentistry. There are currently no recommended effective disease-preventing and modifying remedies. High prevalence rates for musculoskeletal disorders (MSDs) among dentists have been reported in the literature. Complementary and alternative medicine can be helpful in managing and preventing the MSDs. The purpose of this study was to determine if dentists in the western part of India are using complementary and alternative medicine therapies for MSDs, and also to find if those who use complementary and alternative medicine therapies have greater job/career satisfaction compared to conventional therapy (CT) users. Dentists of western India registered under the Dental Council of India (<italic>N</italic> = 2166) were recruited for the study. Data were analyzed using univariate and bivariate analyses and logistic regression. A response rate of 73% (<italic>n</italic> = 1581) was obtained, of which 79% (<italic>n</italic> = 1249) was suffering from MSDs. The use of complementary and alternative medicine or CT was reported by 90% (<italic>n</italic> = 1124) of dentists with MSDs. Dentists using complementary and alternative medicine reported greater health (<italic>P</italic> &#x0003c; 0.001) and carrier satisfaction (<italic>P</italic> &#x0003c; 0.001) and were able to work as many hours they wanted (<italic>P</italic> &#x0003c; 0.001) compared to CT users. Complementary and alternative medicine therapies may improve the quality of life and enhance job satisfaction for a dentist who suffers from MSDs.</p>

<contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Devanand</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Batra</surname><given-names>Renu</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Mahajan</surname><given-names>Shveta</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Bhaskar</surname><given-names>Dara John</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Ankita</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Shiju</surname><given-names>Mohammed</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Yadav</surname><given-names>Ankit</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Chaturvedi</surname><given-names>Mudita</given-names></name><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Gill</surname><given-names>Shruti</given-names></name><xref ref-type="aff" rid="aff8">8</xref></contrib><contrib contrib-type="author"><name><surname>Verma</surname><given-names>Renuka</given-names></name><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Dalai</surname><given-names>Deepak Ranjan</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Rajendra Kumar</given-names></name><xref ref-type="aff" rid="aff9">9</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Dentistry is a mostly a social interaction between a dentist and the patient in their limited job setting and with personal characteristics. A healthy dentist is one of the most important components in a successful dental practice. Like all other professionals, dentists are exposed to occupational health hazards which predispose them to develop a multitude of health problems.[<xref rid="ref1" ref-type="bibr">1</xref>] Maintaining the steady hand and posture by the dentist comes at a cost to the health of the dentist. High frequency of musculoskeletal disorders (MSDs) in dentists has been reported in the literature.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] A recent review of the literature that examined the prevalence and risk factors of MSDs in dentists reported that the prevalence of general musculoskeletal pain in dental professionals ranges between 64% and 93%.[<xref rid="ref5" ref-type="bibr">5</xref>] There are many factors that contribute to MSDs in dental professionals, including repetitive motion, pinch-grasp, vibration, force, and awkward positions, sitting for a long period of time, operator position, poor posture, lack of flexibility and strength, poor ergonomics, and insufficient work breaks.[<xref rid="ref6" ref-type="bibr">6</xref>] It is generally agreed that the physical posture of the dentist should be relaxed while they work. Postures outside of this neutral position are likely to cause musculoskeletal discomfort.[<xref rid="ref7" ref-type="bibr">7</xref>] Dentists can, and do, experience illnesses and problems that can disrupt or impair their practice. Many dentists have reported work stress caused by MSDs. Some have chosen to leave the profession because of their musculoskeletal pain.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] Career and job satisfaction are the indicators that may have an influence on career longevity. Change in the work environment of the dentist might increase his/her career longevity. Complementary and alternative medicine (CAM), as defined by National Centre for Complementary and Alternative Medicine (NCCAM), is a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of conventional medicine. The commonest reason for the use of CAM in the general population is pain. A large number of patients using CAM are those who suffer from chronic musculoskeletal pain. The cause of chronic pain is usually multifactorial and complex. Many studies have reported CAM therapies, including yoga, ayurveda, homeopathy, reiki, acupressure, massage, prayers, and acupuncture, to be effective in managing chronic musculoskeletal pain and other discomforts in the general population.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>]</p><p>There are currently no reports that link musculoskeletal pain, CAM, and career satisfaction among dentists working in western India. Since a large number of dentists all over the world report MSDs, this study was conducted in western India with an aim to determine if dentists are using CAM therapies to manage their MSDs and, if so, to determine if CAM therapies are associated with their job satisfaction and longevity, compared with conventional therapy users.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>The survey was conducted with the approval of the Institutional Review Board of Teerthankar Mahaveer University (TMU). Dentists registered under Indian Dental Association (IDA) were recruited to complete an 21-item questionnaire under 5 domains. A pilot study was conducted among dentists working in the Teerthankar Mahaveer Dental College and Research Centre. Following these pilot tests, the questionnaire was further modified and was uploaded on the web-based survey software. All dentists of western India who are current members of the IDA were recruited to participate. The final version of the questionnaire was formatted using web-based survey software for electronic distribution. Subjects were mailed the link to fill up the web-based questionnaire. This study included all registered dentists residing in western India with their e-mail addresses (<italic>N</italic> = 2166). Dentists were sent the link to their e-mail address for competing the survey. Dentists who participated in the pilot study, dental students, members of the general public, dental hygienists, dental assistants, and others who were not registered dentists were excluded. The questionnaire consisted of five domains: Demographic profile of the dentist, experience with musculoskeletal pain, use of conventional therapies or use of CAM therapies for its management, opinions about CAM and conventional therapies, and job/career satisfaction related to CAM.</p><sec id="sec2-1"><title>Statistical analysis</title><p>Statistical analyses were conducted using SPSS 21. Univariate and bivariate analyses were performed to determine the demographic information, frequently reported areas of location of pain, the number of respondents that used CAM or conventional therapies, types of CAM or conventional therapies frequently used, work disruption caused by MSDs, and job satisfaction by using CAM and conventional therapies. Association between conventional therapy and CAM use in relation to career variables was assessed using Odds Ratio (OR). Independent samples <italic>t</italic>-tests were used to determine the opinions about CAM and conventional therapies for MSD management.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>A total of 2166 survey e-mails were sent electronically, with a response rate of 73% (<italic>N</italic> = 1581). The nonrespondents were assumed to be similar to the respondents based on the notion that the group under study was somewhat a homogeneous group.</p><p>Findings of the demographic status of the dentists showed that a majority of the study population was males (75.7%) and worked primarily in their own private dental clinics (85.7%). A total of 79% (<italic>n</italic> = 1249) reported having MSDs, with the mean duration of pain being 8.3 years (median = 3.5). Other demographic characteristics of respondents are presented in <xref ref-type="table" rid="T1">Table 1</xref>. Frequent areas of location of pain in the body are depicted in <xref ref-type="fig" rid="F1">Figure 1</xref>. Neck and lower back were the most common sites, followed by shoulders, upper back, wrist, elbow/arm, knee, hips, and legs.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Demographic characteristics of the dentists</p></caption><graphic xlink:href="JTCM-4-263-g001"/></table-wrap><fig id="F1" position="float"><label>Figure 1</label><caption><p>Percentage of dentist reported pain by location</p></caption><graphic xlink:href="JTCM-4-263-g002"/></fig><p><xref ref-type="fig" rid="F2">Figure 2</xref> shows work disruption among dentists as a result of MSDs. When comparison was made between individuals who used CAM therapies or conventional therapy alone and those individuals who used both CAM and conventional therapies, the latter group had 4 times lower odds of temporarily quitting work for longer than 1 month [OR = 3.4, 95% confidence interval (CI) =1.7-17.8].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Work disruption among dentist due to MSD</p></caption><graphic xlink:href="JTCM-4-263-g003"/></fig><p><xref ref-type="fig" rid="F3">Figure 3</xref> depicts the use of CAM modalities by dentists. About 81% (<italic>n</italic> = 1012) dentists reported using both CAM and conventional therapies most frequently to manage MSDs. Also, of the 1249 individuals who reported MSDs, 31% (<italic>n</italic> = 388) used CAM therapies alone, 19% (<italic>n</italic> = 238) used conventional therapies alone, and 3% (<italic>n</italic> = 38) did not use any therapy. Dentists&#x02019; degree of pain improved significantly after using CAM therapies versus conventional therapies (<italic>P</italic> = 0.004). Dentists who suffered from musculoskeletal pain agreed 3 times more that CAM therapies were acceptable for MSD management (OR = 3.7, 95% CI = 2.7-3.9) than those with no pain, and were 3 times more likely to use CAM therapies for MSD management (OR = 3.4, 95% CI = 1.4-5.9). <xref ref-type="table" rid="T2">Table 2</xref> depicts the job/career satisfaction among dentists who used CAM therapies and conventional therapies. Dentists who believed in CAM therapies and used them alone had significantly higher odds of agreeing that they were satisfied with their career as a dentist (OR = 3.2, 95% CI = 1.5-5.7) and that it contributed to career longevity (OR = 1.92, 95% CI = 1.4-7.3), increased overall health (OR = 1.67, 95% CI = 1.11-6.1), and improved the working efficiency (OR = 2.37, 95% CI = 1.1-7.3), and thus were satisfied with the job (OR = 1.51, 95% CI = 1.7-7.3) when compared to users of conventional therapies. Older dentists had significantly high rate of CAM usage than younger dentists (OR = 2.17, 95% CI = 1.157-1.007). Dentists who never used CAM reported poorer general health when compared to CAM users (OR = 1.16, 95% CI = 1.06-2.4). There were no statistically significant differences when controlling for race, type of degree earned, and number of years of practice.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>CAM use among dentist with MSD</p></caption><graphic xlink:href="JTCM-4-263-g004"/></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Association between conventional therapy and CAM usage with job/career satisfaction</p></caption><graphic xlink:href="JTCM-4-263-g005"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Recent studies have suggested that CAM usage is increasing worldwide.[<xref rid="ref15" ref-type="bibr">15</xref>] Many different types of CAM therapies, including whole medical systems (homeopathic and naturopathic medicine), mind&#x02013;body medicine (meditation, prayer, and mental healing), biologically based practices (ayurveda/herbal products), manipulative and body-based medicine (chiropractic care and massage), and energy medicine (reiki and therapeutic touch) are practiced worldwide. Many plants are used in various formulations such as decoctions, powders, medicated oils, paste, etc., for the treatment of MSDs either externally or internally. Ayurvedic drugs like <italic>Zingiber officinale</italic>, (&#x0751f;&#x08591; Sh&#x00113;ng Ji&#x00101;ng), <italic>Ricinus communis, Commiphora mukul, Boswellia serrata, Nyctanthes arbor-tristis</italic>, etc., are regularly used in the prescriptions for MSDs. Modern research scientists have conducted many scientific researches to assess the safety, efficacy, and anti-inflammatory potential of these drugs. Deep breathing exercises involve slow, deep breaths through the nose for 10 sec, followed by a complete exhalation for 10 sec for at least 5 cycles. Meditation is a practice in which an individual attempts to keep the mind clear and free from any other thoughts. It causes mental and physical relaxation. It helps individuals to deal with different problems. Chiropractic care deals by manipulation of joints and spine. Chiropractors act by adjusting the spine and sometimes by demonstrating exercises to the individuals. Massage therapist uses the technique of massage to adjust the muscles, which helps in relaxation. Massage is the commonest alternative therapy used for MSDs. Yoga is a combination of relaxation techniques and physical exercises. Yoga practitioners had less muscle weakness than compared to non practitioners.[<xref rid="ref10" ref-type="bibr">10</xref>]. Acupuncture may also be sometimes used for treating MSDs. It involves using thin, metallic needles to penetrate the skin at different anatomical points of the body. There is extensive evidence that musculoskeletal pain is a significant burden in dentistry. Dental operators often cannot avoid prolonged static postures. Even in optimal seated postures, more than one-half of the body's muscles are contracted statically and there is little movement of the vertebral joints. This may result in damaging physiological changes that can lead to back, neck, or shoulder pain or MSDs.</p><p>The term &#x0201c;musculoskeletal disorder&#x0201d; is used to describe a wide range of injuries to the tendons, ligaments, nerves, and supporting structures. Chronic musculoskeletal pain appears after only a few years of clinical practice, or even during second or third year of undergraduate training.[<xref rid="ref16" ref-type="bibr">16</xref>] Dentists are more prone to develop MSDs than any other professionals.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] CAM therapies have been effective in reducing the risk of and treating musculoskeletal pain in the general population for several decades.[<xref rid="ref13" ref-type="bibr">13</xref>] In our study, individuals who used CAM therapies alone were less likely to report temporarily quitting work. Therefore, a dentist who uses CAM therapies may reduce work interruptions caused by musculoskeletal pain. According to the present study, dentists who do not suffer from musculoskeletal pain experience higher job satisfaction when compared to those who suffer from MSDs (<italic>P</italic> = 0.001). Dentists who had MSDs reported negative impact on their career. Dentist using CAM therapies alone had greater career satisfaction compared to those who used conventional therapies alone. Therefore, dentists using CAM therapies for MSD management experienced higher satisfaction and longevity when compared to those using conventional therapies. The most favored CAM therapies among participants in the current study were massage, herbal supplements, and yogic exercises. The sample was predominantly male. Therefore, no effective gender comparison can be made. To date, no studies have examined the use of CAM for MSDs among dentists and its association with career satisfaction; therefore, the results of this study cannot be compared with any other similar study. Similar to other studies, our study also reported maximum musculoskeletal complaints in the neck and lower back region. Most of the dentists (81%) reported use of both CAM and conventional therapies in a complementary way for the treatment of musculoskeletal problems.</p><p>The hectic schedule of the dentists leaves them with no time to practice mind&#x02013;body techniques and other CAM-related modalities to treat their musculoskeletal pain. Various researches have been conducted on the characteristics of CAM users and the determinants of CAM use. Some researches show that utilization of CAM is influenced by an individual's personality, family and friends, and socioeconomic factors such as race/culture, education, and economic level. There are scientifically proven direct links between lifestyle and herbs.[<xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref><xref rid="ref26" ref-type="bibr">26</xref>] Yoga reduces stress and relieves muscular tension or pain. Dentists must practice some sort of exercise in their day-to-day routine, including yoga, to be relieved from the problem of musculoskeletal pain.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Dentists using CAM therapies reported they had greater overall health and more life satisfaction compared to conventional therapy users. CAM education should be incorporated in the dental curriculum to train the budding dentists about better management of MSDs. Education and additional research are needed to promote an understanding of the complexity of the problem and to address the problem's multifactorial nature. Dentist should adopt a holistic way of treating their musculoskeletal complaints. Knowledge and training of alternative therapies is very much required among professionals, especially dentists, so that they can use these therapies as a preventive and treatment modality for chronic pain emerging in day-to-day life and prolong their career.</p></sec>

Sedative-hypnotic Effect of <italic>Ash of Silver</italic> in Mice: A Reverse Pharmacological Study

<p><italic>Ash of silver</italic> is used in traditional systems of medicine for various neurological conditions like insomnias, neuralgias, anxiety disorders, and convulsions. The present study was conducted to evaluate the sedative-hypnotic activity of ash of silver in comparison to pentobarbitone (standard drug) in albino mice. The mice were divided into four groups as follows: Group 1 (control): Gum acacia [GA; 1% per os (p.o.)], group 2 (standard): Pentobarbitone [50 mg/kg intraperitoneal (i.p.)], group 3 (test): <italic>Ash of silver</italic> (50 mg/kg p.o.), and group 4: <italic>Ash of silver</italic> (50 mg/kg p.o.) given 30 min prior to administration of pentobarbitone (50 mg/kg i.p.). Time of onset, recovery, and total duration of loss of righting reflex were studied. <italic>Ash of silver</italic> (test) produced significant sedation (<italic>P</italic> &#x0003c; 0.01) compared to control (GA 1%), but the effect was significantly less compared to that of standard pentobarbitone at the doses used. Also, significant potentiation (<italic>P</italic> &#x0003c; 0.001) of the sedative-hypnotic effect of pentobarbitone was observed with the test drug.</p>

<contrib contrib-type="author"><name><surname>Inder</surname><given-names>Deep</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Pawan</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Traditional systems of medicines have been in use for promotive, preventive, and curative health services since centuries in many parts of the world. Being the oldest traditional system of medicine in India, Ayurveda caters to about 80% of the population in developing countries as per the estimate of World Health Organization (WHO). Despite their wide usage, research in this field is lagging behind with regard to their pharmacologic actions, safety, and efficacy. Ashes or <italic>Bhasmas</italic> used in traditional system of medicine contain heavy metal particles in varying proportions. It is not easy to write off the usage of these preparations just by presuming that heavy metals are toxic. Proper scientific documentation is required to validate the risks and benefits associated with use of such metallic Ayurvedic preparations. There are some specific methods for their detoxification and <italic>Bhasma</italic> preparation, making them suitable for clinical use in therapeutic doses, as claimed by <italic>Rasa Shastra</italic> experts. There is a need to ascertain whether the conventional <italic>Shodhan</italic> (purification) process of Ayurveda is being properly followed or not.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Silver is one among the heavy metals which is considered to be a non-essential accumulative trace element with wide distribution in the body, including the central nervous system, but with no known biological and physiological function.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] In Ayurveda, preparations like <italic>Raupya Bhasma</italic> and <italic>Kusta Nukras</italic> have been used to treat many clinical conditions such as pain, inflammation, insomnia, neuralgias, anxiety disorders, convulsions, memory loss, heat stroke, infections, pro-myelocytic leukemias, sexual disorders, etc., for many centuries. Apart from herbs/shrubs, ashes of silver are prescribed. This system also advocates the use of elemental or metallic preparations.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] Metal <italic>Bhasmas</italic> of gold (e.g., gold disodium thiomalate and auranofin) and silver (<italic>Raupya Bhasma, Kusta Nukras</italic>, etc.) have been used for the treatment of rheumatoid arthritis, acute pro-myelo cytic leukemias, immunostimulation, and as analgesics in painful inflammatory conditions, and are prescribed with accompaniments such as ginger or cumin water, tulsi extract, lemon extract, etc., that have been shown to protect against unwanted toxicity due to various reasons which include high proportions of trace elements and have synergistic or protective effects due to buffering between various constituents or free radical scavenging property. Oxides of heavy metals are usually not toxic, as claimed by <italic>Rasa Shastra</italic>.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Since the safety of <italic>ash of silver</italic> has already been established, reverse pharmacological studies are required to confirm the established facts regarding usage, safety, and efficacy in various clinical conditions mentioned above. Since raw silver ore is considered hazardous for health as mentioned in Ayurvedic literature, it needs to be converted into non-harmful form (<italic>ash of silver</italic>) by the process of trituration, pulverization, and repeated calcinations (at 300&#x000b0;C) for 14 times. Reduced form of silver thus obtained acquires spherical nanostructure with a size of 16 nm without any change in the morphology of silver, and is now called as <italic>ash of silver</italic>.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref9" ref-type="bibr">9</xref>] Nanosize of the silver particle is probably responsible for improving the penetration of silver in brain; hence, <italic>ash of silver</italic> had been used in the past for treatment of various neurological conditions, viz. insomnias, anxiety, and pain.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] Being a heavy metal preparation, <italic>ash of silver</italic> bears cumulative potential after prolonged use and in overdoses, as seen in preliminary animal studies. After certain controversial reports of toxicity due to use of metallic/elemental drugs, it has now been made mandatory (WHO guidelines) that Ayurvedic drugs in any form should be tested for their heavy metal content prior to export, so that heavy metals remain within permissible limits.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>]</p><p>This study was conducted with an aim (a) to explore the sedative-hypnotic effect of <italic>ash of silver</italic>, if any, as claimed in Ayurvedic literature. Further, if <italic>ash of silver</italic> showed sedative-hypnotic effect, the study aimed (b) to observe whether the test drug <italic>ash of silver</italic> was potentiating the sedative-hypnotic effect of pentobarbitone at the doses used in mice when given 30 min prior to the standard drug.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>Swiss albino mice of either sex weighing between 20 and 30 g were screened for the study, after obtaining approval from the Institutional Animal Ethics Committee. Mice were fed on a standard pellet diet and water <italic>ad libitum</italic>, and were housed in polypropylene cages under similar environmental conditions in an animal room that was maintained at 24 &#x000b1; 1&#x000b0;C and 55 &#x000b1; 5% humidity with a 12 h light&#x02013;dark cycle throughout the experiment. In case of oral administration, mice were fasted for 12 h before testing. Plexiglass chamber was used to observe the responses of mice.</p><sec id="sec2-1"><title>Drugs and dosage forms</title><p>The test drug <italic>ash of silver</italic> was procured from M/s Baidyanath Ayurved Bhawan Ltd (Jhansi, India). <italic>Ash of silver</italic> [50 mg/kg per os (p.o.)] was suspended in 1% solution of gum acacia. Gum acacia (1% p.o.), procured from Arora Pharmacy (New Delhi, India), was labeled as cont rol and was administered in a volume of 1 ml/100 g. <italic>Ash of silver</italic> and gum acacia were administered orally using infant feeding pipe with a 1 ml syringe attached at the other end. Standard sedative-hypnotic pentobarbitone [50 mg/kg intraperitoneal (i.p.)] was procured from Nembutal Dainippon Pharmaceutical Co. (Osaka, Japan) and was administered as i.p. injection using 1 ml syringe.</p></sec><sec id="sec2-2"><title>Animals and their grouping</title><p>Animals (mice) were divided into four groups consisting of six animals in each. Study protocol was as follows:</p><p>Group 1: Received vehicle gum acacia (1% p.o.) as control, given in a volume of 1 ml/100 g p.o.</p><p>Group 2: Received pentobarbitone (50 mg/kg i.p.) as the standard drug</p><p>Group 3: Received the test drug <italic>ash of silver</italic> (50 mg/kg p.o.) suspended in 1% solution of gum acacia</p><p>Group 4: Received the test drug <italic>ash of silver</italic> (50 mg/kg p.o.) suspended in 1% solution of gum acacia, following which the standard drug pentobarbitone (50 mg/kg i.p.) was given after 30 min</p><p>The responses of all drugs [in terms of time of onset, time of recovery, and total duration of loss of righting reflex (LORR) in mice] were assessed by continuous observation of animals throughout the experiments from the time of administration of drug in the plexiglass chamber, using a stop watch.</p></sec><sec id="sec2-3"><title>Measurement of the duration of pentobarbital-induced LORR</title><p>The duration of LORR was measured according to the procedures described by Marley <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] Mice were given an i.p. injection of pentobarbitone (50 mg/kg). When the mice became ataxic, they were placed on their backs on a pre-warmed (37&#x000b0;C) pad and the onset, recovery, and total duration of LORR [starting at the time of administration of the test drug (<italic>ash of silver</italic>), the standard drug pentobarbitone (50 mg/kg), and the test drug (<italic>ash of silver</italic>) followed 30 min later by the standard drug pentobarbitone (50 mg/kg)] were noted until they regained their righting reflexes. Mice were presumed to have regained the righting response when they could right themselves three times within 30 sec.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>Findings of the present study are depicted in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Effects of various drug treatments on sleep in mice</p></caption><graphic xlink:href="JTCM-4-268-g001"/></table-wrap><p>All values were expressed as Mean &#x000b1; SEM and analyzed using analysis of variance (ANOVA) followed by Dunnett's &#x0201c;<italic>t</italic>&#x0201d; test. <italic>P</italic> &#x0003c; 0.05 was considered significant.</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>In the present study, we tried to explore the pharmacological effect of <italic>ash of silver</italic> as a sedative-hypnotic and its secondary effect to potentiate the sedative-hypnotic effect of pentobarbitone. Such studies help to fast track drug discovery and development when carried out in selected animal models through screening. Thus, Ayurvedic knowledge and experimental database are able to provide new functional leads, thereby reducing the toxicity of drugs and saving time and money.[<xref rid="ref13" ref-type="bibr">13</xref>]</p><p>In the present study, <italic>ash of silver</italic> was observed to possess sedative effect at a dose of 50 mg/kg (p.o.) in mice. The sedative effect was significant (<italic>P</italic> &#x0003c; 0.01) when compared with the vehicle gum acacia (1% p.o.) in mice. The sedative effect of the test drug <italic>ash of silver</italic> was significantly less (<italic>P</italic> &#x0003c; 0.01) compared to pentobarbitone (50 mg/kg i.p.), the standard drug (<italic>P</italic> &#x0003c; 0.001). Significant potentiation (<italic>P</italic> &#x0003c; 0.001) of the sedative-hypnotic effect of pentobarbitone (50 mg/kg i.p.) was observed with 30 min prior administration of the test drug <italic>ash of silver</italic> (50 mg/kg p.o.).</p><p>The above findings reveal that <italic>ash of silver</italic> had sedative effect at the doses used; therefore, it can be proposed that <italic>ash of silver</italic> might be acting as a sedative-hypnotic owing to its pharmacological effects probably mediated by inhibition of neuropeptide S (NPS) or <italic>N</italic>-methyl-d-aspartate (NMDA)/histamine/5-HT3/dopamine or potentiation of effects mediated through gamma-aminobutyric acid (GABA)/glycine or benzodiazepines (BZDs)/opioid receptors. NPS was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. <italic>In vivo</italic>, NPS produces a unique behavioral profile by increasing wakefulness and exerting anxiolytic-like effects.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>]</p><p>To explore the mechanism of action of <italic>ash of silver</italic> as a sedative, antagonists/blockers need to be administered against the above-mentioned mediators. In our previous study, we tried to explore the analgesic activity of <italic>ash of silver</italic>, which is probably mediated through opioid receptors as it was observed after administering naloxone, the opioid antagonist, although the role of other mediators cannot be ruled out.[<xref rid="ref13" ref-type="bibr">13</xref>] One of the studies has proposed that ashes of heavy metals used in traditional systems of medicine function as a catalyzer by their presence in intestine, plasma, and blood, thereby acting as free radical scavengers.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref11" ref-type="bibr">11</xref>] Ash particles of heavy metals (gold, silver) in calcined form, being insoluble, exist as nanoparticles (16 nm), which are very tiny particles and biocompatible, and therefore can cross the blood&#x02013;brain barrier to exert various central actions as claimed in Ayurvedic literature, viz. analgesic, anti-inflammatory, sedative, anti-anxiety, cognitive, neuroleptic, and antiepileptic.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref20" ref-type="bibr">20</xref>] Lankveld <italic>et al</italic>. and Kim <italic>et al</italic>. have proved the distribution of nanosized silver particles in the central nervous system as well as in other tissues, e.g., liver, kidney and spleen, and intestine, when administered by the oral route.</p><p>As the findings of our study have shown significant potentiation of sedative-hypnotic effect of pentobarbitone with 30 min prior administration of <italic>ash of silver</italic> at the dose used, it can be hypothesized that there is a possibility of <italic>ash of silver</italic> acting as hepatic cytochrome P450 microsomal enzyme inhibitor. Further, it can be hypothesized that <italic>ash of silver</italic> increases the plasma levels of pentobarbitone by inhibiting its hepatic metabolism, so as to potentiate the sedative/anesthetic effect of pentobarbitone. Pentobarbitone is mainly metabolized in the liver. Further studies are required to confirm and establish this fact also by intracerebroventricular injection of <italic>ash of silver</italic> in the brain of mouse/rats.</p><p>Till date, hardly any studies have been conducted to explore the pharmacological effects of <italic>ash of silver</italic> on sedation/sleep in human body, in spite of its wide use in humans in Ayurvedic practice for many centuries. Studies conducted by Nadeem <italic>et al</italic>. on silver preparations showed their interesting anti-anxiety, anti-cataleptic, and anti-aggressive effects. No scientific reports are available to confirm these claims except for some preliminary experimental studies demonstrating slight diminution of discharge frequency in frog nerve-muscle preparation bathed in 3% suspension of <italic>ash of silver</italic>. The anti-anxiety and anti-aggressive effects observed by Nadeem <italic>et al</italic>. support the nerve-soothing properties (nervine tonic) of silver preparations.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>In one study, there is a mention of phytochelatins (PCs), produced from reduced glutathione present in green plants and legumes, which tend to chelate the heavy metals from soil. Therefore, it can be postulated that in the presence of vegetarian diet, a fraction of <italic>ash of silver</italic> is also liable to get chelated if taken with vegetarian food.[<xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] Also, the same theory can be applied to overcome the toxic effects produced by excess dose of <italic>ash of silver</italic>, as mentioned in the study of Inder <italic>et al</italic>. Pharmacokinetic and pharmacodynamic studies need to be carried out to find if there is any interaction of <italic>ash of silver</italic> if taken with vegetarian diet, so as to formulate and revise the dose for human use. The role of free radical scavengers needs to be established, which can help to reduce the adverse effects of <italic>ash of silver</italic>. One of the studies has shown the interaction of <italic>ash of silver</italic> with some biomolecules, proteins, vitamins, etc., thus affecting various physiological reactions.[<xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref><xref rid="ref26" ref-type="bibr">26</xref><xref rid="ref27" ref-type="bibr">27</xref><xref rid="ref28" ref-type="bibr">28</xref>] From the observations and results of the study with the test drug <italic>ash of silver</italic>, it can be postulated that at the doses used in mice (50 mg/kg p.o.), it acts as a mild to moderate sedative and owing to this property, it might have potentiated the duration of LORR effect of pentobarbitone (50 mg/kg i.p.), i.e., synergistic effect. But as both these drugs are metabolized by the liver to a greater extent, inhibition of cytochrome P450 enzymes system in the liver by <italic>ash of silver</italic> cannot be ruled out, which is probably responsible for inhibiting the hepatic metabolism of pentobarbitone resulting in high plasma levels of pentobarbitone, which might have potentiated the sedative-hypnotic effect of pentobarbitone at the doses used in mice.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Ash of silver possesses significant sedative hypnotic potential at a dose used in present animal study. Therefore it can serve as a better alternative as sleep inducing drug with a better safety profile compared to conventional hypnotics. Further studies will provide adequate data to support this evidence.</p></sec>

Effect of Two Herbal Mouthwashes on Gingival Health of School Children

<p>This study aimed at determining the effect of indigenously prepared neem and mango chewing stick mouthwashes on plaque and gingival indices. A sample of 105 children aged 12-15 years was randomized into three groups, namely neem, mango, and chlorhexidine mouthwash groups. All the children were examined at baseline and gingival and plaque indices were recorded. Baseline scores for plaque and gingivitis were fair and moderate, respectively, in all the three groups and there existed no statistically significant difference among them. Ten millilitres each of herbal and chlorhexidine mouthwashes (0.2%) were administered according to the group allocation twice daily for 21 days. Indices were reassessed at 21 days (immediately after intervention) and at 1 month, 2 months, and 3 months after discontinuing the mouthwashes. Statistically significant reduction (<italic>P</italic> &#x0003c; 0.001) in plaque index was found in all the three mouthwash groups at 21 days and at 1 month from discontinuing the mouthwash. Chlorhexidine additionally showed statistically significant reduction in plaque index at 2 months from discontinuing the mouthwash. Statistically significant reduction (<italic>P</italic> &#x0003c; 0.001) in gingival index was found in all the three mouthwash groups at 21 days (immediately after discontinuing the mouthwash) and at 1 and 2 months from discontinuing the mouthwash. To conclude, all the three mouthwashes were effective antiplaque and antigingivitis agents. Chlorhexidine and neem possess equivalent efficacy in reducing plaque, while chlorhexidine has superior antigingivitis properties.</p>

<contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Ratika</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Hebbal</surname><given-names>Mamata</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ankola</surname><given-names>Anil V.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Murugaboopathy</surname><given-names>Vikneshan</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Shetty</surname><given-names>Sindhu Jayasimha</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Research has linked oral microorganisms, particularly those with adherent biofilm properties, to clinically specific oral conditions such as dental caries, periodontal disease, and oral malodor.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Plaque reduction has been the hallmark of preventive dentistry since the advent of antibiotics and the realization that bacteria are possible causative agents of the major dental diseases, caries, and periodontal disease.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Mechanical hygiene procedures such as tooth brushing, interdental brushing, and dental floss are the key methods of plaque control. However, despite the potential for adequate mechanical plaque control, clinical experience and population-based studies demonstrate that such methods are not being employed sufficiently by large numbers of the population.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Chemical methods of reducing plaque, such as mouthwashes, are less technically demanding alternatives to mechanical plaque control.[<xref rid="ref5" ref-type="bibr">5</xref>] Chlorhexidine is the most popular mouthwash which has been recognized by the pharmaceutical industry as the positive control against which the efficacy of alternative antiplaque agents should be measured, and has earned its eponym of gold standard.[<xref rid="ref6" ref-type="bibr">6</xref>] But its long-term usage may result in various side effects.[<xref rid="ref3" ref-type="bibr">3</xref>] An effective substitute to chlorhexidine with all the good qualities and sans its unpleasant effects is highly desirable and has been long awaited.</p><p>Plants have been exploited by man for many centuries as sources of chemotherapeutic and other medicinal drugs due to the presence of various bioactive compounds. These herbal products are not only economical, but also have minimal side effects.</p><p>Brushing with neem and mango twigs and chewing neem leaves and seeds after a meal have been the traditional dental care practices in India. Stems of <italic>Azadirachta indica</italic> (neem) contain substances like nimbin and nimbidin which have anti-inflammatory and broad-spectrum antimicrobial activities.[<xref rid="ref7" ref-type="bibr">7</xref>] The natural C-glucoside xanthone mangiferin, a phenolic compound, has been reported in various parts of <italic>Mangifera indica</italic> leaves, fruits, stem, bark, heartwood, and roots.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] It is known to possess antioxidant, radioprotective, immunomodulatory, antitumor, anti-allergic, anti-inflammatory, antidiabetic, and antimicrobial properties.[<xref rid="ref13" ref-type="bibr">13</xref>] Mangiferin has also demonstrated promising therapeutic potential both in the prevention and treatment of periodontitis.[<xref rid="ref14" ref-type="bibr">14</xref>] <italic>In vitro</italic> studies indicate that neem and mango stick extracts are inhibitory to oral streptococci which are responsible for various oral diseases.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] Literature review revealed very few <italic>in vivo</italic> studies worldwide assessing the effects of neem and mango stick extracts on plaque and gingiva. Hence, the present study was planned to evaluate the effect of neem and mango on plaque and gingival scores in high school children of Belgaum city.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>The present study was a triple-blind randomized controlled field trial conducted to evaluate the effectiveness of two herbal mouthrinses (neem and mango) on plaque and gingival scores of 12-15-year-old school children in Belgaum city. Permission to conduct the study was obtained from the institutional review board of KLE VK Institute of Dental Sciences, Belgaum, Karnataka, India, Deputy Director of Public Instruction (DDPI), Belgaum, and the principal of the selected high school.</p><p>A pilot study was conducted on 10 people to determine the acceptability, palatability, and safety of the mouthwashes. The required sample size was estimated based on the difference in the plaque and gingival scores between the study and control groups. Sample size was calculated based on the minimum difference expected between the two groups, which was 0.7.</p><p>Two examiners were selected to ensure blind evaluation of the study participants. Examiner 1 (principal investigator) selected the schools, obtained permission from them, did primary screening (examination for inclusion and exclusion criteria, which included baseline clinical examination) and selection, collected baseline data, and administered the mouthwashes for 21 days. Examiner 2 recorded the plaque and gingival scores after 21 days (immediately after the intervention) and 1 month, 2 months, and 3 months after discontinuing the mouthwash. Examiner 2 was blinded to the type of mouthwashes admistered. The statistician remained blinded regarding the subject allocation to the three groups. Both the examiners were trained and calibrated before the start of study in the Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, Belgaum under the guidance of a professor in order to limit the intra-examiner and inter-examiner variability. They were reassessed for satisfactory agreement at various time intervals during the clinical examinations. Recording assistants were trained in documenting the readings accurately. The intra-examiner and inter-examiner variabilities were calculated using Kappa statistics. Inter-examiner and intra-examiner variability (Kappa) for plaque index and gingival index ranged from 0.8 to 0.9 and from 0.7 to 0.8, respectively, during all the assessments.</p><p>For obtaining the study sample, two-stage random sampling was done. In the first stage, a list of all the schools was obtained from DDPI, Belgaum. From these schools, one school was selected by lottery method. Written informed consent was obtained from the parents of all the children examined. Children were free to withdraw from the study at any point during the study period.</p><p>During the initial phase of study (before selecting the sample), a 29-item self-designed combination of closed and open-ended questionnaire and assessment form was prepared to collect information regarding socio-demographics, oral hygiene practices, and food habits from all the children aged 12-15 years. All the children were also clinically examined for the inclusion and exclusion criteria, as mentioned later. This form was divided into five parts:</p><p> <list list-type="bullet"><list-item><p>First part (13 questions) pertained to socio-demographic data</p></list-item><list-item><p>Second part (9 questions) dealt with self-reported oral hygiene practices of children</p></list-item><list-item><p>Third part (4 questions) was concerned with food habits</p></list-item><list-item><p>Fourth part (3 questions) was concerned with their use of mouthwashes, medication, and presence of any systemic, chronic diseases</p></list-item><list-item><p>Fifth and last part of this form was designed to record DMFT, plaque and gingival indices.</p></list-item></list> </p><p>Inclusion and exclusion criteria used were as follows.</p><sec id="sec2-1"><title>Inclusion criteria</title><p> <list list-type="bullet"><list-item><p>Free from systemic diseases</p></list-item><list-item><p>Gingival scores were moderate and plaque scores were fair according to the plaque and gingival indices proposed by Silness and Loe[<xref rid="ref19" ref-type="bibr">19</xref>] and Loe and Silness,[<xref rid="ref20" ref-type="bibr">20</xref>] respectively</p></list-item><list-item><p>DMFT scores between 3 and 6</p></list-item><list-item><p>Should not have used mouthwashes for the last 1 month</p></list-item><list-item><p>All the index teeth should be completely erupted</p></list-item><list-item><p>Parents should give informed consent.</p></list-item></list> </p></sec><sec id="sec2-2"><title>Exclusion criteria</title><p> <list list-type="bullet"><list-item><p>Suffering from diseases which might affect the salivary flow</p></list-item><list-item><p>History of antibiotic therapy in the previous 1 month till the start of the study</p></list-item><list-item><p>Retained deciduous teeth</p></list-item><list-item><p>Currently using any mouthwashes or has used mouthwash in the past 1 month</p></list-item><list-item><p>Suffering from any physical disability.</p></list-item></list> </p><p>One hundred and five subjects were randomly selected from the eligible population (who fulfilled the inclusion criteria) and randomized into three groups, namely mango, neem, and chlorhexidine mouthwash groups, having 35 participants each. Randomization was done using lottery method by a person not involved with the study proceedings. All the parameters (including food habits, oral hygiene habits, etc.) were assessed statistically. No statistically significant difference was found among the three groups.</p><p>All the children were administered mango, neem, and chlorhexidine mouthwashes (0.2%) according to the group they were assigned to, twice daily for 21 days. Measured amount (10 ml) of mouthwashes was poured in plastic cups and given to children. All the children were asked to take mouthwash into their mouths and start swishing the mouthwash upon a prompt from the investigator who stood with a stop watch to record the time. After swishing the mouthwash for 30 sec as recorded in the stop watch, children were asked to spit in the nearby wash basin. They were also asked not to eat or drink anything for 30 min. Children were instructed to use 10 ml of mouthwash as prescribed, under parents&#x02019; supervision at night time after dinner. Every participant was provided with 75 ml of their respective mouthwashes for home use on a weekly basis. Before start of the intervention, all the children were instructed to rinse with the mouthwashes given to them in the night, before going to bed, and not to eat or drink anything for atleast half an hour after rinsing. Positive reinforcements were given from time to time. No oral prophylaxis was done prior to commencement of intervention. Children were allowed to follow their individual oral hygiene procedures.</p><p>After 21 days of mouthwash administration, plaque index and gingival index were reassessed. Parameters were reassessed at monthly intervals for 3 months, i.e. at 1 month, 2 months, and 3 months after discontinuing the mouthwash. Children were blinded as to which mouthwash they were receiving. Second examiner was blinded to the group allocation, meaning he did not know which child was assigned to which mouthwash group. The statistician remained blinded regarding the subject allocation to the three groups.</p></sec><sec id="sec2-3"><title>Preparation of mouthwash</title><sec id="sec3-1"><title>Procurement and drying of the tree sticks</title><p>Mango and neem trees are available in abundance in India. The small branches of these trees were freshly cut. Leaves from the concerned branches procured were removed and immediately the branches measuring 4 inches in length were cut. These cut sticks were cleaned thoroughly in a disinfecting solution (2% povidone iodine) and washed in running water for about 10-15 min to remove all traces of dirt and extraneous contaminating material. Final wash was done with distilled water. These branches were dried in direct sunlight keeping each variety on separate sheets of double filter paper. Sticks were covered with filter paper during drying to prevent contamination. Drying was carried out till the sticks were completely dehydrated and became easily breakable. Both types of twigs were stored in separate containers and labeling was done to avoid mixing with other branches during the following procedures.</p></sec><sec id="sec3-2"><title>Preparation of neem and mango sticks powder</title><p>The extracts of the above chewing sticks were prepared for each one separately, starting with the mango sticks. The dried sticks were cut into smaller pieces using a twig cutter and pulverized to fine powder using a Kenstar^&#x000ae; high-speed electric grinder for 15 min. The powder was transferred to separate sterile, airtight plastic containers with lids and each container was labeled with the name of the respective plant. Similar procedure was adopted for the neem sticks. After preparation of powder of each variety, the electric mixer was thoroughly cleaned with distilled water and dried thoroughly before commencing the preparation of the powder of the other variety of chewing sticks. Finally, two containers of the powders of different chewing sticks were obtained which were labeled and kept in cool, dry conditions till further use.</p></sec><sec id="sec3-3"><title>Preparation of mouthwash solution</title><p>Cold maceration technique was employed. The obtained powders of mango and neem were weighed individually into 50 g using Digiweigh^&#x000ae; electronic weighing machine and put into separate sterile containers to which sterile deionized distilled water was added using a measuring jar to make the final volume of 100 ml. The container was then shaken well manually for 5 min to mix the powder well with water before keeping it in the refrigerator at 4&#x000b0;C. The mixture was allowed to soak for 48 h at 4&#x000b0;C in the refrigerator. After 48 h, the mixture was filtered using a filter paper. Sweetening agent (30% sucralose, code E955) and preservative (0.05% sodium benzoate, code 211 and 0.01% sodium methyl paraben, code 218) were added to obtain the final mouthwash.</p></sec></sec><sec id="sec2-4"><title>Statistical analysis</title><p>Data were entered in Microsoft Excel and analyzed using SPSS for Windows, Version 17 (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to calculate frequencies, percentages, and mean values. Analysis of variance (ANOVA) was applied to know whether the differences in the plaque index and gingival index of the three groups being compared were statistically significant or not. Tukey's <italic>post hoc</italic> test was used to know the difference between the pairs of mouthwashes. Student's paired <italic>t</italic>-test was applied to know whether the differences in the plaque index and gingival index of the three groups before and after intervention were statistically significant or not. A <italic>P</italic> value of less than 0.05 was taken as statistically significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>At the onset of the study, there were 105 participants (35 in each group). Final assessment was made on 97 participants as 8 participants dropped out (3 in mango group, 2 in neem group, and 3 in chlorhexidine group). Taste was the main governing factor for children dropping out of herbal mouthwashes group as neem and mango are known for their bitter and astringent taste, respectively (mango = 2, neem = 2). Two participants were excluded from the study on 12^th and 16^th day, respectively, as they acquired throat infection and were put on antibiotics by their physicians (chlorhexidine = 1, mango = 1). One participant dropped out because he had to take a leave of absence from the school (chlorhexidine = 1). One participant was excluded as he revealed that he was not able to comply with home rinsing regimen for all the days.</p><p>There was no statistically significant difference among the three mouthwash groups at baseline with respect to age, sex, plaque and gingival indices [Tables <xref ref-type="table" rid="T1">1</xref> and <xref ref-type="table" rid="T2">2</xref>]. Statistically significant reduction (<italic>P</italic> &#x0003c; 0.001) in plaque index was found in all the three mouthwash groups at 21 days and at 1 month after discontinuing the mouthwash [<xref ref-type="table" rid="T3">Table 3</xref>]. Chlorhexidine additionally showed statistically significant reduction in plaque index (<italic>P</italic> = 0.001) at 2 months of discontinuing the mouthwash. Statistically significant reduction (<italic>P</italic> &#x0003c; 0.001) in gingival index was found in all the three mouthwash groups at 21 days and at 1 and 2 months after discontinuing the mouthwash [<xref ref-type="table" rid="T4">Table 4</xref>]. Chlorhexidine sustained statistically significant reduction in gingival index (<italic>P</italic> = 0.015) at 3 months of discontinuing the mouthwash [<xref ref-type="table" rid="T4">Table 4</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Age and sex distribution in the three mouthwash groups</p></caption><graphic xlink:href="JTCM-4-272-g001"/></table-wrap><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Mean gingival and plaque indices at baseline</p></caption><graphic xlink:href="JTCM-4-272-g002"/></table-wrap><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Comparison of plaque scores at baseline with plaque scores at 21 days and at 1 month, 2 months, and 3 months after discontinuing the mouthwash in different mouthwash groups</p></caption><graphic xlink:href="JTCM-4-272-g003"/></table-wrap><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Comparison of gingival scores at baseline with gingival scores at 21 days and at 1 month, 2 months, and 3 months after discontinuing the mouthwash in different mouthwash groups</p></caption><graphic xlink:href="JTCM-4-272-g004"/></table-wrap><p>There was a statistically significant difference between the plaque scores of mango and neem mouthwashes at 21 days (<italic>P</italic> = 0.036), and at 1 month (<italic>P</italic> &#x02264; 0.001) and 2 months (<italic>P</italic> = 0.027). Mango and chlorhexidine also differed significantly at 1 month (<italic>P</italic> = 0.36) and 2 months (<italic>P</italic> = 0.01) [<xref ref-type="table" rid="T5">Table 5</xref>]. There was a statistically significant difference between the gingival scores of mango and neem mouthwashes at 21 days (<italic>P</italic> &#x0003c; 0.001) and at 1 month (<italic>P</italic> &#x02264; 0.001). Mango and chlorhexidine also differed significantly at 21 days (<italic>P</italic> &#x0003c; 0.001), and at 1 month (<italic>P</italic> = 0.001) and 2 months (<italic>P</italic> = 0.001). Gingival scores of neem and chlorhexidine differed significantly at 2 months (<italic>P</italic> = 0.001) [<xref ref-type="table" rid="T6">Table 6</xref>].</p><table-wrap id="T5" position="float"><label>Table 5</label><caption><p>Comparison of mean plaque scores of different mouthwash groups at baseline, 21 days, and at 1 month, 2 months, and 3 months after discontinuing the mouthwash</p></caption><graphic xlink:href="JTCM-4-272-g005"/></table-wrap><table-wrap id="T6" position="float"><label>Table 6</label><caption><p>Comparison of mean gingival scores of different mouthwash groups at baseline, 21 days, and at 1 month, 2 months, and 3 months after discontinuing the mouthwash</p></caption><graphic xlink:href="JTCM-4-272-g006"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Various studies have shown a high caries and periodontal disease prevalence in India.[<xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref><xref rid="ref26" ref-type="bibr">26</xref><xref rid="ref27" ref-type="bibr">27</xref><xref rid="ref28" ref-type="bibr">28</xref>] This high prevalence of oral diseases may be ascribed to the fact that majority of population gives a low priority to oral health, and basic oral health education and simple interventions are not available, especially to rural and underprivileged strata of the society.[<xref rid="ref29" ref-type="bibr">29</xref>]</p><p>Traditionally, in all parts of the world, mechanical removal of plaque is the most common method for preventing orodental diseases. But at the same time, evidence suggests that mechanical cleaning methods are inadequate.[<xref rid="ref30" ref-type="bibr">30</xref><xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref><xref rid="ref33" ref-type="bibr">33</xref>] Chemical antiplaque agents are a newer concept, but it is gradually taking roots. To large percentages of even the most affluent and developed societies, wholesale use of more expensive chemical antiplaque formulations would be quite restrictive due to high expense or ignorance.[<xref rid="ref34" ref-type="bibr">34</xref>] The World Health Organization estimates that 65-80% of the world's population uses traditional medicine as the primary form of health care.[<xref rid="ref35" ref-type="bibr">35</xref>] This study was an attempt to investigate if indigenously prepared mouthwashes from neem and mango chewing sticks can effectively reduce plaque and gingival scores in the selected population.</p><p>The age group selected to carry out this study was 12-15 years. Children in this age group are vulnerable to both caries and gingival and periodontal problems due to the changes in dietary habits and lifestyle.[<xref rid="ref36" ref-type="bibr">36</xref>] Herbal mouthwashes were prepared based on the findings of an <italic>in vitro</italic> study conducted by Prashant <italic>et al</italic>.[<xref rid="ref18" ref-type="bibr">18</xref>] In this study, 50% extracts of mango and neem chewing sticks were found to be most effective in inhibiting <italic>Streptococcus mutans</italic>. Identical extract was made to be used as a mouthwash in the present study. Artificial sweetening agent was added to make the taste pleasant. No flavoring agent was added as most of the flavoring agents like thymol, menthol, etc., are essential oils which might act as confounders in plaque and gingival assessment, as the essential oils are excellent plaque inhibitors and just as popular as chlorhexidine.[<xref rid="ref37" ref-type="bibr">37</xref>] Sodium benzoate is the sodium salt of benzoic acid. It has long been used as a preservative in foods and other products, and its safety has been established. In the US, sodium benzoate is used at a concentration of 0.03-0.1%.[<xref rid="ref38" ref-type="bibr">38</xref>] Methyl paraben is a methyl ester of p-hydroxybenzoic acid. It is a stable, non-volatile compound that has been used as an antimicrobial preservative in foods, drugs, and cosmetics for over 50 years.[<xref rid="ref39" ref-type="bibr">39</xref>] Both the preservatives were used at a much lower concentration than what was found to inhibit oral bacteria.[<xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref><xref rid="ref42" ref-type="bibr">42</xref><xref rid="ref43" ref-type="bibr">43</xref>] Literature suggests that both neem and mango stick extracts have slightly acidic pH, but the pH is lesser than the tooth critical pH.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref17" ref-type="bibr">17</xref>] The preservative methyl paraben is effective in a wide range of pH (4-8), while sodium benzoate is most effective at pH 4-4.5.[<xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref42" ref-type="bibr">42</xref>]</p><p>A study done by Siswomihardjo <italic>et al</italic>., showed that neem stick extract had higher antibacterial properties than the leaves extract.[<xref rid="ref44" ref-type="bibr">44</xref>] Hence, chewing sticks were preferred to prepare the mouthwash over leaves.</p><p>Chlorhexidine mouthwash was employed as control mouthwash in the present study. Children were made to rinse with 10 ml of mouthwashes for 21 days twice daily &#x02013; once in the morning in school and then again in the night at home after dinner. The time interval between these two rinsings was roughly 12 h. This time table conformed well to the standardized regimen of chlorhexidine mouthwash which has to be used at 12-hourly intervals as it has been shown to suppress salivary bacterial counts for over 12 h.[<xref rid="ref45" ref-type="bibr">45</xref>]</p><sec id="sec2-5"><title>Plaque scores</title><p>After rinsing with the respective mouthwashes, statistically significant differences were found between neem, chlorhexidine, and mango at 21 days, 1 month, and 2 months intervals. Neem and chlorhexidine showed no difference in the mean plaque scores at these time intervals. This implies that neem was equally effective in inhibiting plaque as chlorhexidine. Similar results were obtained by Botelho <italic>et al</italic>., in which mouthwash prepared from neem leaves demonstrated similar efficacy to that of chlorhexidine mouthwash.[<xref rid="ref46" ref-type="bibr">46</xref>] In contrast to our findings, neem extract gel was found to be more effective than chlorhexidine mouthwash in a study done by Pai <italic>et al</italic>.[<xref rid="ref7" ref-type="bibr">7</xref>] In another study conducted by Patel <italic>et al</italic>.,[<xref rid="ref47" ref-type="bibr">47</xref>] neem showed better efficacy in reducing human plaque culture and gram-negative bacteria, compared to commercially available toothpaste. In another study conducted by Sharma <italic>et al</italic>.,[<xref rid="ref48" ref-type="bibr">48</xref>] neem mouthwash reduced plaque and gingival indices, but was not as effective as chlorhexidine.</p><p>When compared with baseline, all the three mouthwashes showed statistically significant decrease in the plaque indices at 21 days and at 1 month after stopping the mouthwash. Only chlorhexidine sustained substantial plaque inhibiting effect till 2 months when compared to the herbal mouthwashes. Plaque indices returned to baseline levels at 3 months evaluation in all the mouthwashes. Neem mouthwash was shown to be effective in reducing plaque indices in the studies conducted by Botelho <italic>et al</italic>. and Sharma <italic>et al</italic>.[<xref rid="ref46" ref-type="bibr">46</xref><xref rid="ref48" ref-type="bibr">48</xref>] Neem gel was effective in reducing plaque scores in 3 and 6 weeks of evaluation in a study conducted by Pai <italic>et al</italic>.[<xref rid="ref7" ref-type="bibr">7</xref>] Prashant <italic>et al</italic>., Wolinsky <italic>et al</italic>., Siswomihardjo <italic>et al</italic>., Bhuiyan <italic>et al</italic>., Almas <italic>et al</italic>., and Subramaniam <italic>et al</italic>. have carried out <italic>in vitro</italic> studies which showed the effectiveness of neem extract against plaque-forming bacteria.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref49" ref-type="bibr">49</xref>]</p></sec><sec id="sec2-6"><title>Gingival scores</title><p>After using the mouthwashes, significant differences in gingival indices were found at 21 days and till 2 months from stopping the mouthwash. Neem and chlorhexidine mouthwashes had equivalent effect on gingival scores at 21 days and at 1 month evaluation, subsequent to which neem showed significantly higher gingival scores at 2 months evaluation when compared to chlorhexidine. Botelho <italic>et al</italic>., conducted a study in which neem and chlorhexidine mouthwashes showed similar improvements in gingival indices after 7 days and 1 month from stopping the mouthwashes.[<xref rid="ref46" ref-type="bibr">46</xref>] In contrast to our findings, Sharma <italic>et al</italic>., found that neem was not as effective as chlorhexidine in reducing gingival indices.[<xref rid="ref48" ref-type="bibr">48</xref>] Another study conducted by Bhat <italic>et al</italic>., found that toothpaste containing neem extract reduced plaque and gingivitis significantly at the end of the 3-month study period.[<xref rid="ref50" ref-type="bibr">50</xref>] No human <italic>in vivo</italic> study examined the effect of mango extract on gingivitis. Carvalho <italic>et al</italic>. found that mangiferin derived from mango prevents periodontitis in Wistar rats.[<xref rid="ref14" ref-type="bibr">14</xref>]</p></sec><sec id="sec2-7"><title>Limitations</title><p>Present study was a short-term study employing a crude extract of neem and mango twigs as mouthrinse. Though significant results were obtained at 21 days and 1 month in the herbal groups, long-term clinical efficacy (6 months as prescribed by American Dental Association) and adverse effects associated with long-term usage could not be assessed.[<xref rid="ref51" ref-type="bibr">51</xref>]</p></sec></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>This study provided sufficient data to suggest that neem and mango extract mouthwashes have a beneficial effect on oral health. Plaque and gingival scores were reduced in both the experimental mouthwash groups to such an extent that it warrants an in-depth evaluation of both mango and neem extracts through long-term studies.</p></sec>

Anticancer Studies of Aqueous Extract of Roots and Leaves of <italic>Pandanus Odoratissimus</italic> f. <italic>ferreus</italic> (Y. Kimura) Hatus: An <italic>In Vitro</italic> Approach

<p>A number of medicinal plant extracts are being used against various diseases in different systems of medicine such as Ayurveda, Unani, and Siddha, but only a few of them have been scientifically explored. The objective of the present study was to explore the dose-dependent <italic>in vitro</italic> anticancer effects of the extracts of <italic>Pandanus odoratissimus</italic> whose scientific documentation as an anticancer agent is lacking despite being used traditionally. The dried parts of roots and leaves were extracted with methanol (MEPO) and water (AEPO). The extracts were then subjected to <italic>in vitro</italic> cytotoxic and antimitotic screening by brine shrimp lethality assay and onion root tip method, respectively. Further, the behavior of the extracts on calu-6 (non-small cell lung cancer cell lines), PBMC (peripheral blood mononuclear cells) and WI (lung fibroblast cell lines) was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay followed by flow cytometric analysis on calu-6 cell lines. AEPO showed significant cytotoxic and antimitotic activities. It showed 100% lethality of brine shrimps at 80 &#x003bc;g/ml and an LC₅₀ of 33.33 &#x003bc;g/ml, which was eightfold higher than that of synthetic standard podophyllotoxin (4.16 &#x003bc;g/ml). AEPO at 10 mg/ml concentration showed significant antimitotic activity by showing 3% mitotic index. which was more than that of standard cyclophosphamide with 4% mitotic index in comparison to control. There was a significant reduction in cell proliferation of calu-6 cells, ranging from 56 to 35%, after 24-48 h of treatment with 200 &#x003bc;g/ml (<italic>P</italic> &#x0003c; 0.001) of AEPO, while AEPO remained unaffected on PBMC and WI-38 cel lines. Cell cycle analysis revealed that AEPO at 50 &#x003bc;g/ml and 100 &#x003bc;g/ml significantly increased the number of cells in sub G0&#x02013;G1 phase, indicating the cells entering in to apoptotic phase. These results suggest that aqueous extract of <italic>P. odoratissimus</italic> possesses better anticancer activity. The plant has the potential to be used in anticancer therapy, and this study scientifically validated the folklore use of this plant.</p>

<contrib contrib-type="author"><name><surname>Raj</surname><given-names>Gunti Gowtham</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Varghese</surname><given-names>Hyma Sara</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kotagiri</surname><given-names>Sarita</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Vrushabendra Swamy</surname><given-names>B. M.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Swamy</surname><given-names>Archana</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Pathan</surname><given-names>Rafi Khan</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>There is a growing interest in the pharmacological evaluation of various plants used in Indian traditional systems of medicine. <italic>Pandanus odoratissimus</italic> f. <italic>ferreus</italic> (Pandanaceae) is one such plant distributed commonly throughout India. In Ayurveda, Unani, and Siddha systems of medicine, the leaves are used for treating backache, rheumatic diseases, epilepsy, wound healing, nervous disorders, loss of appetite, indigestion, constipation, diabetes, infertility, skin diseases, urinary disorders, and fever.[<xref rid="ref1" ref-type="bibr">1</xref>] The plant is known to possess a broad spectrum of medicinal, pharmacologic, and therapeutic properties. Tribals believe that this herb is an effective remedy for a wide range of illnesses.[<xref rid="ref2" ref-type="bibr">2</xref>] Leaves of <italic>Pandanus</italic> plants contain alkaloids such as pandanamine and pandamerilactones with pyrroline-derived structures as the major chemical constituent, and were found to possess antioxidant, anti-inflammatory, and antidiabetic activities.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] Further, the ethanolic extract from <italic>Pandanus amaryllifolius</italic> showed selective cytotoxicity against different human colon, cervical, hepatocellular, and breast cancer cell lines.[<xref rid="ref6" ref-type="bibr">6</xref>] In Ayurveda, a paste of <italic>P. odoratissimus</italic> with sugar is used for treating cancers.[<xref rid="ref7" ref-type="bibr">7</xref>] The pharmacognostic and phytochemical investigation was carried out in the leaves of <italic>P. odoratis simus</italic>.[<xref rid="ref8" ref-type="bibr">8</xref>] The phytochemical constituents present in the <italic>P. odoratissimus</italic> methanol extract were reported to be steroids, saponins, terpenoids, glycosides, tannins, and flavonoids.[<xref rid="ref9" ref-type="bibr">9</xref>] Active principles of the extract of whole plant are 3-(4-(dimethylamino) cinnamoyyl)-4-hydroxycomarin, 3,3&#x02032;-methylenebis (4-hydroxycomarin), erythro-9,10-dihydroxyoctadecanoic acid, octadecanedioic acid, and dihydroagathic acid.[<xref rid="ref10" ref-type="bibr">10</xref>] Scientific evidence in support of the antitumor activity of <italic>P. odoratissimus</italic> f. <italic>ferreus</italic> is lacking despite its use as a potential antitumor agent in traditional system. Hence, it was decided to illustrate the ethnobotanical uses of the plant, and this study aimed to evaluate the dose-dependent cytotoxic, antimitotic, antiproliferative, and apoptotic effects of the methanol and aqueous extracts of <italic>P. odoratissimus</italic> f. <italic>ferreus</italic> (MEPO and AEPO, respectively).</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Chemicals</title><p>Cyclophosphamide, podophyllotoxin, doxorubicin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reagent, Ficoll, ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), propidium iodide (PI), fetal bovine serum (FBS) Media, Dulbecco's modified Eagle's medium (DMEM), and trypsin were obtained from Sigma Aldrich (Bangalore, India) and Himedia Ltd (Mumbai, (India)). All other chemicals and solvents were obtained from Biochem Pharmaceuticals (Ahmedabad, India) and SD Fine Chemicals (Mumbai, India) and were of analytical grade with highest purity.</p></sec><sec id="sec2-2"><title>Collection, authentication, and extract preparation of <italic>P. odoratissimus</italic> f. <italic>ferreus</italic> (Y. Kimura) Hatus</title><p>The plant <italic>P. odoratissimus</italic> f. <italic>ferreus</italic> used for the present study was collected from the forest near Punalur at Kollam district, Kerala during midwinter season of 2012. The plant was identified, confirmed, and authenticated by Dr. M. D. Rajanna, Professor and Head, Department of Botany (No. 3/proj/B-Garden), University of Agricultural Sciences, GKVK, Bangalore, Karnataka, India. A voucher specimen was deposited in the department for future reference.</p><p>The roots and leaves of the plant were shade dried, chopped into small pieces, and powdered by a mechanical mixer. Five hundred grams of the coarse material was extracted with two different solvents, i.e., methanol (2.5 L) and distilled water (2.5 L), separately using Soxhlet extraction apparatus. The solvents were evaporated using a rotary vacuum evaporator (YamatoRE 300, Japan) at 50&#x000b0;C and dried in desiccators.[<xref rid="ref11" ref-type="bibr">11</xref>]</p></sec><sec id="sec2-3"><title>Phytochemical analysis</title><p>The qualitative and the quantitative analyses of the plant's constituents were carried out by the methods described by Trease and Evans.[<xref rid="ref12" ref-type="bibr">12</xref>]</p></sec><sec id="sec2-4"><title>Cytotoxicity assay</title><p>Brine shrimp lethality bioassay was performed as a cytotoxicity assay using the method of Meyer <italic>et al</italic>.[<xref rid="ref13" ref-type="bibr">13</xref>] In this bioassay, the eggs of the brine shrimps, <italic>Artemia salina</italic> Leach, were collected and placed to hatch in a hatching chamber. After 48 h, the larvae (nauplli) from the hatched eggs were observed, collected, and transferred into test tu bes of 10 ml capacity each, containing different concentrations of the extracts. After 24 h of drug exposure, the numbers of dead shrimps were counted. Podophyllotoxin was used as reference standard.</p></sec><sec id="sec2-5"><title>Antimitotic assay</title><p>Antimitotic activity was performed by onion root tip method as described previously by Aprem <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] The root tips of <italic>Allium cepa</italic> are generally used for studying the actions of various compounds on cell division or on chromosomes. Onions of good quality were rooted in water, and the roots were treated with different concentrations of the extracts and standard drug cyclophosphamide for 24 h. The root tips of around 2&#x02013;3 mm were cut and fixed in acetic acid: Alcohol (1:3) and stained with a mixture of acetocarmine and 1 N HCl (9:1). Further, the root tips were squashed and observed under a microscope (45&#x000d7;) to calculate the mitotic index. Mitotic index was calculated using the following formula:</p><p>Mitotic index = number of dividing cells/number of non-dividing cells.</p></sec><sec id="sec2-6"><title>Antiproliferation assay</title><sec id="sec3-1"><title>Tumor cell lines and their maintenance</title><p>The non-small cell lung cancer lines (calu-6) and normal lung fibroblast cell lines (WI-38) were obtained from National Centre for Cell Science (NCCS), Pune, and were processed and maintained in the Cellular and Molecular Biology Section of Department of Biotechnology, Gautham College of Pharmacy, Bangalore. The cell lines were grown and maintained in a humidified incubator at 37&#x000b0;C in an atmosphere of 5% CO₂. DMEM supplemented with 10% FBS, penicillin, and streptomycin was used as the culture medium for these adherent cells. The medium was changed every 2 or 3 days during the experimental period.</p></sec><sec id="sec3-2"><title>Isolation of peripheral blood mononuclear cells</title><p>Isolation and preservation of PBMCs were carried according to the procedure described by Mallone <italic>et al</italic>.[<xref rid="ref15" ref-type="bibr">15</xref>] The isolated and maintained PBMCs were subsequently used for testing the AEPO and MEPO by MTT assay.</p></sec><sec id="sec3-3"><title>MTT assay</title><p>Antiproliferation assay was carried out by the modified method of Lau.[<xref rid="ref16" ref-type="bibr">16</xref>] Cell proliferation was determined using MTT assay, which reflects the normal function of mitochondria and cell viability. Briefly, calu-6, PBMC, and WI-38 cells were seeded onto flat-bottomed 96-well culture plate at a density of 5 &#x000d7; 10⁴ cells/well in DMEM. After 24 h, the cells were washed and placed in a culture medium with different concentrations of MEPO and AEPO, ranging from 25 to 200 &#x003bc;g/ml, for 48 h and 72 h. Next, non-FBS culture medium containing 10% MTT was added to each well of a microtitre plate and the samples were then incubated for 4 h at 37&#x000b0;C. After removing the culture medium, DMSO was added to each well. The absorbance was then read at 570 nm using ELISA reader. The absorbance of control cells (treated with DMSO) was considered as 100%. The percentage cell viability was calculated using the following formula:</p><p>Mean value of [(OD in control group &#x02013; OD in treated group)/OD in control group] &#x000d7;100%.</p></sec></sec><sec id="sec2-7"><title>Apoptotic assay</title><p>A modified method described by Lee and Zhou[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] was used for the evaluation of apoptotic activity. The calu-6 cells were adjusted to a density of 5 &#x000d7; 10⁵ cells/ml using DMEM and added to six-well plate and incubated at 37&#x000b0;C for 24 h. The cells were rinsed with phosphate-buffered saline (PBS) and cultured in the medium along with MEPO and AEPO in a concentration ranging from 25 to 100 &#x003bc;g/ml. After 24 h, the cells were collected by routine enzyme digestion and rinsed with PBS (pH 7.4) twice. The fixed cells were suspended in 70% ethanol at 4&#x000b0;C overnight, centrifuged (1500 rpm, 5 min), and washed with PBS twice. Cells were placed in the dark with PI and RNase A in PBS at 25&#x000b0;C for 30 min. Stained cells were analyzed by flow cytometry at 488 nm laser and 15 mW work rate. The percentage of apoptotic cells was determined using CellQuest and the cell cycle was analyzed by MultiCycle software program. Doxorubicin active against non-small cell lung cancer was used as the positive control.</p></sec><sec id="sec2-8"><title>Statistical analysis</title><p>The values were expressed as Mean &#x000b1; standard error of mean (SEM). The data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett's test using GraphPad Prism software. <italic>P</italic> &#x0003c; 0.05 were considered as statistically significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The results of phytochemical analysis showed the presence of alkaloids (1.2%), flavonoids (4.6%), glycosides (2.6%), and phenolic content (3.1%) in AEPO and alkaloids (1.7%), flavonoids (1.3%), and carbohydrates (2.8%) in MEPO.</p><sec id="sec2-9"><title>Cytotoxicity assay</title><p>The results of cytotoxicity assay, when carried out in triplicates, revealed that AEPO and MEPO caused an increase in percentage death of shrimps in a dose-dependent manner. The LC₅₀ of AEPO was found to be 33.33 &#x003bc;g/ml, which was eightfold higher than that of synthetic standard podophyllotoxin, i.e., 4.16 &#x003bc;g/ml, while MEPO showed lesser response (95.23 &#x003bc;g/ml) in comparison to AEPO [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>The LC50 of AEPO was found to be 33.33 &#x003bc;g/ml, which was eightfold higher than that of synthetic standard podophyllotoxin (4.16 &#x003bc;g/ml). Increase in percentage lethality of shrimps in a dose-dependent manner reveals that AEPO was cytotoxic to shrimps. The numbers on line tips in the figure indicate different concentrations and the scale on the lines indicates percentage death of shrimps</p></caption><graphic xlink:href="JTCM-4-279-g001"/></fig></sec><sec id="sec2-10"><title>Antimitotic activity</title><p>AEPO showed significant reduction in the mitotic index of squashed onion root tips with increase in concentration [<xref ref-type="table" rid="T1">Table 1</xref>]. Antimitotic activity was found to be significant at 10 mg/ml of AEPO (0.03 &#x000b1; 0.02; <italic>P</italic> &#x0003c; 0.05) when compared with the control mitotic index (0.43 &#x000b1; 0.02), and was also more than that of standard cyclophosphamide (0.04 &#x000b1; 0.01; <italic>P</italic> &#x0003c; 0.05).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Effect of cyclophosphamide, AEPO, and MEPO on the mitotic index of onion root tips</p></caption><graphic xlink:href="JTCM-4-279-g002"/></table-wrap></sec><sec id="sec2-11"><title>Antiproliferation assay</title><p>Antiproliferative effect of AEPO on calu-6 revealed significant reduction in cell proliferation at concentrations of 25, 50, 100, and 200 &#x003bc;g/ml (<italic>P</italic> &#x0003c; 0.001) after 24 and 48 h of treatment. AEPO inhibited the proliferation of calu-6 cell lines up to 44% after 24 h of treatment and up to 65% after 48 h of treatment at the highest concentration of 200 &#x003bc;g/ml [<xref ref-type="fig" rid="F2">Figure 2</xref>]. IC₅₀ of AEPO was found to be 153.84 &#x003bc;g/ml, while that of MEPO was found to be &#x0003e;200 &#x003bc;g/ml. Data obtained from MTT assay clearly indicate that AEPO was cytotoxic to human lung cancer cells in a dose- and time-dependent manner, and increasing the incubation time from 24 to 48 h resulted in increased toxicity. The growth rate of WI-38 cell lines was relatively high, indicating no cytotoxicity to normal lung fibroblast cell lines. Studies with PBMCs isolated from whole blood revealed that the critical components in the immune system, such as T cells, B cells and natural killer (NK) cells of lymphocytes and monocytes, remained unaffected by AEPO and MEPO after 48 h of exposure.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Antiproliferative effect of AEPO on calu-6 reveals significant reduction in cell proliferation at 25, 50, 100, and 200 &#x003bc;g/ml (<italic>P</italic> &#x0003c; 0.001) after 24 and 48 h of treatment, whereas the growth rate of WI-38 cell lines was relatively high, indicating no cytotoxicity to normal lung fibroblast cell lines. Studies on PBMCs isolated from whole blood reveal that the critical components of the immune system, such as T cells, B cells, and natural killer (NK) cells of lymphocytes and monocytes, remained unaffected by AEPO and MEPO after 48 h of exposure. AEPO inhibited the proliferation of calu-6 cell lines up to 65% at the highest concentration of 200 &#x003bc;g/ml. IC50 of AEPO was found to be 153.84 &#x003bc;g/ml, while that of MEPO was found to be &#x0003e;200 &#x003bc;g/ml. Data obtained from MTT assay clearly indicate that AEPO was cytotoxic to human lung cancer cells in a dose-and time-dependent manner. Values are Mean &#x000b1; SEM (of triplicates from two independent experiments) analyzed by one-way ANOVA followed by Dunnett's test</p></caption><graphic xlink:href="JTCM-4-279-g003"/></fig></sec><sec id="sec2-12"><title>Apoptotic activity</title><p>Flow cytometric analysis of DNA cell cycle for calu-6 cell lines treated with AEPO showed sub G₀&#x02013;G₁ phase arrest of calu-6 cell lines and the apoptosis was found to be marginal at 50 &#x003bc;g/ml and wide at 100 &#x003bc;g/ml [Figure <xref ref-type="fig" rid="F3">3f</xref> and <xref ref-type="fig" rid="F3">g</xref>], thereby influencing the cells entering G₁ phase, whereas the methanol extract did not show any changes on the cell cycle. These results clearly indicate that AEPO arrested the growth of calu-6 cells and the effect increased with increase in concentration of the drug. On the other hand, doxorubicin, a chemotherapeutic anthracycline antibiotic active against S phase cells, exerted greater effects on exponentially growing cells than on resting cells of non-small cell lung cancer <italic>in vitro</italic> [<xref ref-type="fig" rid="F3">Figure 3h</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Flow cytometric analysis of the DNA cell cycle for calu-6 cell lines treated with AEPO showed sub G0-G1 phase arrest of calu-6 cell lines, which was found to have marginal apoptosis at 50 &#x003bc;g/ml and a wide range of apoptosis at 100 &#x003bc;g/ml (as shown in Figure 2f and g), thereby influencing the cells entering G1 phase. But the methanol extract did not show any changes in the cell cycle. These results clearly indicate that AEPO arrested calu-6 cells, which might increase its potential to arrest with increase in concentration of the drug. (a) Untreated, (b) 25 &#x003bc;g/ml MEPO, (c) 50 &#x003bc;g/ml MEPO, (d) 100 &#x003bc;g/ml MEPO, (e) 25 &#x003bc;g/ml AEPO, (f) 50 &#x003bc;g/ml AEPO, (g) 100 &#x003bc;g/ml AEPO, (h) 25 &#x003bc;g/ml doxorubicin</p></caption><graphic xlink:href="JTCM-4-279-g004"/></fig></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>The discoveries of plant-based anticancer agents have led to significant interest in evaluating various other natural products for their efficacy. Phytochemicals like flavonoids and polyphenols have been reported to act as anticancer agents by regulation of signal transduction pathways of cell growth and proliferation, suppression of oncogenes and tumor formation, induction of apoptosis, modulation of enzyme activity related to detoxification, oxidation, and reduction, stimulation of the immune system and DNA repair, and regulation of hormone metabolism.[<xref rid="ref19" ref-type="bibr">19</xref>] Polyphenols have a protective role in carcinogenesis, inflammation, atherosclerosis, and thrombosis, and have high antioxidant capacity.[<xref rid="ref20" ref-type="bibr">20</xref>] The data obtained in this study show that AEPO contains rich flavonoids and phenols which might contribute to its anticancer effects. AEPO inhibited proliferation of calu-6 cell lines without affecting normal lung fibroblast cells (WI-38) and PBMCs, proving its cytotoxicity to only human lung cancer cells in a dose- and time-dependent manner. Effect of AEPO and MEPO on PBMCs remained unaffected, indicating their safety on immune cells like T cells, B cells, and NK cells. The cytotoxicity may be due to the release of cytochrome c from mitochondria, leading to apoptosis.[<xref rid="ref21" ref-type="bibr">21</xref>] Control of the cell cycle is accomplished by the coordinated interaction of cyclins with their respective cyclin-dependent kinases (CDKs) to form active complexes and drive cells into the next phase at the appropriate time. Any disorder in the cell cycle may result in genomic instability and apoptosis. Bcl-2 family proteins are important regulators of apoptosis. The family comprises both anti-apoptotic (e.g.,, Bcl-2) and pro-apoptotic (e.g., Bax) proteins with opposing biological functions.[<xref rid="ref22" ref-type="bibr">22</xref>] Apoptosis in cells might occur through complex mechanisms.[<xref rid="ref23" ref-type="bibr">23</xref>] It has been suggested that apoptosis may occur by disruption of mitochondrial function and induces lysosomal damage as the first target which leads to other cellular events including reactive oxygen species (ROS) production andoxidative damage,[<xref rid="ref24" ref-type="bibr">24</xref>] lysosomal damage, lipid peroxidation, DNA strand breaks, gene expression, chromosomal aberrations, inhibition of DNA repair processes.[<xref rid="ref25" ref-type="bibr">25</xref>] Apoptosis of calu-6 cell lines by AEPO might be due to any of the reported reasons. Onion root tip cells are in the active stage of division and can be used to study the effects of various compounds on cell division or chromosomes by the number of dividing and non-dividing cells which gives the mitotic index. Reduction in mitotic index caused by AEPO indicates inhibition of actively dividing cells. Brine shrimp lethality assay is proposed as a simple bioassay for natural product research and this procedure determines the LC₅₀ values of active compounds in brine medium. Effect of AEPO on brine shrimps reveals its cytotoxic potential.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>The above results suggest that the aqueous extract of roots and leaves of <italic>P. odoratissimus</italic> f. <italic>ferreus</italic> possesses cytotoxic, antimitotic, antiproliferative, and apoptotic effects. The plant has the potential to be used in cancer therapy, and this study scientifically validated the traditional use of this plant.</p></sec>

Total Phenol Content and <italic>In Vitro</italic> Antioxidant Potential of <italic>Helicanthus elastica</italic> (Desr.) Danser-A Less-explored Indian Mango Mistletoe

<p>Natural products are an important source of antioxidant molecules like tannins, phenolic compounds, flavonoids, etc., <italic>Helicanthus elastica</italic> (Desr.) Danser (Loranthaceae) is one such plant belonging to the category of mistletoe, and grows commonly on the mango trees in India. In the present study, an attempt has been made to assess the antioxidant properties of the plant. Ethanol extract of H. elastica growing on mango tree was studied using different <italic>in vitro</italic> models. Shade-dried whole plant material was extracted with ethanol by cold percolation. Fifty milligrams of the alcohol extract of <italic>H. elastica</italic> was weighed and dissolved in 10 ml of methanol. The resultant 5 mg/ml solution was suitably diluted to obtain different concentrations. Total phenol content, reducing power assay, and scavenging of free radicals like nitric oxide, hydroxyl, hydrogen peroxide, and 1,1-diphenyl-2-picrylhydrazyl were studied by standardized <italic>in vitro</italic> chemical methods using ascorbic acid as the standard. The total phenol content of the plant was found to be 1.89% w/w. The extract showed good reducing power as well as scavenging of free radicals (nitric oxide, hydroxyl, superoxide anion, and hydrogen peroxide) at concentrations ranging from 5 to 100 &#x003bc;g/ml. The study revealed the antioxidant potential of <italic>H. elastica</italic>.</p>

<contrib contrib-type="author"><name><surname>Sunil Kumar</surname><given-names>Koppala Narayana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Saraswathy</surname><given-names>Ariyamuthu</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Amerjothy</surname><given-names>Swaminathan</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Susan</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Ravishankar</surname><given-names>Basaviah</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Antioxidants can be broadly defined as &#x0201c;any substance, which when present at low concentration compared to that of an oxidizable substrate, significantly prevents or delays any oxidation of that substrate.&#x0201d;[<xref rid="ref1" ref-type="bibr">1</xref>] Oxidizable substrates include almost anything found in foods and living tissues, including proteins, carbohydrates, and DNA. The body has developed several endogenous antioxidant systems to deal with the production of reactive oxygen species (ROS). These systems can be divided into enzymatic and non-enzymatic groups. The enzymatic antioxidants include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). The non-enzymatic antioxidants include the lipid-soluble vitamins [vitamin E and vitamin A or provitamin A (&#x003b2;-carotene), the water-soluble vitamin C, and glutathione. When ROS are generated in living system, a wide variety of antioxidants come into play, such as tocopherol, ascorbic acid, SOD, GSH-Px, catalase, ceruloplasmin, flavonoids, uric acid, and several others. The relative importance and efficacy of these depends on which ROS are involved, how and where they are generated, and which target of damage is selected. Thus, an antioxidant may protect against the free radicals in one system but fails to protect other systems. Natural products are an important source of antioxidant molecules. Search for these molecules from traditional herbs is an ongoing process. <italic>Helicanthus elastica</italic> (Desr.) Danser (Loranthaceae) is one such plant belonging to the category of mistletoe, and grows commonly on the mango trees in India. This hemiparasite was found to be a rich source of phenolic compounds;[<xref rid="ref2" ref-type="bibr">2</xref>] hence, an attempt was made to assess the antioxidant properties of the plant in the present study. The ethanol extract of <italic>H. elastica</italic> growing on mango tree was studied using different <italic>in vitro</italic> models.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Preparation of extract</title><p>Fresh plants of the mistletoe growing on <italic>Mangifera indica</italic> L. were collected during flowering during August 2009 from Kasaragod District of Kerala. Both host and the mistletoe were authenticated and the voucher specimen of the plant (no. 00637) was deposited at the pharmacognosy department of Captain Srinivasa Murti Drug Research Institute for Ayurveda, Chennai. The shade-dried whole plant material including the parasitic roots found on the surface of host was extracted with 90% ethanol by cold percolation. Fifty milligrams of the alcohol extract of <italic>H. elastica</italic> was weighed and dissolved in 10 ml of methanol. The resultant 5 mg/ml solution was suitably diluted to obtain different concentrations (50, 100, 150, 250, 500, 1000, 1500, and 2500 &#x003bc;g/ml) of the plant extracts and used for the following studies. Ascorbic acid (vitamin C) was used as the standard for comparison in the present study.</p></sec><sec id="sec2-2"><title>Total phenol content</title><p>Five grams of the sample was extracted with 50 ml of alcohol. The extract was concentrated and weighed. One milliliter of Folin's reagent was diluted with 1 ml of water. Twenty grams of Na₂CO₃ was dissolved in 100 ml of water at 70-80&#x000b0;C and cooled overnight. The clear liquid was decanted before use. Ten milligrams of tannic acid was dissolved in 100 ml of water afresh. Standard curve was obtained by taking the standard solution at concentrations of 0.2, 0.4, 0.6, 0.8, and 1 ml. After adding the reagents add sample, make up with water, phenols reagent, followed by Na₂CO₃), the mixture was incubated at room temperature for 40 min in dark and the blue color developed was read at 725 nm in a UV spectrophotometer. The phenol content was estimated from the calibration curve of standard tannic acid which was obtained by plotting concentration versus absorbance.[<xref rid="ref3" ref-type="bibr">3</xref>]</p></sec><sec id="sec2-3"><title>Reducing power assay</title><p>About 0.75 ml of different concentrations of the extract were mixed with 0.75 ml of phosphate buffer (0.2 M, pH 6.6) and 0.75 ml of potassium ferricyanide (1% v/v) and the mixture was incubated at 50&#x000b0;C for 20 min. The reaction was stopped by adding 0.75 ml of 10% trichloroacetic acid and centrifuged at 800 rpm speed for 10 min. About 1.5 ml of the supernatant was mixed with 1.5 ml distilled water and 0.1 ml ferric chloride (0.1%). This mixture was incubated at room temperature for 10 min and the absorbance was measured at 700 nm with UV-visible spectrophotometer. Higher absorbance of the reaction mixture indicates the greater reducing power.[<xref rid="ref4" ref-type="bibr">4</xref>]</p></sec><sec id="sec2-4"><title>Free radical scavenging</title><sec id="sec3-1"><title>Nitric oxide scavenging activity</title><p>Two milliliters of 10 mM of sodium nitroprusside in 0.5 ml of phosphate-buffered saline (pH 7.4) was mixed with 0.5 ml of varying concentrations of the extract and the mixtures incubated at 25&#x000b0;C for 2&#x000bd; h. From the incubated mixture, 0.5 ml was taken out and 1 ml of 0.33% sulfanilic acid was added. This was allowed to stand at room temperature for 5 min. Then, 1 ml of 0.1% of naphthyl ethylenediamide dichloride was added, mixed, and incubated at room temperature for 30 min. The absorbance of the mixture was measured at 540 nm with UV-visible spectrophotometer.[<xref rid="ref5" ref-type="bibr">5</xref>]</p></sec><sec id="sec3-2"><title>Hydroxyl radical scavenging activity</title><p>Two series of tubes were taken. In the first set, 60 &#x003bc;l of 1 mM ferrous chloride, 90 &#x003bc;l of 1 mM 1,10-bathophenanthroline, and 2.4 ml of 0.2 M phosphate-buffered saline (pH 7.4) were taken and 150 &#x003bc;l of 0.17 M hydrogen peroxide was added to initiate the reaction. This set was labeled as blank. In the second set, before adding hydrogen peroxide, 1.5 ml of varying concentrations of the extract was added. After incubation at room temperature for 5 min, the absorbance of the mixture was measured at 560 nm with UV-visible spectrophotometer.[<xref rid="ref6" ref-type="bibr">6</xref>]</p></sec><sec id="sec3-3"><title>Superoxide anion radical scavenging activity</title><p>One milliliter of 156 &#x003bc;M nitroblue T and 1 ml of 468 &#x003bc;M of nicotinamide adenine dinucleotide were mixed with 0.5 ml of varying concentrations of the extract. To this mixture was added 100 &#x003bc;l of phenazine methosulfate and the solution was incubated at room temperature for 5 min. The absorbance was measured at 560 nm with UV-visible spectrophotometer. Decreased absorbance of the reaction mixture indicated increased superoxide anion generation.[<xref rid="ref7" ref-type="bibr">7</xref>]</p></sec><sec id="sec3-4"><title>Hydrogen peroxide radical scavenging activity</title><p>One milliliter of varying concentrations of the extract and 1 ml of 0.1 M H₂O₂ were mixed, followed by addition of two drops of 3% ammonium molybdate, 10 ml of 2 M H₂SO₄, and 7 ml of 1.8 M potassium iodide. This reaction mixture was titrated with 5.09 mM of sodium thiosulfate until the disappearance of the yellow color.[<xref rid="ref8" ref-type="bibr">8</xref>]</p></sec><sec id="sec3-5"><title>DPPH scavenging</title><p>1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable free radial with purple color (absorbs at 517 nm). If free radicals have been scavenged, DPPH will generate yellow color. This assay uses this character to show the free radical scavenging activity. The extract was dissolved in methanol at a concentration of 1 mg/ml, which was then used to determine its antioxidant activity. Blank used was methanol, control was methanol + DPPH, and test was methanol + DPPH + sample [100 &#x003bc;l extract (1 mg/ml)].[<xref rid="ref9" ref-type="bibr">9</xref>]</p></sec></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The total phenol content of the plant was found to be 1.89% w/w. The results of antioxidant activity of various concentrations ranging from 50 to 2500 &#x003bc;g/ml of the alcoholic extracts of <italic>H. elastica</italic> showed that the free radical scavenging effect of the tested extract was concentration dependent. The results obtained for the reducing power assay are presented in <xref ref-type="fig" rid="F1">Figure 1</xref>. The alcoholic extract produced marked and concentration-dependent increase in the reducing power [<xref ref-type="fig" rid="F1">Figure 1</xref>]. It was observed that the extract was effective in inhibiting the nitric oxide radical scavenging activity [<xref ref-type="fig" rid="F2">Figure 2</xref>]. The tested extract caused moderate inhibition of NO formation. The observed activity was not concentration dependent-moderate inhibition of NO formation was observed at 25 and 100 &#x003bc;g/ml. The inhibition observed at higher concentration level was less in comparison to the effect observed at lower concentration level. It was observed that <italic>H. elastica</italic> extract was effective in scavenging hydroxyl radical [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The IC₅₀ value was found to be 37.42 &#x003bc;g/ml. The effect was not concentration dependent at the concentration level studied. Moderate inhibition was observed at 25 &#x003bc;g/ml, whereas at higher concentration level (100 &#x003bc;g/ml), the inhibitory effect was almost absent. The alcoholic extract of <italic>H. elastica</italic> produced significant superoxide anion radical scavenging activity. The IC₅₀ value was found to be 218.03 &#x003bc;g/ml. The activity was concentration dependent up to a concentration of 800 &#x003bc;g/ml; above this concentration, the scavenging activity was found to be lesser [<xref ref-type="fig" rid="F4">Figure 4</xref>]. The IC₅₀ value was found to be 17.96 &#x003bc;g/ml. The extract exhibited concentration-dependent inhibition of hydrogen peroxide formation [<xref ref-type="fig" rid="F5">Figure 5</xref>]. The tested extract produced remarkable decrease in the free radical formation at the concentration of 1 mg/ml [<xref ref-type="fig" rid="F6">Figure 6</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Reducing power assay of alcoholic extract of <italic>Helicanthus elastica</italic></p></caption><graphic xlink:href="JTCM-4-285-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Nitric oxide radical scavenging activity of alcoholic extract of <italic>Helicanthus elastica</italic></p></caption><graphic xlink:href="JTCM-4-285-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Hydroxyl radical scavenging activity of alcoholic extract of <italic>Helicanthus elastica</italic></p></caption><graphic xlink:href="JTCM-4-285-g003"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Superoxide anion radical scavenging activity of alcoholic extract of <italic>Helicanthus elastica</italic></p></caption><graphic xlink:href="JTCM-4-285-g004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Hydrogen peroxide scavenging assay of alcoholic extract of <italic>Helicanthus elastica</italic></p></caption><graphic xlink:href="JTCM-4-285-g005"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p>DPPH scavenging assay of alcoholic extract (1 mg/ml) of <italic>Helicanthus elastica</italic> (BHT, butylated hydroxytoluene)</p></caption><graphic xlink:href="JTCM-4-285-g006"/></fig></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Evaluation of putative drugs, especially plant products, for antioxidant activity is considered important. The antioxidant activity is normally initially screened in <italic>in vitro</italic> conditions and is followed by evaluation under <italic>in vivo</italic> conditions. In the present study, the alcoholic extract was subjected to antioxidant property evaluation in a series of test paradigms representing generation of different types of ROS. It is well known that superoxide is generated in cells during oxidative stress, mainly from decomposition of lipid peroxides or by spontaneous dismutation of super oxide.[<xref rid="ref10" ref-type="bibr">10</xref>] Peroxynitrite is another potent oxidant formed by the interaction of superoxide with nitric oxide under various pathophysiological conditions. Nitric oxide is one of the smallest and most diffusible signal molecules known,[<xref rid="ref11" ref-type="bibr">11</xref>] and is also a very active molecule involved in many and diverse biological pathways. Nitric oxide can have both positive and negative effects depending on the concentrations reached in the diseases.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Analysis of the results obtained indicates a remarkable and concentration-dependent reducing activity that is almost equal to that of ascorbic acid at 25 &#x003bc;g/ml. This clearly indicates that the extract contains active principle(s) with good antioxidant potential. This was further confirmed in the DPPH assay. In general, the assay systems measured the scavenging activity against free radicals as a whole, followed by assessing the effect on different individual free radicals. The nitric oxide scavenging activity exhibited by the extract was not concentration dependent. Maximum inhibition of around 35.5% was observed at 100 &#x003bc;g/ml concentration, which is almost half of that shown by ascorbic acid. A moderate inhibition of around 59% was obtained for hydroxyl radical formation (56.06% for ascorbic acid), but the IC₅₀ value was found to be lower at 37.42 &#x003bc;g/ml. With regard to superoxide formation, which along with hydroxyl radical is considered as the most reactive of free radicals, good inhibition (up to 80%) was observed, but with comparatively higher IC₅₀ value of 218.03 &#x003bc;g/ml. Ascorbic acid did not show any inhibition in this system. The extract exhibited concentration-dependent inhibition of hydrogen peroxide formation up to a dose of 5 &#x003bc;g/ml, which was almost equivalent to that of ascorbic acid (61.26%), and at a concentration above this, the inhibitory effect was found to be decreased. The results obtained indicate the presence of inhibitory effect on hydrogen peroxide formation at a comparatively low dose level. The results obtained indicate a good potential of antioxidant activity. However, it remains to be ascertained using <italic>in vivo</italic> systems whether the observed effect would translate into meaningful antioxidant effect in an intact animal.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>The extract showed good reducing power as well as efficient scavenging of free radicals (nitric oxide, hydroxyl, superoxide anion, and hydrogen peroxide) at concentrations ranging from 5 to 100 &#x003bc;g/ml. The study revealed the antioxidant potential of <italic>H. elastica</italic> which is attributed to its high phenol content.</p></sec>

A Validation Study of Homeopathic Prescribing and Patient Care Indicators

<p>A preliminary version of the homeopathic prescribing and patient care indicators was available. The instrument was modified further in this study with an intention to address formally its validity and reliability, audit prescriptions, identify areas of sub-optimal prescribing, and highlight target areas for improving the quality of practices. A cross-sectional study with record analysis was conducted on systematically sampled 377 patients of Mahesh Bhattacharyya Homeopathic Medical College and Hospital (MBHMC and H), Howrah, West Bengal, India. The outcome measures were homeopathic prescribing indicators (6 items) and patient care indicators (5 items). Individualized homeopathic prescriptions predominated in the encounters. Areas demanding immediate attention were extremely poor labeling of drugs dispensed from the hospital pharmacy, improper record of case history and disease diagnosis, ongoing therapies, and investigational findings in the prescriptions. Internal consistency of the overall instrument was estimated to be good (Cronbach's alpha: Prescribing indicators 0.752 and patient care indicators 0.791). The prescribing indicators, except items 1 and 3, reflected acceptable item-corrected total correlations &#x02013; Pearson's r from 0.58 (95% CI: 0.52-0.65) to 0.74 (95% CI: 0.69-0.78). The patient care indicators, except item 2, showed acceptable correlations &#x02013; Pearson's r from 0.40 (95% CI: 0.31-0.48) to 0.82 (95% CI: 0.78-0.85). The instrument also showed high discriminant validity (prescribing indicators <italic>P</italic> &#x0003c; 0.0001 and patient care indicators <italic>P</italic> &#x0003c; 0.0001). Improper prescribing practice was quite rampant and corrective measures are warranted. The developed indicators appeared to be validated and reliable; however, they are amendable for further development.</p>

<contrib contrib-type="author"><name><surname>Koley</surname><given-names>Munmun</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Saha</surname><given-names>Subhranil</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ghosh</surname><given-names>Shubhamoy</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Nag</surname><given-names>Goutam</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kundu</surname><given-names>Monojit</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Mondal</surname><given-names>Ramkumar</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Purkait</surname><given-names>Rajib</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Patra</surname><given-names>Supratim</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>As medical practice has become more complex, the scope of the term &#x0201c;prescription&#x0201d; has been broadened to include clinical outcome assessments, disease diagnosis, and reporting of investigations performed relevant to optimizing the safety or efficacy of medical treatment. [<xref rid="ref1" ref-type="bibr">1</xref>] In a prescription audit study, these parameters may be evaluated for their presence or absence; the number of absent parameters directly correlates to the inconsistencies in the prescriptions and raises medico-legal concern. The indicators may be used to measure the impact of the interventions undertaken and problems in performance. They can help health planners, managers, and researchers to make basic comparisons between healthcare and prescribing practices in different areas or at different time periods. [<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>A preliminary version of the indicator instrument was developed which was pilot-tested and implemented on 600 samples as well. [<xref rid="ref3" ref-type="bibr">3</xref>] The instrument was modified further in this study. This study shall address formally the validity and reliability of this newly developed instrument, audit prescriptions, and intend to identify sub-optimal levels of prescribing and highlight target areas for improving the quality of prescribing and patient care practices.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Setting and design</title><p>A cross-sectional, prospective, institutional, observational, prescription and record analysis study was conducted in January 2014 on 377 patients visiting different outpatient clinics of Mahesh Bhattacharyya Homeopathic Medical College and Hospital (MBHMC and H), Howrah, West Bengal, India.</p></sec><sec id="sec2-2"><title>Participation criteria</title><p>Inclusion criteria were patients 18 years and above, completing their physician's and pharmacist's consultation, giving written informed consent, and being ready to share their prescription information. Exclusion criteria were patients who were too sick for consultation, unable to read patient information sheets, unwilling to stay after the doctor's visit, and not giving consent to join the survey.</p></sec><sec id="sec2-3"><title>Sample size</title><p>The sample size was determined as 377 [margin of error 5%, confidence level 95%, population size 13,500 (monthly average patient turnover of the hospital in 2013), and response distribution estimated to be 50%.] Systematic sampling method was used for recruitment of the patients. Sampling fraction was estimated (and approximated) to be 5/6 (<italic>n</italic>/<italic>N</italic>; <italic>n</italic> = required sample size of 377; <italic>N</italic> = average number of out-patient patients every day, i.e. 450); 5 was decided as the sampling unit by simple random sampling, and thus every 5th patient was interviewed.</p></sec><sec id="sec2-4"><title>Study instrument</title><p>The prescribing indicators consisted of six items &#x02013; a single item (single individualized medicine per encounter) provided with &#x0201c;yes&#x0201d;/&#x0201c;no&#x0201d; options and five items provided with a 5-point agreement Likert scale (strongly agree: 5; agree: 4; uncertain: 3; disagree: 2; strongly disagree: 1; does not apply: 0), which were proper record of case history and disease diagnosis, proper record of patient identification, good legibility of prescription, proper record of ongoing therapy (if any), and proper record of investigations (if any). There were five patient care indicators &#x02013; drugs properly dispensed as per prescription, drugs adequately labeled, patient understands the directions given in prescription and has a knowledge of correct dosage and follow-up, patient understands what to do in adverse events, and patients satisfied with the care they received &#x02013; all ascribed with similar 5-point Likert scale to assess agreement. Agreement ratings were arrived at by a consensus among the six research assistants. Maximum obtainable score for prescribing indicators was either 26 or 16 and that of patient care indicators was 25.</p></sec><sec id="sec2-5"><title>Methodology</title><p>The audit involved documentation of current drug regimens and analysis of case notes. No identifiable information of the patients was required, ensuring anonymized protection of patient's privacy. The modified version of the instrument was pilot-tested on 10 randomly selected patients for length, clarity, language, relevance, overall adequacy, and whether the content reflected what it purports to assess. The instrument appeared to be satisfactory and ready for field-testing.</p><p>The study protocol was approved by the Institutional Ethics Committee of MBHMC and H. Patient information sheets were provided to the participants to achieve full cooperation. Though the survey did not intend to intervene anyway with the treatment being provided by the institutional doctors, written consent was obtained from all the participants. The survey matter was also explained verbally to all the participants by the research assistants. The filled-in questionnaires by the research assistants were concealed by putting those inside opaque envelopes, which were sealed at the survey site. All these were subjected to data analysis.</p></sec><sec id="sec2-6"><title>Statistical analysis</title><p>Different computational websites were used for the purpose.</p><p>Descriptive analysis was presented in the form of absolute values, percentages, and mean values. <italic>P</italic> values less than 0.05 for a two-tailed test were considered as statistically significant. The instrument was tested for item-corrected total correlations (Pearson's r), internal consistency or reliability (Cronbach's alpha coefficient), and discriminant validity [by comparing the mean scores obtained by the different indicators of the instrument using one-way analysis of variance (ANOVA)].</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>Survey participants mostly spanned the age group of 41-55 years (<italic>n</italic> = 132, 35%). Most of the participants were females (<italic>n</italic> = 220, 58.4%), had a level of education of 10^th -12^th standard (<italic>n</italic> = 163, 43.2%), were urban residents (<italic>n</italic> = 278, 73.7%), married (<italic>n</italic> = 231, 61.3%), had a monthly family income of less than 10,000 Indian rupees (INR) (<italic>n</italic> = 234, 62.1%), and were dependent (<italic>n</italic> = 160, 42.4%). Self-reported health status was good in most of the respondents (<italic>n</italic> = 136, 36.1%), and rheumatologic complaints were the most frequently encountered conditions (<italic>n</italic> = 57, 15.1%) [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Socio-demographic characteristics of the survey participants (<italic>N</italic>=377)</p></caption><graphic xlink:href="JTCM-4-289-g001"/></table-wrap><p>Majority of the homeopathic encounters were individualized (97.4%), and record of patients&#x02019; identification in the prescription (83.8%) was quite satisfactory. Legibility of the prescriptions was moderate (57%). Proper records of case history and disease diagnosis (46.7%), ongoing therapies (39%), and laboratory investigational findings (34.9%) in the prescription were not up to the mark, and these are the areas requiring immediate attention and urgent corrective measures. Among the patient care indicators, labeling of drugs was extremely poor (only 2.9%). Other indicators reflected satisfactory patient care (82.8-98.7%) [<xref ref-type="table" rid="T2">Table 2</xref>].</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Results on indicators at a glance (<italic>N</italic>=377)</p></caption><graphic xlink:href="JTCM-4-289-g002"/></table-wrap><p>The face and content validity of the indicators were already established in an earlier study. [<xref rid="ref3" ref-type="bibr">3</xref>] Internal consistency or reliability of the items considered and the overall instrument was estimated to be good (Cronbach's alpha: Prescribing indicators 0.752 and patient care indicators 0.791). The prescribing indicators, except items 1 and 3, reflected acceptable item-corrected total correlations &#x02013; Pearson's r from 0.58 (95% CI: 0.52-0.65) to 0.74 (95% CI: 0.69-0.78). The patient care indicators, except item 2, showed acceptable correlations &#x02013; Pearson's r from 0.40 (95% CI: 0.69-0.78) to 0.82 (95% CI: 0.78-0.85). The instrument also showed high discriminant validity (prescribing indicators: <italic>F</italic> = 280.209, <italic>P</italic> &#x0003c; 0.0001; patient care indicators: <italic>F</italic> = 1825.596, <italic>P</italic> &#x0003c; 0.0001; one-way ANOVA) [Tables <xref ref-type="table" rid="T3">3</xref> and <xref ref-type="table" rid="T4">4</xref>].</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Item-corrected total correlations and internal consistency of the indicators</p></caption><graphic xlink:href="JTCM-4-289-g003"/></table-wrap><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Discriminant validity of the items of the instrument</p></caption><graphic xlink:href="JTCM-4-289-g004"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>This study identified sub-optimal levels of prescribing and highlighted target areas for improving the quality of prescribing and patient care practices. Urgent corrective measures are warranted in areas like proper recording of case history and disease diagnosis, ongoing therapies, and laboratory investigational findings in the prescription, and labeling of drugs dispensed from the pharmacy. In this cross-sectional study, systematic sampling method was adopted to minimize selection bias and increase the generalizability of the findings. Internal consistency of the instrument might further be improved by rephrasing the few items (prescribing indicators 1 and 3 and patient care indicator 2) that had relatively low item-corrected total correlations. Further validation in other samples and more specific statistical (Rasch) analyses are required to confirm whether the sequence of the items requires readjustment in future.</p><p>The prescribing indicators of homeopathy are performances derived from the prescription records and case notes. They are not absolute measures, and so, poor performance, as evaluated, should be an indication for investigation and not automatic castigation. Indicators are not exact measures; there will be some variation for good reasons, reflecting the difficulty of any guideline being relevant to all cases. Additionally, inevitable incorporation of central tendency bias and acquiescence bias arising from the use of Likert scale responses into the analysis could not be eliminated. Furthermore, the study was undertaken in an India-based homeopathic school, making the generalizability of the results unclear. Future similar studies investigating the matter under question are welcome from other homeopathic schools in India and abroad. However, at present, as no standardized and validated measures for homeopathic prescribing exist, we believe that these indicators are the best available tool purporting the purpose.</p><p>In comparison with the previous study, [<xref rid="ref3" ref-type="bibr">3</xref>] individualized homeopathic encounters increased from 85.6 to 97.4% (Yates&#x02019; Chi-square = 33.67; <italic>P</italic> two-tailed &#x0003c; 0.0001), reflecting higher intention to practice the &#x0201c;classic&#x0201d; form of homeopathy. However, all other prescribing indicators reflected a dismal performance &#x02013; record on ongoing therapies decreased from 59.4 to 39% (Yates&#x02019; Chi-square = 15.33; <italic>P</italic> &#x0003c; 0.0001), record of patients&#x02019; identification from 100 to 83.8% (Yates&#x02019; Chi-square = 100.8; <italic>P</italic> &#x0003c; 0.0001), record of investigations decreased from 68.8 to 34.9% (Yates&#x02019; Chi-square = 24.97; <italic>P</italic> &#x0003c; 0.0001), and legibility decreased from 92.2 to 57% (Yates&#x02019; Chi-square = 167.9; <italic>P</italic> &#x0003c; 0.0001). Case records and diagnosis were merged together as one indicator in this study. Results were somewhat similar in patient care indicators also. An increase was observed in the following: Proper dispensing of drugs (from 92.3 to 98.7%; Yates&#x02019; Chi-square = 5.87; <italic>P</italic> = 0.015), patients&#x02019; knowledge of dosage (from 94.3 to 97.1%; Yates&#x02019; Chi-square = 3.38; <italic>P</italic> = 0.066), patients&#x02019; knowledge of adverse events (from 74.5 to 82.8%; Yates&#x02019; Chi-square = 8.64; <italic>P</italic> = 0.003), and satisfaction (from 86.5 to 94.7%; Yates&#x02019; Chi-square = 15.9; <italic>P</italic> &#x0003c; 0.0001). Labeling of drugs further dropped from 5.8 to 2.9% (Yates&#x02019; Chi-square = 3.01; <italic>P</italic> = 0.083).</p><p>Wide deviations from the given set of standards in any homeopathic practice setting can not be considered acceptable and should be subjected to investigation and action. If these data are to be used for national benchmarking, practice settings achieving a low standard should be encouraged to achieve at least the standards of the better performing settings.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Through studies using these newly developed indicators for homeopathy, it may be possible to evaluate the conditions of services offered by an institution. Thus, the indicators can be used to help the healthcare settings obtain better organizational structure, improve pharmaceutical prescribing, and raise the overall level of homeopathic healthcare practices in West Bengal, India. Some indicators may need to be revised or updated, especially to improve the internal consistency. Thus, a prescription management regulation needs to be promulgated for homeopathic practitioners.</p></sec>

Effects of Goshajinkigan, Hachimijiogan, and Rokumigan on Mechanical Allodynia Induced by Paclitaxel in Mice

<p>Peripheral neuropathy is a major dose-limiting side effect of the chemotherapeutic agent paclitaxel. This study examined whether the three related traditional herbal formulations, goshajinkigan (GJG; &#x0725b;&#x08eca;&#x0814e;&#x06c23;&#x04e38; Ni&#x000fa; Ch&#x00113; Sh&#x000e8;n Q&#x000ec; W&#x000e1;n), hachimijiogan (HJG; &#x0516b;&#x05473;&#x05730;&#x09ec3;&#x04e38; B&#x00101; W&#x000e8;i D&#x000ec; Hu&#x000e1;ng W&#x000e1;n), and rokumigan (RMG; &#x0516d;&#x05473;&#x04e38; Li&#x000f9; W&#x000e8;i W&#x000e1;n), would relieve paclitaxel-induced mechanical allodynia in mice. A single intraperitoneal injection of paclitaxel (5 mg/kg) induced mechanical allodynia, which peaked on day 14 after injection. On day 14 after paclitaxel injection, oral administration of GJG (0.1-1.0 g/kg) produced a significant inhibition of established allodynia, but HJG and RMG did not affect the allodynia. Repeated oral administration of GJG (0.1-1.0 g/kg) starting from the day after paclitaxel injection did not affect allodynia development, but significantly inhibited allodynia exacerbation. Repeated oral administration of HJG produced a slight inhibition of allodynia exacerbation, but that of RMG did not. These results suggest that prophylactic administration of GJG is effective in preventing the exacerbation of paclitaxel-induced allodynia. The herbal medicines <italic>Plantaginis Semen</italic> (&#x08eca;&#x0524d;&#x05b50; Ch&#x00113; Qi&#x000e1;n Z&#x001d0;) and <italic>Achyranthis Radix</italic> (&#x0725b;&#x0819d; Ni&#x000fa; X&#x0012b;), which are present in GJG but not in HJG, may contribute to the inhibitory action of GJG on the exacerbation of paclitaxel-induced allodynia.</p>

<contrib contrib-type="author"><name><surname>Andoh</surname><given-names>Tsugunobu</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kitamura</surname><given-names>Ryo</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Fushimi</surname><given-names>Hirotoshi</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Komatsu</surname><given-names>Katsuko</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Shibahara</surname><given-names>Naotoshi</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Kuraishi</surname><given-names>Yasushi</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Paclitaxel, an anti-microtubule agent originally isolated from the bark of the Pacific yew tree (<italic>Taxus brevifolia</italic>), is widely used to treat solid neoplasms such as ovarian, breast, and lung cancers.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] However, paclitaxel causes peripheral neuropathy, which is characterized by pain and allodynia, with a stocking-and-glove distribution.[<xref rid="ref3" ref-type="bibr">3</xref>] The peripheral neuropathy is a major dose-limiting side effect of paclitaxel. Several drugs, such as gabapentin and amifostine, were tested and failed to relieve paclitaxel-induced peripheral neuropathy in patients.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>]</p><p>Goshajinkigan (GJG; &#x0725b;&#x08eca;&#x0814e;&#x06c23;&#x04e38; Ni&#x000fa; Ch&#x00113; Sh&#x000e8;n Q&#x000ec; W&#x000e1;n) is a traditional herbal formulation consisting of 10 herbal medicines [<italic>Rehmanniae Radix</italic> (&#x05730;&#x09ec3; D&#x000ec; Hu&#x000e1;ng), <italic>Achyranthis Radix</italic> (&#x0725b;&#x0819d; Ni&#x000fa; X&#x0012b;), <italic>Corni Fructus</italic> (&#x05c71;&#x08331;&#x08438; Sh&#x00101;n Zh&#x0016b; Y&#x000fa;), <italic>Dioscoreae Rhizoma</italic> (&#x05c71;&#x085e5; Sh&#x00101;n Y&#x000e0;o), <italic>Plantaginis Semen</italic> (&#x08eca;&#x0524d;&#x05b50; Ch&#x00113; Qi&#x000e1;n Z&#x001d0;), <italic>Alismatis Rhizoma</italic> (&#x06fa4;&#x07009; Z&#x000e9; Xi&#x000e8;), <italic>Poria</italic> (&#x0832f;&#x082d3; F&#x000fa; Ling), <italic>Moutan Cortex</italic> (&#x07261;&#x04e39;&#x076ae; M&#x001d4; D&#x00101;n P&#x000ed;), <italic>Cinnamoni Cortex</italic> (&#x06842;&#x076ae; Gu&#x000ec; P&#x000ed;), and <italic>Processi Aconiti Radix</italic> (&#x09644;&#x05b50; F&#x000f9; Z&#x001d0;)]. GJG is used for the treatment of several neurological symptoms including pain and dysesthesia (unpleasant abnormal sensation). It has been demonstrated to be effective against chemotherapy-induced peripheral neuropathy in cancer patients.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Recent studies have shown that GJG does not attenuate the antineoplastic action of chemotherapeutic agents.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] Excluding <italic>Achyranthis Radix</italic> and <italic>Plantaginis Semen</italic>, hachimijiogan (HJG; &#x0516b;&#x05473;&#x05730;&#x09ec3;&#x04e38; B&#x00101; W&#x000e8;i D&#x000ec; Hu&#x000e1;ng W&#x000e1;n) consists of the same herbal medicines found in GJG, and rokumigan (RMG; &#x0516d;&#x05473;&#x04e38; Li&#x000f9; W&#x000e8;i W&#x000e1;n) consists of all herbal medicines found in HJG, excluding <italic>Cinnamoni Cortex</italic> (&#x06842;&#x076ae; Gu&#x000ec; P&#x000ed;) and <italic>Processi Aconiti Radix</italic> (&#x09644;&#x05b50; F&#x000f9; Z&#x001d0;). There is no clinical evidence of the effects of HJG and RMG on chemotherapy-induced peripheral neuropathy. In this study, we investigated the effects of GJG, HJG, and RMG on paclitaxel-induced mechanical allodynia.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Animals</title><p>Male C57BL/6NCr mice (Japan SLC Ltd, Hamamatsu, Japan) were used. All animals were 6 weeks old at the start of the experiments. They were housed in a room with controlled temperature (21-23&#x000b0;C), humidity (45-65%), and a 12-h light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.). Food and water were provided <italic>ad libitum</italic>. Experiments were performed after obtaining approval from the animal care committee of the University of Toyama.</p></sec><sec id="sec2-2"><title>Drugs</title><p>Paclitaxel purchased from Sigma-Aldrich (St Louis, MO, USA) was dissolved in vehicle [physiological saline containing 10% Cremophor^&#x000ae; EL (Sigma-Aldrich) and 10% ethanol]. Paclitaxel and the vehicle were administered intraperitoneally; the dose (5 mg/kg) was selected from a published report[<xref rid="ref11" ref-type="bibr">11</xref>] and was in accordance with the recommended clinical dose of 210 mg/m² body surface area, which corresponds to a dose of 5.9 mg/kg in a person of 170 cm body height and 60 kg weight. Dried extracts of GJG (Lot. No. 2080107010, 2009), HJG (Lot. No. 2090007010, 2009), and RMG (Lot. No. 2090087010, 2009) were obtained from Tsumura and Co., Ltd (Tokyo, Japan). These dried extracts were dissolved in 5% gum arabic and were administered orally once on day 14 after the paclitaxel injection or once daily from the day after paclitaxel injection is given.</p></sec><sec id="sec2-3"><title>Behavioral experiments</title><p>Mechanical allodynia in the hind paw was evaluated using a von Frey filament (North Coast Medical Inc., Morgan Hill, CA, USA), as described.[<xref rid="ref11" ref-type="bibr">11</xref>] After an acclimation period of at least 30 min, a fine von Frey filament with a bending force of 0.69 mN was applied perpendicularly against the central part of the plantar hind paw and was held for 1-3 s with it slightly bent. Responses to the stimuli were scored as follows: 0, no reaction; 1, lifting of the hind paw; and 2, licking and flinching of the hind paw. A stimulus of the same intensity was applied three times alternately to each hind paw at intervals of several seconds, and the average response score served as the allodynia score (the maximum score was 2). When the effects of single administration of dried extracts of traditional formulations were examined on day 14 after paclitaxel injection, mechanical allodynia was evaluated before and after the administration of traditional formulations. When the effects of repeated administrations of the dried extracts were examined, mechanical allodynia evaluation was performed before each administration of traditional formulations.</p></sec><sec id="sec2-4"><title>Statistical analysis</title><p>All data are presented as mean &#x000b1; standard error of the mean. Statistical significance was analyzed using two-way repeated measures analysis of variance (ANOVA) and <italic>post hoc</italic> Holm-Sidak multiple comparison test. A <italic>P</italic> &#x0003c; 0.05 was considered statistically significant. The statistical analyses were performed using SigmaPlot&#x02122; graphing and statistical software version 11 (Systat Software, Inc., Chicago, IL, USA).</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-5"><title>Paclitaxel-induced mechanical allodynia</title><p>A single intraperitoneal injection of paclitaxel (5 mg/kg) caused mechanical allodynia, which became apparent on day 4 after injection, peaked on day 14, and almost subsided by day 21 [<xref ref-type="fig" rid="F1">Figure 1a</xref> main effect of paclitaxel treatment, <italic>F</italic>_1,10 = 87.676, <italic>P</italic> &#x0003c; 0.001; interaction between paclitaxel treatment and time, <italic>F</italic>_30,300 = 12.286, <italic>P</italic> &#x0003c; 0.001 (two-way repeated measures ANOVA)].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Effects of single administration of goshajinkigan (GJG), hachimijiogan (HJG), and rokumigan (RMG) on the established mechanical allodynia after paclitaxel injection. (a) Development of mechanical allodynia after a single injection of paclitaxel. Mice were injected intraperitoneally with paclitaxel (5 mg/kg) or vehicle on day 0. GJG (b), HJG (c), RMG (d), or vehicle (5% gum arabic) was orally administered on day 14 after paclitaxel injection. Data are presented as mean and standard error of the mean (<italic>n</italic> = 5-6). *<italic>P</italic> &#x0003c; 0.05 compared to vehicle (Holm-Sidak multiple comparisons)</p></caption><graphic xlink:href="JTCM-4-293-g001"/></fig></sec><sec id="sec2-6"><title>Effects of single administration of GJG, HJG, and RMG on paclitaxel-induced mechanical allodynia</title><p>Three formulations were administered orally on day 14 after paclitaxel injection. Single administration of GJG (0.1-1.0 g/kg) exerted a relatively short-lasting but significant dose-dependent inhibition on the established mechanical allodynia; two-way repeated measures ANOVA revealed a significant main effect of GJG (<italic>F</italic>_3,20 = 3.611, <italic>P</italic> = 0.031) [<xref ref-type="fig" rid="F1">Figure 1b</xref>]. Single administration of HJG and RMG did not affect the established mechanical allodynia at doses of 0.1-1.0 g/kg [Figure <xref ref-type="fig" rid="F1">1c</xref> and <xref ref-type="fig" rid="F1">d</xref>].</p></sec><sec id="sec2-7"><title>Effects of prophylactic administration of GJG, HJG, and RMG on paclitaxel-induced mechanical allodynia</title><p>In this series of experiments, three formulations were administered daily from the day after paclitaxel injection. Repeated administration of GJG (0.1-1 g/kg) did not affect the initial development of mechanical allodynia induced by paclitaxel, but significantly inhibited the exacerbation of allodynia from day 9 after paclitaxel injection, in comparison with the vehicle, although no clear dose-dependency was observed [<xref ref-type="fig" rid="F2">Figure 2a</xref>; interaction between paclitaxel treatment and time, <italic>F</italic>_42,252 = 1.889, <italic>P</italic> = 0.002 (two-way repeated measures ANOVA)]. Similarly, repeated administration of HJG (0.1-1 g/kg) did not affect the initial development of mechanical allodynia, but the highest dose of HJG (1 g/kg) significantly inhibited allodynia on day 14 after the injection, in comparison with the vehicle [<xref ref-type="fig" rid="F2">Figure 2b</xref>; interaction between paclitaxel treatment and time, <italic>F</italic>_42,266 = 1.504, <italic>P</italic> = 0.03 (two-way repeated measures ANOVA)]. Repeated administration of RMG (0.1-1 g/kg) did not affect the mechanical allodynia induced by paclitaxel [<xref ref-type="fig" rid="F2">Figure 2c</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Effects of prophylactic administration of goshajinkigan (GJG), hachimijiogan (HJG), and rokumigan (RMG) on paclitaxel-induced mechanical allodynia. Paclitaxel (5 mg/kg) was injected intraperitoneally in mice, and GJG (a), HJG (b), RMG (c), or vehicle (5% gum arabic) was administered orally once daily from the day after the paclitaxel injection. Data are presented as mean and standard error of the mean (<italic>n</italic> = 5-6). *<italic>P</italic> &#x0003c; 0.05 compared to vehicle (Holm-Sidak multiple comparisons)</p></caption><graphic xlink:href="JTCM-4-293-g002"/></fig></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Single oral administration of GJG (0.1-1 g/kg) exerted a dose-dependent inhibition on the established mechanical allodynia after paclitaxel injection, whereas the GJG-related herbal formulations HJG and RMG did not affect the established allodynia. The recommended clinical daily dose of GJG granules is 7.5 g, which contains 4.5 g of dried GJG extract. Given that the body weight is 60 kg, the dose of the dried GJG extract is 0.075 g/kg. Thus, the antiallodynic dose (1 g/kg) in mice with paclitaxel-induced neuropathy was 13 times higher than the recommended clinical dose. Recently, we have demonstrated that single oral administration of GJG (0.3 and 1.0 g/kg) inhibits the established mechanical allodynia after the injection of oxaliplatin, a platinum chemotherapeutic agent.[<xref rid="ref12" ref-type="bibr">12</xref>] The inhibitory effect of 1 g/kg GJG on paclitaxel-induced allodynia (present study) was less than that of 0.3 g/kg GJG on oxaliplatin-induced allodynia.[<xref rid="ref12" ref-type="bibr">12</xref>] The inhibitory action of GJG on oxaliplatin-induced allodynia is at least in part mediated by the activation of the descending noradrenergic and serotonergic systems,[<xref rid="ref12" ref-type="bibr">12</xref>] and in our preliminary experiment, single administration of milnacipran, a serotonin&#x02013;noradrenaline reuptake inhibitor, markedly inhibited the established allodynia after oxaliplatin injection (data not shown). In contrast, single administration of milnacipran does not affect the established allodynia after paclitaxel injection.[<xref rid="ref13" ref-type="bibr">13</xref>] These findings, taken together, raise the possibility that a low efficacy of GJG in paclitaxel-induced allodynia is due to the decreased activity of the descending noradrenergic and serotonergic systems. It is possible that paclitaxel decreases the activity of the descending noradrenergic and/or serotonergic systems.</p><p>Prophylactic repeated administration of GJG inhibited paclitaxel-induced mechanical allodynia. GJG consists of the following 10 herbal medicines: <italic>Rehmanniae Radix</italic> (&#x05730;&#x09ec3; D&#x000ec; Hu&#x000e1;ng), <italic>Corni Fructus</italic> (&#x05c71;&#x08331;&#x08438; Sh&#x00101;n Zh&#x0016b; Y&#x000fa;), <italic>Dioscoreae Rhizoma</italic> (&#x05c71;&#x085e5; Sh&#x00101;n Y&#x000e0;o), <italic>Alismatis Rhizoma</italic> (&#x06fa4;&#x07009; Z&#x000e9; Xi&#x000e8;), <italic>Poria</italic> (&#x0832f;&#x082d3; F&#x000fa; Ling), <italic>Moutan Cortex</italic> (&#x07261;&#x04e39;&#x076ae; M&#x001d4; D&#x00101;n P&#x000ed;), <italic>Cinnamoni Cortex</italic> (&#x06842;&#x076ae; Gu&#x000ec; P&#x000ed;), <italic>Processi Aconiti Radix</italic> (&#x09644;&#x05b50; F&#x000f9; Z&#x001d0;), <italic>Achyranthis Radix</italic> (&#x0725b;&#x0819d; Ni&#x000fa; X&#x0012b;), and <italic>Plantaginis Semen</italic> (&#x08eca;&#x0524d;&#x05b50; Ch&#x00113; Qi&#x000e1;n Z&#x001d0;); HJG consists of the first 8 of these herbal medicines and RMG consists of the first 6 of these medicines. The results showed that HJG had only a slight effect at the highest dose tested and RMG did not have a prophylactic effect, suggesting that <italic>Plantaginis Semen</italic> and/or <italic>Achyranthis Radix</italic> play a role in the prophylactic activity of GJG. The underlying mechanisms are unclear, and allodynia-preventing substances have not yet been isolated from the extracts of these two herbal medicines. However, one possibility is that the activity of the scavengers of reactive oxygen species is involved in the antiallodynic activity. Although a single administration is ineffective, prophylactic administration of the reactive oxygen species scavenger N-tert-butyl-a-phenylnitrone suppresses paclitaxel-induced mechanical allodynia.[<xref rid="ref14" ref-type="bibr">14</xref>] Since <italic>Plantaginis Semen</italic> and <italic>Achyranthis Radix</italic> have antioxidant activity,[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>] this action may be involved in the prevention of an exacerbation of paclitaxel-induced allodynia. Another possibility is the improvement of decreased blood flow. Paclitaxel, but not vincristine, decreases peripheral blood flow.[<xref rid="ref11" ref-type="bibr">11</xref>] The prostaglandin E1 analog limaprost, which counteracts this reduction in blood flow without affecting normal blood flow,[<xref rid="ref18" ref-type="bibr">18</xref>] exerts prophylactic inhibition on allodynia induced by paclitaxel, but not by vincristine.[<xref rid="ref11" ref-type="bibr">11</xref>] In this study, prophylactic administration of GJG prevented mechanical allodynia induced by paclitaxel. In contrast, in our preliminary experiments, prophylactic administration of GJG did not prevent vincristine-induced allodynia. GJG and its constituent <italic>Achyranthis Radix</italic> invigorate blood circulation.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref>] Taken together, these findings raise the possibility that the prevention of the decrease in peripheral blood flow contributes to the prophylactic activity of GJG.</p><p>Prophylactic administration of HJG, but not RMG, showed slight antiallodynic action at the highest dose tested in paclitaxel-treated mice. Thus, it is possible that <italic>Cinnamoni Cortex</italic> and <italic>Processi Aconiti Radix</italic> also have prophylactic antiallodynic activity. Similar to <italic>Achyranthis Radix</italic>, <italic>Cinnamoni Cortex</italic> and <italic>Processi Aconiti Radix</italic> invigorate blood circulation.[<xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref>] However, the prophylactic antiallodynic activity of HJG was weaker than that of GJG. Thus, although <italic>Cinnamoni Cortex</italic> and <italic>Processi Aconiti Radix</italic> may have some prophylactic antiallodynic activity, <italic>Plantaginis Semen</italic> and <italic>Achyranthis Radix</italic> may play important roles in the antiallodynic action of GJG.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Prophylactic administration of GJG prevents an exacerbation of paclitaxel-induced mechanical allodynia. Since GJG also has acute antiallodynic activity, it may be useful for the prevention of paclitaxel-induced peripheral neuropathy. Although the underlying mechanisms remain unclear, <italic>Plantaginis Semen</italic> and/or <italic>Achyranthis Radix</italic> may contribute to the antiallodynic activity of GJG.</p></sec>

The Antinociceptive Effects of Tualang Honey in Male Sprague-Dawley Rats: A Preliminary Study

<p>Tualang honey (&#x08702;&#x0871c; F&#x00113;ng M&#x000ec;) is known to have anti-inflammatory property, but its antinociceptive property has not been extensively investigated. In this study, we examined the preemptive effects on administering different doses of Tualang honey and prednisolone on the nociceptive response in male Sprague-Dawley rats. Thirty-five male Sprague-Dawley rats were randomized into five groups (<italic>n</italic> = 7) and each group received either distilled water, Tualang honey (0.2, 1.2 or 2.4 g/kg) or prednisolone (10 mg/kg) for 10 days. The response to noxious thermal stimulus was assessed using tail flick test on Day 10. The well-being of the rats was also assessed by monitoring their food intake and body weight. Data were analyzed using one-way Analysis of Variance (ANOVA) with <italic>post-hoc</italic> Scheffe's test and <italic>P</italic> value less than 0.05 was considered significant. In tail flick test, the tail flick latency time was significantly higher in the groups that received 1.2 g/kg and 2.4 g/kg of Tualang honey and 10 mg/kg of prednisolone, compared to the control group (<italic>P</italic> &#x0003c; 0.05). There was significant reduction in the total food pellet intake in the groups receiving prednisolone and Tualang honey (1.2 g/kg and 2.4 g/kg) compared to controls; however, the body weight gain was only significantly reduced in the prednisolone group. All the parameters were not significantly affected in the group receiving 0.2 g/kg of Tualang honey. In conclusion, preemptive administration of Tualang honey (1.2 g/kg and 2.4 g/kg) and prednisolone (10 mg/kg) had reduced the pain responses. The reduced weight gain in the prednisolone group is an unwanted effect due to its metabolic and central actions. Further studies are required to confirm the antinociceptive effects and elucidate the mechanism of antinociceptive action of Tualang honey in the rats.</p>

<contrib contrib-type="author"><name><surname>Aziz</surname><given-names>Che Badariah Abd</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Ismail</surname><given-names>Che Aishah Nazariah</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hussin</surname><given-names>Che Maraina Che</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Mohamed</surname><given-names>Mahaneem</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib>

Journal of Traditional and Complementary Medicine

<sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Honey (&#x08702;&#x0871c; F&#x00113;ng M&#x000ec;) is a natural product produced by honeybees. It is used to treat various conditions including infertility, respiratory and gastrointestinal symptoms.[<xref rid="ref1" ref-type="bibr">1</xref>] Previous reports have shown the anti-inflammatory and antinociceptive properties of honey. Honey was reported to reduce pain from burns and reduce the local pain rapidly.[<xref rid="ref2" ref-type="bibr">2</xref>] The reduction in pain might be attributed to honey's ability to reduce plasma prostaglandins (thromboxane B₂, PGE₂, and PGF_2a) in normal human subjects and animal models.[<xref rid="ref3" ref-type="bibr">3</xref>] Apart from this, other studies have demonstrated that honey reduces the release of nitrous oxide, histamines, and cytokines such as tumor necrosis factor-alpha (TNF-&#x003b1;), which may reduce inflammation and pain.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>There are several types of honey, including Tualang and Gelam honey, in Malaysia. Both types of honey have been shown to have antioxidant and anti-inflammatory activities.[<xref rid="ref4" ref-type="bibr">4</xref>] The phenolic and flavonoid compounds in Tualang honey contribute to its antioxidant activity and might have a role in its anti-inflammatory and probably its antinociceptive effects.[<xref rid="ref5" ref-type="bibr">5</xref>] An established anti-inflammatory agent, prednisolone, has been used to treat inflammatory diseases and was found to be useful in painful conditions such as bladder pain syndrome and arthralgia.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] However, it has various side effects including immune suppression, insulin resistance, skeletal muscle wasting, and osteoporosis.[<xref rid="ref8" ref-type="bibr">8</xref>]</p><p>The analgesic and anti-inflammatory effects of other types of honey, e.g. Gelam and Manuka honey, were reported in various studies, but the present study is among the first few studies which investigated the analgesic effects of Tualang honey.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref9" ref-type="bibr">9</xref>] It is not known whether Tualang honey's antinociceptive property is dose dependent and whether its antinociceptive property is comparable to prednisolone. Therefore, in the present study, we hypothesized that the antinociceptive effects of Tualang honey were dose dependent and comparable to prednisolone. The first aim of the study was to examine the effects of different doses of Tualang honey and prednisolone administration on nociceptive responses induced by noxious thermal stimuli in male Sprague-Dawley rats. In this study, the well-being of the rats was also assessed by monitoring their food intake and body weight. As previous reports have shown various effects of prednisolone and Tualang honey on these parameters, the second aim was to determine whether administration of Tualang honey and prednisolone altered the food pellet intake and body weight in the rats studied.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>This was an experimental study performed in the Physiology Laboratory, Universiti Sains Malaysia (USM) Health Campus, Kelantan. Experiments were performed between 08:00 and 16:00 hours. The research was approved by the USM Animal Ethical Committee [USM/Animal Ethics Approval/2010/(63)(266)].</p><sec id="sec2-1"><title>Animals</title><p>The rats were obtained from Animal Research and Service Centre (ARASC), USM Health Campus. Thirty-five Sprague-Dawley male rats, weighing from 250 to 300 g (8-10 weeks old) were used in this study. They were kept under 12-h light dark cycle and permitted free access to food pellets and water. All the rats were housed individually and allowed to acclimatize at least for 4 days in the physiology laboratory before beginning the experiment.[<xref rid="ref12" ref-type="bibr">12</xref>] The experiment was performed between 08:00 and 16:00 hours and behavior test was conducted in the morning.[<xref rid="ref12" ref-type="bibr">12</xref>] The testing room was consistently maintained at 22&#x000b0;-24&#x000b0;C. The body weight and total food pellet intake of the animals were recorded daily during the experiment. The changes in body weight and total food pellet consumption were calculated.</p></sec><sec id="sec2-2"><title>Tualang honey and prednisolone administration</title><p>Tualang honey (&#x08702;&#x0871c; F&#x00113;ng M&#x000ec;) was supplied by Federal Agricultural Marketing Authority (FAMA), Ministry of Agriculture and Agro-based Industry, Malaysia. All the animals were force-fed using a gavage needle in order to ensure that the accurate amount of distilled water, Tualang honey, or prednisolone was administered. The doses of Tualang honey chosen (0.2 g/kg, 1.2 g/kg, and 2.4 g/kg body weight) were based on the doses of Tualang honey according to a previous report by Mohamed <italic>et al</italic>.[<xref rid="ref13" ref-type="bibr">13</xref>] Meanwhile, the dose of prednisolone (10 mg/kg) was chosen from a study by Nakamura <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] based on its effectiveness in reducing inflammatory symptoms. Tualang honey or prednisolone was dissolved in distilled water to obtain the required concentration. The treatment was given daily for 10 days before conducting the tail flick test.</p></sec><sec id="sec2-3"><title>Tail flick test</title><p>This test measured nociceptive reflex response when stimulated with noxious heat using Tail Flick Analgesia Meter (IITC, Woodlands Hill, California, USA). The test followed the protocol used by Bannon and Malmberg.[<xref rid="ref15" ref-type="bibr">15</xref>] The light beam used for this test was standardized at 8 units. On the 10^th day, tail flick test was conducted 1 hour after administration of treatment. The rats were acclimatized to the test environment for 30 min before the experiment. The rat was positioned in a Plexiglas restrained tube and three areas were selected to conduct the test, which were at 30, 40, and 50 mm from the tip of the tail. Three minutes interval was given in between two tests to reduce the effects of tissue damage at each site. The time when the tail flicked away from the light beam source was taken and the responses were averaged for each rat to increase the accuracy of the test. Ten seconds was the cut-off time to avoid any tissue damage.</p></sec><sec id="sec2-4"><title>Statistical analysis</title><p>Data were analyzed using Statistical Package for Social Sciences version 19 software (IBM, New York, United States). One-way Analysis of Variance (ANOVA) with <italic>post-hoc</italic> Scheffe's test was used to analyze the changes in rats&#x02019; body weight, food consumption, and tail flick responses. All the data were expressed as mean &#x000b1; standard error of mean (SEM). <italic>P</italic> values less than 0.05 were considered as significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-5"><title>Total consumption of food pellets</title><p>The data analysis of food intake by one-way ANOVA revealed significant difference between all groups [<italic>F</italic> (4,30) = 30.996, <italic>P</italic> &#x0003c; 0.001]. <italic>Post-hoc</italic> Scheffe's test demonstrated a significant reduction of food pellet intake in rats receiving Tualang honey (&#x08702;&#x0871c; F&#x00113;ng M&#x000ec;) at 1.2 g/kg and 2.4 g/kg compared to the control group (<italic>P</italic> &#x0003c; 0.05) [<xref ref-type="table" rid="T1">Table 1</xref>]. There was a significant reduction in prednisolone group compared to groups administered different doses of Tualang honey and the control group (<italic>P</italic> &#x0003c; 0.05).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Total food intake and changes in body weight of rats (mean&#x000b1;SEM) following 10 days of treatment (<italic>n</italic>=7)</p></caption><graphic xlink:href="JTCM-4-298-g001"/></table-wrap></sec><sec id="sec2-6"><title>Changes in body weight of rats</title><p>The analysis with one-way ANOVA revealed a significant difference in the changes of animal body weight during treatment in the groups [<italic>F</italic> (55,4) = 25.173, <italic>P</italic> &#x0003c; 0.001]. <italic>Post-hoc</italic> Scheffe's test demonstrated a significant reduction in weight gain in the group that received preemptive administration of prednisolone compared to the control and Tualang honey groups (<italic>P</italic> &#x0003c; 0.01). There was no significant difference between the groups receiving different doses of Tualang honey and the control group [<xref ref-type="table" rid="T1">Table 1</xref>].</p></sec><sec id="sec2-7"><title>Tail flick test</title><p>The results of tail flick test, as illustrated in <xref ref-type="table" rid="T2">Table 2</xref>, reveal that there was a significant difference between the groups [<italic>F</italic> (25,4) = 3.914, <italic>P</italic> &#x0003c; 0.05]. <italic>Post-hoc</italic> Scheffe's test demonstrated a significant increase in the tail flick latency in the groups administered Tualang honey at 1.2 g/kg and 2.4 g/kg and prednisolone, compared to the control group (<italic>P</italic> &#x0003c; 0.05). There was no significant difference in the latency time between the groups administered 0.2 g/kg of Tualang honey and the control group.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Tail flick test in all treatment groups (<italic>n</italic>=7)</p></caption><graphic xlink:href="JTCM-4-298-g002"/></table-wrap></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>In the present study, the results showed that there was significant reduction in total food intake and body weight gain in the prednisolone group compared to the control group. The groups that received 1.2 and 2.4 g/kg of Tualang honey (&#x08702;&#x0871c; F&#x00113;ng M&#x000ec;) had reduced total food intake, but the weight gain was not significantly reduced. The parameters were not significantly affected in the group that received 0.2 g/kg of Tualang honey. In the tail flick test, the tail flick latency was prolonged in the groups that received 1.2 g/kg and 2.4 g/kg of honey and 10 mg/kg of prednisolone.</p><p>Tualang honey contains fructose (39.84%), glucose (25.29%), and maltose (3.17%), in addition to aforementioned phenolic and flavonoid compounds.[<xref rid="ref16" ref-type="bibr">16</xref>] Hence, the reduced food pellet consumption in the groups that received 1.2 g/kg and 2.4 g/kg of Tualang honey could be partly attributed to its constituent, fructose. Studies have shown that low or moderate doses of fructose intake were associated with reduced leptin levels, while high dose of fructose intake increased the leptin levels.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] Leptin inhibits the release of neuropeptide Y and agouti-related peptide (NPY and AgRP, respectively) in hypothalamic arcuate nucleus and stimulates the synthesis of an appetite suppressant, &#x003b1;-melanocyte stimulating hormone.[<xref rid="ref19" ref-type="bibr">19</xref>] The doses (1.2 g/kg and 2.4 g/kg) used in the study were probably sufficient to suppress the release of NPY, and AgRP, and stimulate the release of appetite suppressant. Furthermore, honey consumption may alter the meal-induced response of ghrelin (hunger hormone) and anorexigenic hormone, peptide tyrosine-tyrosine 3-36 (PYY_3-36).[<xref rid="ref20" ref-type="bibr">20</xref>]</p><p>Although there was a significant reduction in the food intake in the Tualang honey groups (1.2 g/kg and 2.4 g/kg), the body weight of the rats was not significantly affected. Similar findings were also reported in male rats supplemented with 1.2 g/kg Tualang honey for 13 weeks.[<xref rid="ref21" ref-type="bibr">21</xref>] It was suggested that there are adequate calories in Tualang honey which might contribute to the normal body weight gain in the groups administered honey despite the reduced total food pellet intake. Other studies which have demonstrated alteration in the body weight gain were conducted either on obese, diabetic, or hypertensive subjects.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref16" ref-type="bibr">16</xref>]</p><p>The present study has shown that the group that received 10 days of prednisolone treatment had reduced total food intake and body weight gain. Although glucocorticoid therapy is usually associated with weight gain, its administration may not alter or may lead to reduction in the parameter.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] Studies have reported that pretreatment with a lower dose of prednisolone was associated with increase in food intake, while higher dose was associated with a decrease in feeding.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] The dose used in the present study is considered high and its administration may lead to hyperinsulinemia, glucolipid metabolic disturbances, and probably down-regulating mRNA expression levels of the orexigenic neuropeptides, NPY and AgRP, and anorexigenic neuropeptide, cocaine- and amphetamine-regulated transcript (CART), in the hypothalamus in the rats.[<xref rid="ref22" ref-type="bibr">22</xref>] The interactions of these factors probably contribute to the reduction in food intake and body weight. Several studies have demonstrated the stimulatory effects of glucocorticoids on leptin secretion in both human and animal subjects. High leptin level would reduce NPY/AgRP expression and stimulate the release of appetite suppressant, &#x003b1;-melanocyte stimulating hormone.[<xref rid="ref24" ref-type="bibr">24</xref>] These mechanisms will lead to a decrease in food intake as seen in the present study. However, the leptin level was not measured in the present study due to financial constraint. The effects of prednisolone (10 mg/kg) and Tualang honey (1.2 g/kg and 2.4 g/kg) on leptin level will be investigated in the future.</p><p>The reduction in body weight found in the present study might be due to the reduced weight of the organs and reduced muscle bulk, which are the side effects of prednisolone treatment. Hull <italic>et al</italic>. have demonstrated a significant reduction in the total body weight and the weight of several organs including spleen, thymus, bursa, muscle, testis, and oviduct in Japanese quail following glucocorticoid treatment.[<xref rid="ref25" ref-type="bibr">25</xref>] The study has shown that the weight reduction was significantly correlated with the dose of glucocorticoid administered. The reduced muscle bulk was attributed to increased protein catabolism and impaired protein synthesis.[<xref rid="ref26" ref-type="bibr">26</xref>]</p><p>In the present study, the groups that received 1.2 g/kg and 2.4 g/kg of Tualang honey had shown a significant increase in tail flick latency time. The increase in reaction time of the rats shows that Tualang honey at the doses given has analgesic activities. The antinociceptive effects of Tualang honey might be contributed partly by its action on opioid receptors in the spinal cord. However, other mechanisms might also be involved.[<xref rid="ref27" ref-type="bibr">27</xref>] A report has shown that the antinociceptive effects of Nigerian honey samples (Idanre and Ewu honey) were reversed with the administration of naloxone, an opioid antagonist, but the effects of other Nigerian honey samples (Jigawa, Ile-Ife, and Umudike) were not reversed, suggesting other mechanisms which might come into play (e.g. inhibition of voltage-gated Na⁺ channels, stimulation of noradrenergic inhibitory system and/or serotonergic system).[<xref rid="ref27" ref-type="bibr">27</xref>] The analgesic properties of Tualang honey could also be contributed by its antioxidant property (53.06 &#x000b1; 0.41 mg of ascorbic acid equivalent per gram of Tualang honey).[<xref rid="ref5" ref-type="bibr">5</xref>] Reports have shown the role of oxidative stress in the development of pain/hyperalgesia, and vitamin C, one of the antioxidants in Tualang honey, has been shown to inhibit nociceptive transmission by interacting at the level of glutamate receptors in the central nervous system.[<xref rid="ref28" ref-type="bibr">28</xref>] The lowest dose used (0.2 g/kg) probably was not sufficient to give a central effect and did not significantly alter the latency time, compared to controls.</p><p>In the present study, the antinociceptive effect in the tail flick test was also evident in the group that received prednisolone. The reduction in pain behavior is probably related to a decrease in calcitonin gene&#x02013;related peptide (CGRP) and an increase in B2-gamma-aminobutyric acid (GABAB₂) receptor expression in the spinal cord.[<xref rid="ref29" ref-type="bibr">29</xref>] CGRP is one of the neurotransmitters involved in the nociceptive transmission in the spinal cord dorsal horn, while GABA is an inhibitory neurotransmitter that inhibits the transmission. The presence of nuclear glucocorticoid receptor immunoreactivity in a large number of spinal cord nerve cells which have substance <italic>P</italic> or CGRP immunoreactivity (IR) suggests that a glucocorticoid, prednisolone, may modulate the nociceptive transmitting system, which may lead to a decrease in pain response.[<xref rid="ref30" ref-type="bibr">30</xref>]</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>Preemptive administration of Tualang honey (1.2 and 2.4 g/kg) and prednisolone (10 mg/kg) is capable of altering the feeding habits and modulating the nociceptive responses in male Sprague-Dawley rats. The preliminary study provides novel knowledge regarding the possible effects of Tualang honey in pain modulation and its protective effects against weight alteration in normal rats. The weight alteration by prednisolone is an unwanted effect due to its metabolic and central actions. Further studies are required to confirm the antinociceptive effects and elucidate the mechanisms of antinociceptive action of Tualang honey in rats.</p></sec>

Passive appendages generate drift through symmetry breaking

<p>Plants and animals use plumes, barbs, tails, feathers, hairs and fins to aid locomotion. Many of these appendages are not actively controlled, instead they have to interact passively with the surrounding fluid to generate motion. Here, we use theory, experiments and numerical simulations to show that an object with a protrusion in a separated flow drifts sideways by exploiting a symmetry-breaking instability similar to the instability of an inverted pendulum. Our model explains why the straight position of an appendage in a fluid flow is unstable and how it stabilizes either to the left or right of the incoming flow direction. It is plausible that organisms with appendages in a separated flow use this newly discovered mechanism for locomotion; examples include the drift of plumed seeds without wind and the passive reorientation of motile animals.</p>

<contrib contrib-type="author"><name><surname>L&#x00101;cis</surname><given-names>U.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Brosse</surname><given-names>N.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ingremeau</surname><given-names>F.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Mazzino</surname><given-names>A.</given-names></name><xref ref-type="aff" rid="a3">3</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Lundell</surname><given-names>F.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kellay</surname><given-names>H.</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Bagheri</surname><given-names>S.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Linn&#x000e9; Flow Centre, KTH Royal Institute of Technology, Department of Mechanics</institution>, 10044 Stockholm, <country>Sweden</country></aff><aff id="a2"><label>2</label><institution>Department of Mechanical and Aerospace Engineering, Princeton University</institution>, Princeton, New Jersey 08544, <country>USA</country></aff><aff id="a3"><label>3</label><institution>Department of Chemical, Civil and Environmental Engineering (DICCA), University of Genova</institution>, 16145 Genova, <country>Italy</country></aff><aff id="a4"><label>4</label><institution>INFN and CINFAI Consortium, Genova Section</institution>, 16146 Genova, <country>Italy</country></aff><aff id="a5"><label>5</label><institution>Universit&#x000e9; de Bordeaux, Laboratoire Ondes et Mati&#x000e8;re d'Aquitaine (UMR 5798 CNRS), 351 cours de la Lib&#x000e9;ration</institution>, 33405 Talence, <country>France</country></aff>

Nature Communications

<p>Aerial and aquatic animals have developed distinct and complex mechanisms to move through air and water with little resistance<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. To efficiently manipulate the surrounding flow, they combine both active and passive methods<xref ref-type="bibr" rid="b3">3</xref>. By actively flapping, undulating or oscillating appendages, the animal generates forces that displace the surrounding fluid, which in turn pushes the animal in the desired direction. The contribution to locomotion through passive mechanisms, on the other hand, is much harder to identify. This is because the function of a non-smooth compliant skin<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>, hair, feathers and other passive protrusions are not only related to the movement of the animal, but also to other features, such as sensation, protection and insulation. The advantage of passive locomotion techniques is that no energy needs to be expended by the animal; instead the energy is extracted through a complex interaction with the environment. Forces are generated through instabilities that are often nucleated at the boundary of the fluid and elastic structures<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>. Passive mechanisms are also the only way for non-motile organisms to disperse.</p><p>When a body travels through a fluid at sufficiently high speed, there is a difference in pressure between the front and rear surfaces of the object. As explained by Prandtl&#x02019;s boundary layer theory in 1905<xref ref-type="bibr" rid="b8">8</xref>, this pressure drag&#x02014;which is often undesired for locomotion&#x02014;is a consequence of flow separation (that is, a region where the flow becomes detached from the surface of the body and has the form of vortices and eddies). In this manuscript, we show that a separated region behind a body may in fact be exploited to aid locomotion. This becomes possible when an appendage of simple shape is added to the body. As we will show, the resting position of a short protuberance in a separated flow is unstable in the same way as an inverted pendulum is unstable under gravity. The protuberance stabilizes at an angle (up to 40 degrees) either to the left or right of its resting position and, as a consequence, a net drift/lift force (transverse to drag force) is generated. Although, not explicitly demonstrated in this paper, it is likely that this symmetry breaking has implications for locomotion<xref ref-type="bibr" rid="b9">9</xref>; the trailing part of a cephalopod shell<xref ref-type="bibr" rid="b10">10</xref>, the tail of a gliding tadpole<xref ref-type="bibr" rid="b11">11</xref>, the hind-wing tails of the swallowtail butterfly<xref ref-type="bibr" rid="b12">12</xref> and the pop-up feathers of many birds<xref ref-type="bibr" rid="b13">13</xref> are a few examples of appendages that are susceptible to this instability. We use experiments and numerical simulations to show the existence of an inverted-pendulum-like (IPL) instability under a wide range of conditions (steady/unsteady flows, rigid/elastic protrusions and fixed/falling objects) and that it generates rotation and drift of the body. We then unravel the mechanism with a simple model&#x02014;which will justify the term IPL&#x02014;that provides quantitative prediction of the induced rotation and drift.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Experiments of a rigid plate attached to a cylinder</title><p>To show evidence of the IPL instability, we carried out experiments in which a soap film<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref> flows vertically (due to gravity) at a constant velocity <italic>&#x003c5;</italic>=1.9&#x02009;m&#x02009;s^&#x02212;1 between two wires (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). Inside the soap film, we placed a circular cylinder of diameter <italic>D</italic>=6.3&#x02009;mm with a clamped rigid splitter plate of length <italic>L</italic>. The body is free to rotate around the centre of the cylinder. When the plate is longer than the critical length of <italic>L</italic>_c=(4.0&#x000b1;0.2)<italic>D</italic>, the body will&#x02014;in the presence of any perturbation&#x02014;always restore to a steady symmetric straight position (<xref ref-type="fig" rid="f1">Fig. 1c</xref>). However, for a shorter plate (<italic>L</italic>=1.0<italic>D</italic>) the body stabilizes at an angle of 16 degrees to the right of the incoming stream (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). It is of equal probability that the plate settles to the right or left of the incoming stream of flow. In <xref ref-type="fig" rid="f1">Fig. 1d</xref>, we show how the time-averaged turn angle <italic>&#x003b8;</italic> depends on the splitter-plate length. A clear transition is observed from a symmetric state to an asymmetric one at a critical threshold.</p><p>To show the existence of a drift force <italic>F</italic>_d on the body as a result of the symmetry breaking, we fixed the same object on a &#x02018;loose&#x02019; pendulum made of a thin nylon wire (<xref ref-type="fig" rid="f2">Fig. 2a</xref>). This wire crosses the film perpendicularly through a small hole drilled in the centre of the cylinder. If the object&#x02014;which is free to rotate under the imposed conditions&#x02014;turns, we expect a non-zero drift force on the object that will induce a displacement of the equilibrium position of the pendulum. <xref ref-type="fig" rid="f2">Figure 2b,c</xref> shows snapshots of the cylinder with a splitter plate of length <italic>L</italic>=2.1<italic>D</italic> for low and high film velocities, respectively. We observe that when the flow velocity is high, the average turn angle <italic>&#x003b8;</italic> is non-zero (asymmetric state) and the whole object has drifted sideways, resulting in a new equilibrium position of the pendulum (<xref ref-type="supplementary-material" rid="S1">Supplementary Movies 1</xref> and <xref ref-type="supplementary-material" rid="S1">2</xref>). Moreover, we observe from <xref ref-type="fig" rid="f2">Fig. 2c</xref> that the disk drifts in the same direction as the splitter plate is tilted.</p><p>We calculate the corresponding drift angle<xref ref-type="bibr" rid="b2">2</xref> <italic>&#x003b1;</italic>, which can be obtained from the ratio of the drift force to the drag force, that is,</p><p><disp-formula id="eq1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e346" xlink:href="ncomms6310-m1.jpg"/></disp-formula></p><p>We estimated <italic>F</italic>_drag by fixing the cylinder to the end of a calibrated cantilever perpendicular to the soap-film plane and measuring its deflection (the procedure is depicted in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>). We estimated the drift force <italic>F</italic>_d from a force-balance equation (see Methods: soap film experiments of hanging body). The average drift angle as a function of the splitter-plate length is shown in <xref ref-type="fig" rid="f2">Fig. 2d</xref>. We observe that short plates that have a non-zero turn-angle (<italic>&#x003b8;</italic>) also have a non-zero drift angle <italic>&#x003b1;</italic>, confirming the instability-induced forcing. Moreover, as we approach short and long appendage limits (<italic>L</italic>&#x02192;0 and <italic>L</italic>&#x02192;<italic>&#x0221e;</italic>, respectively), the drift angle tends to zero. It is also observed that there exists an optimal value of splitter-plate length for maximum drift force. The existence of an optimal configuration may be an important factor in evolution of tails and appendages of motile animals, because it is often desirable to move in a specific direction as fast as possible.</p></sec><sec disp-level="2"><title>Simulations of a free-falling cylinder with a rigid plate</title><p>We complement our experiments with two-dimensional numerical simulations of a free-falling cylinder with a splitter plate clamped to its rear end. This allows us to investigate two orders of magnitude lower Reynolds numbers <italic>Re</italic>=<italic>UD</italic>/<italic>&#x003bd;</italic> (<italic>U</italic> being the descent speed of the body, and &#x003bd; the kinematic viscosity of the fluid) as well as to demonstrate how the instability generates a lateral motion. We found that when the body with a splitter plate shorter than a critical length is released, the body rotates an angle <italic>&#x003b8;</italic> towards a new equilibrium, and drifts at a constant angle <italic>&#x003b1;</italic> with respect to the straight vertical path. Whereas <italic>&#x003b8;</italic> is due to the symmetry-breaking-induced torque, the drift angle <italic>&#x003b1;</italic> is a manifestation of the induced transverse force. <xref ref-type="fig" rid="f3">Figure 3a</xref> shows an instantaneous snapshot of the unsteady vorticity field forming behind a falling body (<italic>Re</italic>=156) during steady drift. A constant drift angle in an unsteady wake could not have been anticipated from fixed-body experiments, as it is well-known that freely falling bodies may have highly non-trivial descent paths due to wake-induced oscillations<xref ref-type="bibr" rid="b17">17</xref>.</p><p>Next, we show how the turn and drift angles depend on the splitter-plate length for a steady wake (<italic>Re</italic>=45). In <xref ref-type="fig" rid="f3">Fig. 3b</xref>, a distinct bifurcation from a straight position of the plate (<italic>&#x003b8;</italic>=0) to a skewed position (<italic>&#x003b8;</italic>&#x02260;0) is observed. <xref ref-type="fig" rid="f3">Figure 3c</xref> shows that plates with a non-zero turn angle have an oblique path (<italic>&#x003b1;</italic>&#x02260;0). The drift angle in <xref ref-type="fig" rid="f3">Fig. 3c</xref> displays the same features as the drift angle from our soap-film experiments in <xref ref-type="fig" rid="f2">Fig. 2d</xref>, namely, that as <italic>L</italic>&#x02192;0 the drift angle tends to zero and that there exists an optimal length for maximum drift. Despite that the Reynolds number of the computations is several orders of magnitude smaller than the experiments (<italic>Re</italic>=12,000), we have a good qualitative agreement between the two for both turn and drift angles.</p></sec><sec disp-level="2"><title>A theoretical model</title><p>Having established that symmetry breaking is prevalent in the presence of rigid plates for fixed/falling bluff bodies with steady/unsteady wakes (<italic>Re</italic>=45&#x02013;12,000), we now develop a model that uncovers the underlying instability mechanism. Consider an inverted pendulum system as shown in <xref ref-type="fig" rid="f4">Fig. 4a</xref> that is confined between two walls and free to rotate. Due to the offset between centre-of-mass and fixation point (centre of cylinder), the symmetric straight configuration is unstable and it relaxes to either side of the supporting walls in the presence of any small perturbation. We claim that the physical mechanism for the instability of the symmetric straight configuration of a bluff body with a compact appendage placed in a free stream is similar to the inverted pendulum. The pressure (instead of gravity) in the recirculation zone behind the bluff body acts as the destabilizing force (<xref ref-type="fig" rid="f4">Fig. 4b</xref>).</p><p>Consider a steady and uniform free stream <italic>U</italic> and a body consisting of a rigid splitter plate clamped at one point to a circular cylinder (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). The body is free to rotate around the centre of the cylinder, but it may not translate. We assume that the flow is equal to <italic>U</italic> everywhere, except inside a confined back-flow region behind the cylinder. Inside this region the flow, denoted by <italic>U</italic>_R, is uniform, steady and in opposite direction to <italic>U</italic>. We also assume that the shape of the back-flow region is in the form of an ellipse (Methods: Back-flow region).</p></sec><sec disp-level="2"><title>Normal forces on the plate</title><p>An inclined plate in a free stream experiences a normal pressure force that depends on the inclination angle <italic>&#x003b8;</italic>. To find a steady state in our model, part of the splitter plate must be outside the back-flow region and exposed to the free stream <italic>U</italic>, whereas the remaining part is inside the back-flow region and experiences a uniform reversed flow <italic>U</italic>_R. The length of splitter plate inside the back-flow region is given by a function <italic>B</italic>(<italic>&#x003b8;</italic>), which depends on the turn angle <italic>&#x003b8;</italic>. We will show that it is the competition between the forces acting on these two parts of the plate that determines the stability of the system. Since the instability exists under steady conditions (as shown using numerical simulations in <xref ref-type="fig" rid="f3">Fig. 3b,c</xref>), we assume steady forces. Further neglecting viscous forces<xref ref-type="bibr" rid="b18">18</xref>, the total force on each part of splitter plate due to the fluid can be modelled as</p><p><disp-formula id="eq2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e518" xlink:href="ncomms6310-m2.jpg"/></disp-formula></p><p>inside the back-flow region and</p><p><disp-formula id="eq3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e523" xlink:href="ncomms6310-m3.jpg"/></disp-formula></p><p>outside the region (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). Here, <italic>A</italic> is a force law calibration coefficient and <italic>&#x003c1;</italic>_f is the fluid density. The constant coefficient <italic>k</italic>&#x0003e;0 describes the (averaged) magnitude of the force density on the inner part of the splitter plate relative to the outer part. It is shown in the Methods section (Normal force on an inclined plate) that our model of the forces is a special case of a commonly used model for describing the forces on a freely falling plate<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>.</p></sec><sec disp-level="2"><title>The onset of instability</title><p>We further assume that the splitter plate is sufficiently thin, such that its weight can be neglected. As a result, the centre-of-mass coincides with the pivot point and the total torque around this point is</p><p><disp-formula id="eq4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e549" xlink:href="ncomms6310-m4.jpg"/></disp-formula></p><p>where <inline-formula id="d33e552"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e553" xlink:href="ncomms6310-m5.jpg"/></inline-formula> and <inline-formula id="d33e555"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e556" xlink:href="ncomms6310-m6.jpg"/></inline-formula>. In our model the condition for the equilibrium state is zero torque. We thus seek turning angles <italic>&#x003b8;</italic>, for which the total torque on the body vanishes. This condition is satisfied for the trivial straight position <italic>T</italic>(0)=0 and for two non-trivial skewed positions <italic>T</italic>(&#x000b1;<italic>&#x003b8;</italic>_s)=0.</p><p>The linear stability of the trivial solution is determined by the sign of the first-order term of the Taylor expansion of <italic>T</italic>(<italic>&#x003b8;</italic>) around <italic>&#x003b8;</italic>=0, that is,</p><p><disp-formula id="eq7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e586" xlink:href="ncomms6310-m7.jpg"/></disp-formula></p><p>which results in the following condition for instability</p><p><disp-formula id="eq8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e591" xlink:href="ncomms6310-m8.jpg"/></disp-formula></p><p>When <xref ref-type="disp-formula" rid="eq8">equation (5)</xref> holds, any small deviation from the zero angle <italic>&#x003b8;</italic>=&#x000b1;<italic>&#x003b5;</italic>, induces a torque in the direction away from the zero angle. Setting the left-hand side of expression (5) to zero and solving a quadratic equation gives the critical length <inline-formula id="d33e603"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e604" xlink:href="ncomms6310-m9.jpg"/></inline-formula> of the splitter plate for the onset of instability,</p><p><disp-formula id="eq10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e608" xlink:href="ncomms6310-m10.jpg"/></disp-formula></p><p>This condition is independent of the coefficient <italic>A</italic>. The two empirical parameters <italic>k</italic> and <inline-formula id="d33e617"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e618" xlink:href="ncomms6310-m11.jpg"/></inline-formula> can be chosen based on wake measurements (see Methods: Model parameters).</p><p>At <inline-formula id="d33e623"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e624" xlink:href="ncomms6310-m12.jpg"/></inline-formula>, the straight position <italic>&#x003b8;</italic>=0 loses its stability, and two attracting states <italic>&#x003b8;</italic>=&#x000b1;<italic>&#x003b8;</italic>_s appear via a pitchfork bifurcation. These non-trivial equilibria can be found by setting the expression in curly brackets of <xref ref-type="disp-formula" rid="eq4">equation (4)</xref> to zero, which results in a quadratic equation for <italic>&#x003b8;</italic>_{<italic>s</italic>},</p><p><disp-formula id="eq13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e649" xlink:href="ncomms6310-m13.jpg"/></disp-formula></p><p>This condition corresponds to a geometrical problem and can be solved analytically for <italic>&#x003b8;</italic>_{<italic>s</italic>} (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 1</xref> and <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 9</xref>). In <xref ref-type="fig" rid="f3">Fig. 3b</xref>, we compare the predictions of the analytical model with the turn angle of a free-falling body (<italic>Re</italic>=45), where we assert that our model captures the bifurcation with respect to <inline-formula id="d33e671"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e672" xlink:href="ncomms6310-m14.jpg"/></inline-formula> very well. In <xref ref-type="fig" rid="f1">Fig. 1d</xref>, we compare the turn angle based on the steady force law (<xref ref-type="disp-formula" rid="eq2">equations (2)</xref> and <xref ref-type="disp-formula" rid="eq3">(3)</xref>) with the time-averaged turn angle of the soap-film experiments at <italic>Re</italic>=12,000. We observe a good agreement, which indicates that a steady model is sufficient to capture the instability threshold. In the limit of zero splitter-plate length <inline-formula id="d33e686"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e687" xlink:href="ncomms6310-m15.jpg"/></inline-formula>, our model predicts that the stable position is at the angle &#x000b1;<italic>&#x003b8;</italic>₀, for which the flow separates from the body surface (<italic>&#x003b8;</italic>₀&#x02248;55 degrees for a cylinder). This is in agreement with our experimental (<xref ref-type="fig" rid="f1">Fig. 1d</xref>) and numerical (<xref ref-type="fig" rid="f3">Fig. 3b</xref>) results, where the angle <italic>&#x003b8;</italic> does not approach zero as <inline-formula id="d33e710"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e711" xlink:href="ncomms6310-m16.jpg"/></inline-formula>. Our study thus indicates that an arbitrary small protrusion&#x02014;but larger than the scales of surface irregularities of the cylinder surface&#x02014;will make the cylinder turn and stabilize at an angle for which the boundary layer on the body detaches from the surface.</p></sec><sec disp-level="2"><title>Drift induced by the instability</title><p>Our model also predicts the drift angle for a falling body. Due to the asymmetric pressure field at skewed equilibrium turn angles, a non-zero net force, <italic>F</italic>_d, in the transverse direction to the free stream acts on the object. We may decompose this drift force into a part acting on the cylinder body <italic>F</italic>_cylinder,d and a part acting on the splitter plate <italic>F</italic>_plate,d. The latter contribution can be obtained by projecting forces (<xref ref-type="disp-formula" rid="eq2">equations 2</xref> and <xref ref-type="disp-formula" rid="eq3">3</xref>) on the direction orthogonal to the free stream. The former contribution can be taken into account by assuming <italic>F</italic>_cylinder,d=<italic>A</italic>_c<italic>F</italic>_plate,d, because for any angle <italic>&#x003b8;</italic>, the cylinder and the plate experience the same pressure field (validation of this assumption based on numerical computations is reported in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2a</xref>). Thus, the total drift force on the object can be written as</p><p><disp-formula id="eq17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e763" xlink:href="ncomms6310-m17.jpg"/></disp-formula></p><p>where <italic>&#x000c3;</italic>=<italic>A</italic>(1+<italic>A</italic>_c). This parameter is determined by calibration with numerical simulations (see Methods: Model parameters).</p><p>For a freely falling object, the force acting on the body from the fluid must be balanced by the gravitational force (<xref ref-type="fig" rid="f5">Fig. 5a</xref>). The turn angle <italic>&#x003b8;</italic>_s with respect to the direction of movement in the freely falling case is the same as the turn angle <italic>&#x003b8;</italic>_s with respect to the direction of the free stream in the static case (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). The drift angle <italic>&#x003b1;</italic> in the former case thus corresponds to the force angle in the latter case, given by</p><p><disp-formula id="eq18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e801" xlink:href="ncomms6310-m18.jpg"/></disp-formula></p><p>After inserting <xref ref-type="disp-formula" rid="eq17">equation (8)</xref> into this expression for <italic>F</italic>_d with <italic>&#x000c3;</italic>=<italic>C</italic>_D/4, we obtain</p><p><disp-formula id="eq19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e822" xlink:href="ncomms6310-m19.jpg"/></disp-formula></p><p>From this expression one finds the drift angle is in the same direction as the splitter plate is tilted (<xref ref-type="supplementary-material" rid="S1">Supplementary Note 2</xref>). The direction of drift force can be explained by the fact that the pressure force outside the back-flow region has a larger lever arm than the force inside the region, and thus generates a larger torque <italic>T</italic>_out. To balance <italic>T</italic>_out with the torque on the plate inside the back-flow region, a larger force inside the back-flow region is required to compensate for the smaller lever arm. As such, the force inside the back-flow region is larger than outside and thus determines the drift direction. This is in agreement with our numerical findings; in <xref ref-type="fig" rid="f3">Fig. 3c</xref> we compare the drift angle <italic>&#x003b1;</italic> obtained from (10) with the drift angle from numerical simulations of a freely falling body, where we again observe a good agreement. The drift observed in the soap-film experiments of the hanging body is compared to (10) in <xref ref-type="fig" rid="f2">Fig. 2d</xref>.</p><p>So far, we have presented evidence of the IPL instability and its consequence for locomotion in two dimensions and for rigid appendages. Our final results show that the IPL instability is also present and meaningful in three dimensions and for flexible appendages.</p></sec><sec disp-level="2"><title>IPL instability for flexible appendages</title><p>Flexible appendages, often observed on plants and animals, are also susceptible to an IPL instability. We performed soap-film experiments of circular cylinder of diameter <italic>D</italic>=6.88&#x02009;mm fixed (no rotation or translation allowed) at its centre with a flexible filament (silk) attached to its rear end (see Methods: Soap-film experiments of a fixed body). When the filament is sufficiently long, the inertial and elastic forces of the filament interact with the fluid pressure, causing the filament to flap (<xref ref-type="fig" rid="f6">Fig. 6b</xref>). This archetype of fluid&#x02013;structure interaction problem is often used as a model of flag fluttering<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>. The time-average mean position of the long filament is a vertical straight position, and therefore the flapping is symmetric. However, due to the IPL instability, filaments shorter than a critical length (<italic>L</italic>_c=3.3<italic>D</italic>) have an asymmetric mean position (<xref ref-type="fig" rid="f6">Fig. 6a</xref>) that is sustained for all times. This is manifested by a non-zero average angle <italic>&#x003b8;</italic> formed by the vertical centre axis with the straight line connecting the filament anchor point to its tail position. The elastic appendage undergoes the same type of bifurcation (<xref ref-type="fig" rid="f6">Fig. 6c</xref>) as observed for the splitter-plate configuration, and is surprisingly well captured by our rigid-plate model.</p></sec><sec disp-level="2"><title>IPL instability for three-dimensional bodies</title><p>Using direct numerical simulations, we show that a three-dimensional object undergoes an IPL instability with an ensuing side force on the body. The object under consideration has a density ratio <italic>&#x003c1;</italic>_s/<italic>&#x003c1;</italic>_f =5 and is composed of a sphere of diameter <italic>D</italic> and a thin elliptic-shaped sheet (<xref ref-type="fig" rid="f7">Fig. 7a&#x02013;c</xref>). The sheet is attached to the sphere and protrudes a maximum length of <italic>L</italic>=0.8<italic>D</italic> from the rear stagnation point of the sphere (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>). The object is subject to a constant free stream <italic>U</italic> in the streamwise direction <italic>x</italic>. We allow the body to rotate around the transverse axis <italic>y</italic> and to translate in the <italic>yz</italic>-plane. The three degrees of freedom of the body can be described by the angles <italic>&#x003b8;</italic>, <italic>&#x003b1;</italic> and <italic>&#x003b3;</italic>. The two former angles correspond to the turn angle and to the drift angle in the <italic>zx</italic>-plane (<xref ref-type="fig" rid="f7">Fig. 7b</xref>), that is, <italic>&#x003b8;</italic> is the deviation of the sheet from the direction of movement in the <italic>zx</italic>-plane and <italic>&#x003b1;</italic> is the angle formed between the velocity of the object in the <italic>z</italic>-direction and the free-stream velocity <italic>U</italic> in the <italic>x</italic>-direction. Similarly, the angle formed between the velocity in the <italic>y</italic>-direction and <italic>U</italic> is denoted by <italic>&#x003b3;</italic>.</p><p>At <italic>Re</italic>=<italic>UD</italic>/<italic>&#x003bd;</italic>=200 a steady axisymmetric wake<xref ref-type="bibr" rid="b23">23</xref> forms behind the sphere alone (no sheet attached). Due to symmetry, the sphere neither rotates nor drifts (<xref ref-type="fig" rid="f7">Fig. 7d,e</xref>). According to the IPL model and our two-dimensional investigations, we expect that by adding an appropriate protrusion to the sphere, the object will rotate and experience a non-zero transverse force. Indeed, in the presence of the elliptic sheet, we observe that after a transient time, the sphere stabilizes at a turn angle of <italic>&#x003b8;</italic>=&#x02212;8.5 degrees (<xref ref-type="fig" rid="f7">Fig. 7d</xref>) and drifts with a constant velocity in the <italic>zx</italic>-plane with an angle of <italic>&#x003b1;</italic>=4.5 degrees (<xref ref-type="fig" rid="f7">Fig. 7e</xref>). We observe a zero drift in the <italic>xy</italic>-plane, that is, <italic>&#x003b3;</italic>=0.0 degrees (<xref ref-type="fig" rid="f7">Fig. 7e</xref>). The drift is a consequence of the IPL instability: any small perturbation causes the sheet to move away from the straight unstable position (<italic>&#x003b8;</italic>=0) and to settle on a skewed stable angle <italic>&#x003b8;</italic>_s. The new equilibrium breaks the symmetry of the wake (<xref ref-type="fig" rid="f7">Fig. 7b</xref>) in the <italic>zx</italic>-plane, which in turn induces a side force on the body in the <italic>z</italic>-direction, making it drift. Note that the trait of the IPL-induced movement&#x02014;the direction of drift and the direction that the appendage is titled in are the same&#x02014;is present. Although the chosen three-dimensional appendage triggers the IPL instability, its size and shape have not been optimized to yield maximum drift.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>There exist many motile animals with splitter-plate-shaped appendages<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. While the presence of a splitter plate has for a long time been associated to a &#x02018;trick&#x02019; to reduce drag on bodies<xref ref-type="bibr" rid="b2">2</xref>, it has not until the present work been associated to the generation of rotation and drift. We can postulate two requirements that need to be fulfilled for an organism to make use of the IPL instability for locomotion. First, when the organism moves in the fluid, a separated region has to be formed around its body. This requirement excludes very small organisms, where fluid inertia is negligible, that is, the Stokes flow regime. Second, the passive appendage needs to be sufficiently short, such that a significant portion of its area is exposed to a reversed flow. We have shown that the instability is a two-dimensional mechanism, but that the induced drift is equally significant in three dimensions as in two. We expect that the consequences for locomotion can be even more significant if the three-dimensional shape of an appendage is optimized to yield maximum drift. In particular, it would be interesting to investigate whether these optimized shapes resemble appendages that have evolved naturally.</p><p>In conclusion, we identify a new mechanism for locomotion; within a biologically well-inhabited domain of parameters (<italic>Re</italic>=45&#x02013;12,000, covering steady and unsteady wakes) moving two- or three-dimensional bodies with short rigid or flexible protrusions are likely to undergo an IPL instability. We believe that these results form a foundation, from which scientists can discover the existence of the IPL instability in various forms in nature. The beauty of this passive locomotion technique is that no energy needs to be expended by the animal; instead the existing energy in the flow is used.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Soap-film experiments of a fixed body</title><p>The experiment is performed with a gravity-driven soap film located at KTH in Stockholm (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The typical size of the test section is 1.2&#x02009;m long and 8&#x02009;cm wide. The fluid velocity is varied between 1 and 3&#x02009;m&#x02009;s^&#x02212;1, and the corresponding film thickness varies from 1 to 4&#x02009;&#x003bc;m. The fluid velocity&#x02014;measured with Laser Doppler velocimetry&#x02014;is close to uniform at the centre of the channel (the variation of the fluid velocity was below &#x000b1;2.5% over 70% of the channel span). The diameter of the cylinder is around 6.5&#x02009;mm and the length of the filament/splitter plate varies from 6 to 50&#x02009;mm. For the cylinder&#x02013;flexible filament system, a fixed cylinder made of plexiglass puncture the film; the filament&#x02014;a silk fibre with a diameter of 0.25&#x02009;mm&#x02014;pass through a hole at the back of the cylinder. The bending stiffness of the silk filament is determined by measuring its Young&#x02019;s modulus with a tensile test. Using the area moment of inertia of the filament, the bending stiffness is found to be 0.04&#x02009;erg&#x02009;cm^&#x02212;1. The cylinder&#x02013;splitter plate are made with a plastic sheet (0.1&#x02009;mm thick); the cylinder is free to rotate around a solid axis passing through its centre. For the visualization of the flow a low-pressure sodium lamp is used, and the resulting interference fringes are filmed with a high-speed camera at 500&#x02009;Hz, the resolution of the camera is 2,048 &#x000d7; 2,048 pixels, 1,500 images are taken for each test corresponding to a recorded time of 1.5&#x02009;s. To determine the position of the end of the filament/splitter plate the recorded grey-scale images are binarized in black and white images using a threshold in which the filament/splitter plate is black and the background is white. The position of the end of the filament/splitter plate are then averaged in time to determine the mean position and turn angle of the filament/splitter plate.</p></sec><sec disp-level="2"><title>Soap-film experiments of a hanging body</title><p>The experiment is performed with a gravity-driven soap film in Bordeaux. The setup in terms of the typical size of the test section, the fluid velocity and film are the same as in the soap-film experiments of the fixed body, except that for the visualization of the flow a white lamp is used. We use cylinder with diameter <italic>D</italic>=7&#x02009;mm, to which splitter length with length <italic>L</italic>=2.1<italic>D</italic> is attached. We fixed it on a &#x02018;loose&#x02019; pendulum (see <xref ref-type="fig" rid="f2">Fig. 2a</xref>) made of a thin nylon wire. This wire crosses the film perpendicularly through a small hole drilled in the centre of the disk. The system, consisting of a disk and splitter plate, is free to rotate under such conditions. We estimate the drift force <italic>F</italic>_d from a force-balance equation. When the pendulum has reached equilibrium, the following forces act on the cylinder and splitter-plate system (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4b</xref>): the drift force <bold>F</bold>_d, the drag on the system <bold>F</bold>_drag (we approximate it with the drag of cylinder alone, see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 1</xref>), the weight of the disk <bold>P</bold> and the tension of the wire <bold>T</bold>. At equilibrium, the torque around the fixation point of the pendulum is zero. Neglecting the wire weight (which means underestimating the drift force), this condition leads to,</p><p><disp-formula id="eq20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1108" xlink:href="ncomms6310-m20.jpg"/></disp-formula></p><p>where <italic>L</italic>_p is the length of the pendulum and <italic>&#x003b2;</italic> is the deviation angle of the pendulum. We may calculate <italic>F</italic>_d as</p><p><disp-formula id="eq21"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1126" xlink:href="ncomms6310-m21.jpg"/></disp-formula></p><p>where <italic>&#x003b4;</italic> is the displacement (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 4b</xref>) between the position given by the symmetric state (<xref ref-type="fig" rid="f2">Fig. 2b</xref>) and the position of the disk in the asymmetric state (<xref ref-type="fig" rid="f2">Fig. 2c</xref>). Thus, to determine <italic>F</italic>_d, we measured <italic>&#x003b4;</italic> and <italic>P</italic> and estimated <italic>F</italic>_drag. Using those measurements, we obtain the force angle, shown in <xref ref-type="fig" rid="f2">Fig. 2d</xref>.</p></sec><sec disp-level="2"><title>Numerical simulations of a two-dimensional body</title><p>We discretize the two-dimensional incompressible Navier&#x02013;Stokes equations with a staggered-grid, finite-volume formulation using a second-order semi-implicit time integration scheme. The no-slip boundary condition is enforced at Lagrangian points by appropriate regularized surface forces<xref ref-type="bibr" rid="b24">24</xref>. A uniform grid size of <italic>h</italic>=1/25 dimensionless length units is sufficient to reproduce previous work<xref ref-type="bibr" rid="b25">25</xref> on freely falling circular cylinder in terms of trajectory. The dimensionless length is the diameter of the cylinder, <italic>D</italic>. The equations for the motion of the rigid body are coupled to fluid solver implicitly. This ensures numerical stability at density ratios between solid and fluid as low as <italic>&#x003c1;</italic>_s/<italic>&#x003c1;</italic>_f=1.0001. The resulting linear equation system is solved using approximation of block-LU decomposition<xref ref-type="bibr" rid="b24">24</xref> and direct solver<xref ref-type="bibr" rid="b26">26</xref>. The freely falling cylinder with the splitter plate is placed in a large computational box with no-slip boundary conditions imposed at the walls. The box dimensions depend on the duration of transient behaviour; typical size used is 40<italic>D</italic> in width and 110<italic>D</italic> in height. A uniform cartesian grid covers a large region, typical size is 20<italic>D</italic> wide and 90<italic>D</italic> high. In the region between the uniform mesh and domain boundary, the mesh is expanded smoothly. The no-slip boundary condition at the box wall is always at least 10<italic>D</italic> away from the body. The time-dependent simulation is continued until it reaches a terminal motion. The angle of drift is obtained using linear regression of the trajectory in the terminal regime. The obtained angle is compared with corresponding simulation of the cylinder alone, using the same mesh and the same initial conditions. The cylinder alone has a small drift (around 0.3 degrees) due to boundary effects. To increase the accuracy, we subtract the unphysical drift from the results of cylinder with splitter plate for all lengths. The angle of splitter-plate orientation is determined as a mean over terminal regime.</p></sec><sec disp-level="2"><title>Back-flow region</title><p>In the proposed model, the back-flow region defines the boundary between two sections of the plate; the two sections experience a total normal force in opposite directions. Thus, the underlying assumption of our model is that the normal force on the plate from the fluid changes sign at some distance from the cylinder surface. Indeed, our two-dimensional numerical computations verify that when a plate of a given length stabilizes at a particular angle, there exists a point on the plate for which the normal force changes sign. In <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5a</xref>, we show with black stars these points for different plate lengths. Ideally, these points would define the function <italic>B</italic>(<italic>&#x003b8;</italic>), however, one does not always have a detailed information about force distribution over an appendage available. Therefore, in what follows we suggest a way to approximate the function <italic>B</italic>(<italic>&#x003b8;</italic>) with measurements of the wake in the absence of a plate.</p><p>In <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5b</xref> streamlines (black lines) of the flow past a two-dimensional circular cylinder at <italic>Re</italic>=45 are shown. The unperturbed wake consists of two steady symmetrical vortices and the length of the recirculation bubble <italic>L</italic>_w is around 2.5<italic>D</italic> (measured from the rear stagnation point). In the same figure, we show contours (green lines) of zero azimuthal velocity <italic>u</italic>_{<italic>&#x003b8;</italic>}, defined as</p><p><disp-formula id="eq22"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1252" xlink:href="ncomms6310-m22.jpg"/></disp-formula></p><p>where <italic>&#x000ea;</italic>_{<italic>&#x003b8;</italic>}=(&#x02212;cos<italic>&#x003b8;</italic>, sin<italic>&#x003b8;</italic>) is the azimuthal unit vector (as defined in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5d</xref>), and <bold>u</bold>=(<italic>u</italic>_{<italic>x</italic>},<italic>u</italic>_{<italic>y</italic>}) is the velocity field. Excluding the vertical centre line, we observe that <italic>u</italic>_{<italic>&#x003b8;</italic>} =0 encloses a region with a length about 1.2<italic>D</italic>. This region is compared with the one obtained from the numerical force distribution (black stars) in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 5c</xref>, where we observe that the two regions are similar in shape. We have approximated this shape with a half ellipse.</p></sec><sec disp-level="2"><title>Normal force on an inclined plate</title><p>Our starting point for modelling the forces on the splitter plate attached to the cylinder surface is the same as the force model for freely falling rigid plates<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>. To simplify the problem of a moving plate in a still fluid, we consider the plate in a translating coordinate system as shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6a</xref>. This transformation results in a fixed plate subject to an incoming free stream. The direction of the free stream is from bottom to top (in <italic>&#x00177;</italic> direction) to better resemble a free-falling motion. Then, following the work of Andersen <italic>et al.</italic><xref ref-type="bibr" rid="b27">27</xref>, we write the drag force <bold>F</bold>_D and lift force <bold>F</bold>_L on the plate as</p><p><disp-formula id="eq23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1329" xlink:href="ncomms6310-m23.jpg"/></disp-formula></p><p><disp-formula id="eq24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1332" xlink:href="ncomms6310-m24.jpg"/></disp-formula></p><p>where <italic>&#x003c1;</italic>_f is the density of the fluid, <italic>a</italic> is the length of the plate, <italic>A</italic>, <italic>E</italic>, <italic>C</italic>_T and <italic>C</italic>_R are constants, <inline-formula id="d33e1360"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1361" xlink:href="ncomms6310-m25.jpg"/></inline-formula> is the angular velocity and <italic>U</italic>=|<bold>U</bold>| is the flow speed. The drag force is an empirical formulation proportional to velocity square and the turn angle, as suggested by Wang <italic>et al.</italic><xref ref-type="bibr" rid="b28">28</xref> The lift force is based on the empirical model for circulation</p><p><disp-formula id="eq26"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1376" xlink:href="ncomms6310-m26.jpg"/></disp-formula></p><p>developed by Pesavento and Wang<xref ref-type="bibr" rid="b29">29</xref>. The above model of forces may be used to describe the motion of the plate (with additional models of added mass and the plate inertia). The coefficients <italic>A</italic>, <italic>E</italic>, <italic>C</italic>_T and <italic>C</italic>_R are usually calibrated to fit experimental or numerical data of the trajectory of the falling body.</p><p>The normal force component can be obtained by projection of lift and drag forces</p><p><disp-formula id="eq27"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1402" xlink:href="ncomms6310-m27.jpg"/></disp-formula></p><p>where the normal unit vector <inline-formula id="d33e1405"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1406" xlink:href="ncomms6310-m28.jpg"/></inline-formula> is defined in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>. We simplify this expression by assuming steady conditions and zero contribution from viscous forces (see <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref> and associated discussion below). In a steady configuration, <inline-formula id="d33e1414"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1415" xlink:href="ncomms6310-m29.jpg"/></inline-formula>, which renders the second term on the right-hand side in the lift force (15) zero. In the absence of viscous forces, the tangential force component <bold>F</bold>_{<italic>&#x003c4;</italic>} is zero. When <italic>&#x003b8;</italic>=0,</p><p><disp-formula id="eq30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1429" xlink:href="ncomms6310-m30.jpg"/></disp-formula></p><p>which results in the condition <italic>E</italic>=<italic>A</italic>. When <italic>&#x003b8;</italic>&#x02260;0, we have</p><p><disp-formula id="eq31"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1444" xlink:href="ncomms6310-m31.jpg"/></disp-formula></p><p>where the trigonometric identities sin2<italic>&#x003b8;</italic>=2sin<italic>&#x003b8;</italic> cos<italic>&#x003b8;</italic> and 1&#x02212;cos2<italic>&#x003b8;</italic>=2sin²<italic>&#x003b8;</italic> have been used. This expression is zero for <italic>C</italic>_T=<italic>A</italic>.</p><p>Inserting the conditions <inline-formula id="d33e1475"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1476" xlink:href="ncomms6310-m32.jpg"/></inline-formula>, <italic>C</italic>_{<italic>T</italic>}=<italic>A</italic> and <italic>E</italic>=<italic>A</italic> in (16), the normal force component (which now is also the total force on the plate) can be written as</p><p><disp-formula id="eq33"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1495" xlink:href="ncomms6310-m33.jpg"/></disp-formula></p><p>To model the forces on a plate attached to the rear end of a cylinder, we assume that a section of the plate (near the cylinder surface) with length <italic>a</italic>₁=<italic>B</italic>(<italic>&#x003b8;</italic>) experiences a reversed flow <inline-formula id="d33e1509"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1510" xlink:href="ncomms6310-m34.jpg"/></inline-formula>; the outer section of the plate of length <italic>a</italic>₂=<italic>L</italic>&#x02212;<italic>B</italic>(<italic>&#x003b8;</italic>) is exposed to the incoming free stream <bold>U</bold>₁=<italic>U&#x00177;</italic>. As shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6b</xref>, the inclined plate experiences uniform steady flows from two directions. Then, by considering the two sections separately, we arrive with the model of normal forces in <xref ref-type="disp-formula" rid="eq2">equations (2)</xref> and <xref ref-type="disp-formula" rid="eq3">(3)</xref>. In this model, <italic>k</italic> defines a constant scaling between the force on the outer side and the inner side of the splitter plate.</p><p>Let us assess our force model (<xref ref-type="disp-formula" rid="eq2">equations (2)</xref> and <xref ref-type="disp-formula" rid="eq3">(3)</xref>) by comparing the torque <italic>T</italic>(<italic>&#x003b8;</italic>) around the body from our model (<xref ref-type="disp-formula" rid="eq4">equation (4)</xref>) with the torque extracted from numerical simulations of a body fixed at various turn angles (<italic>Re</italic>=45). <xref ref-type="supplementary-material" rid="S1">Supplementary Figure 7</xref> shows that by calibrating <italic>A</italic> appropriately (in this case <italic>A</italic>=<italic>C</italic>_D/16=0.094), our force law provides a reasonable model of the numerically computed torque. Moreover, in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 7</xref>, we report on the magnitude of the viscous torque in the generated total torque. We observe that the contribution of the viscous component is smaller than the pressure component; using pressure force alone to compute torque introduces less than one degree error in the equilibrium turn angle. This observation underlies our assumption of zero contribution of viscous forces to the total force. Note, however, that viscosity is necessary to induce boundary layer separation. Thus, while we neglect the viscous component in the total force acting on the plate, our model takes viscosity into account implicitly by modelling a back-flow region.</p></sec><sec disp-level="2"><title>Model parameters</title><p>Here, we propose to determine parameters <italic>B</italic>_max, <italic>k</italic> and <italic>&#x003b8;</italic>₀ from measurements of the wake behind the body without an appendage and to calibrate the coefficient <italic>&#x000c3;</italic> with measurement of the drift force on the body. Let us start with <italic>B</italic>_max by considering the azimuthal velocity of the unperturbed wake, which is zero along the centre line. For a two-dimensional flow the derivative of the azimuthal velocity on centre line is given by</p><p><disp-formula id="eq35"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1616" xlink:href="ncomms6310-m35.jpg"/></disp-formula></p><p>where the continuity equation (&#x02202;<italic>u</italic>_{<italic>x</italic>}/&#x02202;<italic>x</italic>=&#x02212;&#x02202;<italic>u</italic>_{<italic>y</italic>}/&#x02202;<italic>y</italic>) and coordinate relation (&#x02202;/&#x02202;<italic>&#x003b8;</italic>=&#x02212;<italic>r</italic>&#x02202;/&#x02202;<italic>x</italic> at <italic>&#x003b8;</italic>=0) have been used. In <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8a,b</xref> we see that the derivative of the azimuthal velocity changes sign along the centre line. One can conclude that the point on the centre line, where the derivative of the azimuthal velocity changes sign, corresponds to the point where the centre line intersects with the (off-centre) zero isoline of azimuthal velocity. This is the point that we suggest to use for determining the value of <inline-formula id="d33e1653"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1654" xlink:href="ncomms6310-m36.jpg"/></inline-formula>. In <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8a</xref>, we show &#x02202;_{<italic>&#x003b8;</italic>}<italic>u</italic>_{<italic>&#x003b8;</italic>} for the flow past a cylinder at <italic>Re</italic>=45, where one observes that <inline-formula id="d33e1672"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1673" xlink:href="ncomms6310-m37.jpg"/></inline-formula>. In <xref ref-type="fig" rid="f3">Fig. 3b</xref>, using the slightly smaller value of <inline-formula id="d33e1679"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1680" xlink:href="ncomms6310-m38.jpg"/></inline-formula> provided the same critical value <italic>L</italic>_{<italic>c</italic>} as the full numerical simulations. Thus by combining measurements of the cylinder wake without a protrusion and our model, one may predict the critical value for bifurcation with a 90% accuracy. Regarding the soap-film experiments, we measure the vertical velocity component along the centre line, and use its time-averaged value to obtain &#x02202;_{<italic>&#x003b8;</italic>}<italic>u</italic>_{<italic>&#x003b8;</italic>} from <xref ref-type="disp-formula" rid="eq35">equation (20)</xref>. The derivative of the azimuthal velocity obtained from experiments is shown in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8b</xref>, where a change of sign is observed at <inline-formula id="d33e1704"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1705" xlink:href="ncomms6310-m39.jpg"/></inline-formula>; for model predictions in <xref ref-type="fig" rid="f2">Fig. 2d</xref>, <xref ref-type="fig" rid="f1">Fig. 1d</xref> and <xref ref-type="fig" rid="f6">Fig. 6c</xref>, the length was <inline-formula id="d33e1716"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1717" xlink:href="ncomms6310-m40.jpg"/></inline-formula>. Thus, the accuracy of wake measurements is around 70%.</p><p>We propose to obtain the value <italic>k</italic> by estimating the forcing inside and outside of the back-flow region. Using the definitions of <inline-formula id="d33e1724"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1725" xlink:href="ncomms6310-m41.jpg"/></inline-formula> and <inline-formula id="d33e1727"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1728" xlink:href="ncomms6310-m42.jpg"/></inline-formula> and choosing <italic>L</italic>=<italic>L</italic>_w as a representative splitter-plate length, we may estimate <italic>k</italic> from</p><p><disp-formula id="eq43"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1744" xlink:href="ncomms6310-m43.jpg"/></disp-formula></p><p>We aim to estimate <italic>k</italic> from a wake without an appendage. It is not possible to measure these forces directly, since they do not exist before the introduction of the splitter plate; therefore we suggest to use measurements of the azimuthal velocity close to the straight position (<italic>&#x003b8;</italic>=0) to estimate the force ratio. Since <italic>u</italic>_{<italic>&#x003b8;</italic>} approaches zero as <italic>&#x003b8;</italic>&#x02192;0, we assume direct proportionality</p><p><disp-formula id="eq44"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1764" xlink:href="ncomms6310-m44.jpg"/></disp-formula></p><p>where</p><p><disp-formula id="eq45"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1769" xlink:href="ncomms6310-m45.jpg"/></disp-formula></p><p>are the average azimuthal velocities inside and outside of the BFR. The limit can be found as</p><p><disp-formula id="eq46"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1775" xlink:href="ncomms6310-m46.jpg"/></disp-formula></p><p>Evaluating the above integrals for the numerical profile (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8a</xref>), we obtain <italic>k</italic>=0.83. We used a slightly larger value of <italic>k</italic>=0.9 in our model. The discrepancy is small when taking into account the number of assumptions employed to derive the model. The integral of the experimental profile (<xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 8b</xref>) results in <italic>k</italic>=0.18, whereas the value <italic>k</italic>=1.0 was used in the model predictions in <xref ref-type="fig" rid="f2">Fig. 2d</xref>, <xref ref-type="fig" rid="f1">Fig. 1d</xref> and <xref ref-type="fig" rid="f6">Fig. 6c</xref>. We are thus able to determine the order of magnitude of the forcing coefficient <italic>k</italic> from local measurements of one velocity component.</p><p>Moreover, from measurements we observe that the angle for which the boundary layer separates from the cylinder is close to <italic>&#x003b8;</italic>₀&#x02248;55 degrees for the range of Reynolds numbers under investigation.</p><p>Finally, to determine the coefficient <italic>&#x000c3;</italic>, we calibrate our model with the drift force <italic>F</italic>_d extracted from numerical simulations at <italic>Re</italic>=45 of the whole body (cylinder and splitter plate) fixed at various turn angles. The numerical force is compared with the drift force (<xref ref-type="disp-formula" rid="eq17">equation (8)</xref>) predicted from our model in <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 2b</xref> for <italic>&#x000c3;</italic>=<italic>C</italic>_D/4, where <italic>C</italic>_D=1.5 is the drag coefficient of the whole body.</p></sec><sec disp-level="2"><title>Numerical simulations of a three-dimensional body</title><p>We use the open-source software OpenFOAM<xref ref-type="bibr" rid="b30">30</xref> to solve the three-dimensional incompressible Navier&#x02013;Stokes equations. A sphere is placed in a computational box, which is <italic>L</italic>_{<italic>x</italic>}=40<italic>D</italic> long, <italic>L</italic>_{<italic>y</italic>}=30<italic>D</italic> wide and <italic>L</italic>_{<italic>z</italic>}=40<italic>D</italic> high. Here <italic>D</italic> denotes the sphere diameter, which also serves as a reference unit length. The coordinates for the sphere centre are (15,15,20)<italic>D</italic>. A uniform velocity is imposed at the inflow and a Neumann condition at the outflow, whereas on the lateral sides of the box a slip boundary condition is enforced. No-slip boundary condition are imposed on the object. A hexahedral-dominant mesh is generated using the utilities blockMesh and snappyHexMesh. The former generates a uniform cartesian mesh, while the latter inserts the geometry of the body and refines the mesh locally. The final mesh consists of around 750,000 cells. The chosen solver is pimpleFoam and its extension for dynamic meshes pimpleDyMFoam. This solver makes use of a blend of PIMPLE and PISO algorithms to handle the pressure&#x02013;velocity coupling, together with an adaptive choice of the timestep under a maximum Courant number condition. To compute the rigid motion of the body, we use the sixDoFRigidBodyMotion solver, which also handles the dynamic mesh (by rotating and stretching the cells according to the body motion<xref ref-type="bibr" rid="b31">31</xref>). The coupling between the fluid and the body is solved with an explicit scheme, that is, using the so-called weak-coupling approach. To validate the solver, we performed a simulation of the fixed sphere at <italic>Re</italic>=200 and obtained a good agreement in terms of the wake length and drag coefficients with previous studies<xref ref-type="bibr" rid="b23">23</xref>. We complement the sphere with an elliptic sheet (length behind sphere is 0.8<italic>D</italic>, thickness of sheet is 0.1<italic>D</italic>). The design of the appendage is shown in the <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 3</xref>. We use the same mesh settings and the same computational box, which was verified using the simulation of the sphere alone. The length of the simulation is limited due to large displacement of mesh towards the end of simulation, when the body approaches the boundaries of the box. To partially overcome this limitation, we fix the body for the first 50 time units and then allow it to move. In this way, most of the transient dynamics of the flow can be simulated when the object is at the initial position and no mesh deformation is required.</p></sec></sec><sec disp-level="1"><title>Author contributions</title><p>S.B. and A.M. conceived the original idea. S.B. initiated and supervised the research. N.B. performed the soap-film experiments of the fixed cylinder with feedback from F.L. F.I. and H.K. performed the soap-film experiments of the free-hanging cylinder. U.L. performed the numerical simulations of the two-dimensional free-falling cylinder. A.M. supervised the numerical simulations of the three-dimensional sphere. U.L. created the theoretical model, which he further developed with feedback from all authors. All authors analysed data. S.B. and U.L. wrote the paper.</p></sec><sec disp-level="1"><title>Additional information</title><p><bold>How to cite this article:</bold> L&#x00101;cis, U. <italic>et al.</italic> Passive appendages generate drift through symmetry breaking. <italic>Nat. Commun.</italic> 5:5310 doi: 10.1038/ncomms6310 (2014).</p></sec><sec sec-type="supplementary-material" id="S1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Figures 1-9 and Supplementary Notes 1-2</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s1.pdf"/></supplementary-material><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Movie 1</title><p>This movie shows soap-film experiments of a disc and splitter plate suspended to a pendulum as the film velocity is gradually increased. Initially, for low velocities, the body is in a symmetric state, but as the velocity is increased, the system becomes unstable. This results in a turn of the body as well as - because the disk splitter hang freely - a drift to the right (in the same direction as the tilt of the splitter).</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s2.mov"/></supplementary-material><supplementary-material id="d33e30" content-type="local-data"><caption><title>Supplementary Movie 2</title><p>The experimental setup is the same as in Supplementary Movie 1, but at a fixed film velocity for which the disk plus splitter is always in an asymmetric state. Initially, the splitter points to right. A needle is then brought close to the splitter plate and causes a perturbation of the flow. The splitter plate then changes angle and points to the left. The disk slowly drifts to the left as expected.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s3.mov"/></supplementary-material><supplementary-material id="d33e36" content-type="local-data"><caption><title>Supplementary Movie 3</title><p>In this movie we show a contour plot of the vorticity field (levels from -3.0 to 3.0) obtained from numerical simulations of a freely falling body (main paper Fig.3a) at Re = 156, L= 1.0 and &#x003c1;= 1.01. To demonstrate the drift of the body, the initial position is marked with red dot and the trajectory with a red line. </p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s4.mov"/></supplementary-material><supplementary-material id="d33e42" content-type="local-data"><caption><title>Supplementary Movie 4</title><p>We show soap-film experiments (film velocity v = 2 m/s) of a silk thread of length L = 6.6D attached to the rear of a cylinder of diameter D = 6.88 mm. The time average position of the flapping filament is a straight vertical line, rendering its motion symmetric.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s5.mov"/></supplementary-material><supplementary-material id="d33e48" content-type="local-data"><caption><title>Supplementary Movie 5</title><p>Same experiment as in Supplementary Movie 4, but now with a silk thread of length L = 1.26D, which is below the critical value for instability Lc = 3.3D. The motion of the filament is constraint to the right side of the recirculation zone.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s6.mov"/></supplementary-material><supplementary-material id="d33e54" content-type="local-data"><caption><title>Supplementary Movie 6</title><p>The animation shows vorticity contours of a three-dimensional sphere in the zx-plane. To model a free-falling object, a free-stream is imposed in the xdirection, and the object is allowed to translate in y and z directions as well as to rotate around the y-axis. The three degree-of-freedom rigid-body motion is enabled at t = 50, where it is observed that the body experience a very small drift to right (order 0.1 degrees) due to transient effects as well as asymmetries arising due to numerical approximations (in particular the computational grid).</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s7.mov"/></supplementary-material><supplementary-material id="d33e60" content-type="local-data"><caption><title>Supplementary Movie 7</title><p>Same numerical experiment as in Supplementary Movie 6, but now with an elliptic-shaped sheet of length L = 0.8D attached to the sphere. A clear drift to the left (order 1 degree) is observed. The appendage points to the left, as expected when bodies experience an IPL instability.</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="ncomms6310-s8.mov"/></supplementary-material></sec>

Hypoxia and fibrosis in chronic kidney disease: crossing at pericytes

<p>Chronic kidney disease (CKD) is placing an increasing burden on patients and societies because no decisive therapy has been established. Tubulointerstitial lesions accompanied by fibrosis, inflammatory cells, and capillary rarefaction not only characterize, but also aggravate renal dysfunction in CKD. In this setting, renal cells, particularly tubular cells, suffer from hypoxia caused by the imbalance of blood perfusion and oxygen demand despite their adaptive responses represented by upregulation of hypoxia-inducible factors (HIFs). Fibrosis is a pathological state characterized by excess extracellular matrix (ECM) deposition, which is also a hallmark and causative factor of many chronic diseases including CKD. Recent studies have suggested that the dominant origin of ECM-producing myofibroblasts (MFs) may be pericytes, which are indispensable cells for maintaining proper capillary functions, as they wrap capillaries and stabilize them through a fine-tuned interplay with endothelial cells. During fibrosis, pericytes are activated and detach from capillaries before conversion into MFs, which compromises capillaries and worsens hypoxia. We also discuss how hypoxia and HIFs affect fibrogenesis. Given that hypoxia is caused by insufficient angiogenesis and that fibrosis results from pericyte loss, restoration of pericytes should be an intriguing target for overcoming both hypoxia and fibrosis. We propose the deactivation of MFs to recover lost pericytes as a promising therapy for CKD.</p>

<contrib contrib-type="author"><name><surname>Kawakami</surname><given-names>Takahisa</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Mimura</surname><given-names>Imari</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Shoji</surname><given-names>Kumi</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Tanaka</surname><given-names>Tetsuhiro</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Nangaku</surname><given-names>Masaomi</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Division of Nephrology and Endocrinology, The University of Tokyo</institution>, Bunkyo-ku, Tokyo, <country>Japan</country></aff>

Kidney International Supplements

<p>Fibrosis is a pathological state with excessive deposition of extracellular matrix (ECM), which is commonly accompanied by chronic diseases in many organs, including the kidneys. Production of ECM occurs as a reaction against injury, and fibrosis itself is intrinsically a process to promote tissue repair. However, when it occurs in excess, it can deteriorate the functions of the affected organ. Therefore, fibrosis is not only an outcome of the injury, but also a mechanism leading to a further damage.^{<xref ref-type="bibr" rid="bib1">1</xref>}</p><p>In the kidney, tubulointerstitial fibrosis with injured tubules and inflammatory leukocytes is a hallmark of chronic kidney disease (CKD) with renal dysfunction, regardless of its cause. As the glomerular filtration rate, which indicates kidney function, continually declines, CKD results in end-stage renal disease necessitating renal replacement therapy. However, decreases in the glomerular filtration rate correlate with tubulointerstitial lesions better than with glomerular lesions, even in glomerulonephritis.^{<xref ref-type="bibr" rid="bib2">2</xref>} Collectively, the literature suggests that tubulointerstitial injury is a common feature of renal dysfunction, and a leading candidate as a target for CKD therapies.</p><p>How does tubulointerstitial injury occur? One of the most important mechanisms is tissue hypoxia.^{<xref ref-type="bibr" rid="bib3">3</xref>} Rarefaction of peritubular capillaries can also be observed in almost all kidney diseases, decreasing the oxygen supply and inducing hypoxia in tubulointerstitial areas. Furthermore, in fibrosis, pericytes, a constituent of peritubular capillaries, detach from vessels and produce ECM, which compromises the capillaries.^{<xref ref-type="bibr" rid="bib1">1</xref>} Therefore, tubulointerstitial injury, hypoxia, and fibrosis are intricately related to one another, which is mediated by capillary rarefaction.</p><p>In this review, we verify the critical roles of hypoxia in the pathophysiology of CKD, briefly summarize the mechanisms of fibrosis, and discuss the effects of hypoxia in the context of fibrosis. Thereafter, we debate the potential of pericytes as a promising target for therapy in CKD, as it can ameliorate capillary loss as well as tissue hypoxia.</p><sec><title>HYPOXIA IN CKD</title><p>The kidney is physiologically hypoxic despite its plentiful blood supply (as much as 20&#x00025; of the cardiac output in humans), because an oxygen shunt is present between arteries and veins that run in close proximity.^{<xref ref-type="bibr" rid="bib3">3</xref>} Therefore, it is reasonable to consider that erythropoietin-producing cells reside in the kidney, where they can sensitively detect hypoxia due to anemia. Physiological hypoxia in the kidney has been demonstrated in mammals using pimonidazole measurements and in hypoxia-monitoring transgenic mice and rats created by exploiting the hypoxia-inducible factor (HIF) system.^{<xref ref-type="bibr" rid="bib4">4</xref>, <xref ref-type="bibr" rid="bib5">5</xref>} Augmented kidney hypoxia in CKD has also been confirmed in patients using blood oxygen-level&#x02013;dependent magnetic resonance imaging,^{<xref ref-type="bibr" rid="bib6">6</xref>} as well as in animal models using the methods mentioned above.^{<xref ref-type="bibr" rid="bib5">5</xref>}</p><p>In CKD, hypoxia occurs in tubulointerstitial areas through multiple mechanisms. First, glomerulosclerosis leads to a reduction of flow in downstream peritubular capillaries, which can be further compromised by constriction of the efferent arterioles of glomeruli and peritubular capillaries themselves due to activation of the renin&#x02013;angiotensin system. Second, distortion and loss of peritubular capillaries due to fibrosis decreases blood perfusion. Third, ECM deposition by fibrogenesis widens the distance between capillaries and tubules, diminishing the efficiency of oxygen diffusion.</p><p>Following exposure to hypoxia, cells induce adaptive responses to survive the harsh environment. Among them, the HIF transcription factors have crucial roles (<xref ref-type="fig" rid="fig1">Figure 1</xref>).^{<xref ref-type="bibr" rid="bib7">7</xref>} HIFs are heterodimers composed of an &#x003b1; subunit, including HIF-1&#x003b1;, HIF-2&#x003b1;, and HIF-3&#x003b1;, and a common &#x003b2; subunit, HIF-1&#x003b2;. Despite their constitutive expression, the &#x003b1; subunits of HIF are degraded by proteasomes and are not functional under normoxic conditions. Prolyl hydroxylases (PHDs) hydroxylate the conserved proline residues of HIF-&#x003b1; using oxygen, recruiting the von-Hippel-Lindau protein, which is a recognition component of E3 ubiquitin ligase. This results in the ubiquitination of the modified HIF-&#x003b1; subunits, and eventually leads to their proteasomal degradation. Under hypoxic conditions, HIF-&#x003b1; escapes post-translational modification by PHDs, forms a heterodimer with HIF-1&#x003b2;, and promotes the expression of its target genes. Its targets include glycolytic enzymes, which allow anaerobic ATP production, and angiogenic factors, including vascular endothelial growth factors (VEGFs), which can promote new vessel formation to increase oxygen supply.</p><p>Despite such cellular-adaptive mechanisms, hypoxia causes tubular cell injury and death, which advances CKD.^{<xref ref-type="bibr" rid="bib8">8</xref>} The proximal tubules are considered more susceptible to hypoxia because they solely depend on aerobic oxidative metabolism.^{<xref ref-type="bibr" rid="bib9">9</xref>} Damaged tubular cells can, in turn, worsen glomerular lesions through tubular obstruction and maladaptive tubuloglomerular feedback. They can also induce interstitial fibrosis, as described below. Thus, tubular injury aggravates two leading causes of tubular hypoxia, glomerular injury and interstitial fibrosis, which forms a vicious cycle and advances CKD.</p><p>Therefore, these findings suggest that enhanced HIF activity might protect the kidney against hypoxic injury and suppress the progression of CKD. In fact, activation of HIFs by the systemic administration of an inhibitor of PHDs has been shown to mitigate tubular injury and capillary rarefaction in various CKD models.^{<xref ref-type="bibr" rid="bib10">10</xref>} These results suggest that inhibitors of PHDs may have therapeutic potential in CKD.</p><p>Recent studies have also demonstrated that hypoxia can cause epigenetic changes in cells. For example, hypoxic conditions alter the transcription of some genes through changes in the chromatin conformational structure and histone modification changes.^{<xref ref-type="bibr" rid="bib11">11</xref>} Such epigenetic alterations in hypoxic renal cells may affect the pathophysiology of CKD, and may be newly emerging targets for therapy.</p></sec><sec><title>MECHANISMS OF FIBROSIS</title><p>In chronic diseases, tissue damage persists without resolution, often along with inflammatory leukocytes and fibrosis, as some repair mechanisms may be insufficient or maladaptive. Therefore, it is reasonable to consider recurrent and persistent injury to epithelial cells, which fulfill the predominant physiological functions in tissues, as the prime element that initiates and sustains fibrogenesis. Epithelial cell death can provoke pro-inflammatory responses through damage-associated molecular pattern molecule receptors, and lead to fibrosis. Cellular stress in epithelial cells also induces activation of innate immunity through the production of cytokines and chemokines.^{<xref ref-type="bibr" rid="bib1">1</xref>}</p><p>The cells that actually produce pathologic ECM are called myofibroblasts (MFs), which are activated mesenchymal cells that express &#x003b1;-smooth muscle actin. Because epithelial-to-mesenchymal transition was proposed as a dominant mechanism that gives rise to MFs, their origin has been debated for decades. However, recent studies indicate that the dominant origin of MFs is not epithelial cells, but mesenchymal cells in most organs, including the kidneys, liver, lungs, skin, and spinal cord.^{<xref ref-type="bibr" rid="bib1">1</xref>} In the kidney, some studies have shown that most of these mesenchymal fibroblasts are attached to the microvasculature and have proven to be pericytes.^{<xref ref-type="bibr" rid="bib12">12</xref>} Other studies, however, argue that these mesenchymal progenitor cells are &#x02018;fibroblasts', although they did not investigate their anatomical relationship with vessels.^{<xref ref-type="bibr" rid="bib13">13</xref>, <xref ref-type="bibr" rid="bib14">14</xref>} Pericytes are cells that wrap around the endothelial tubes of capillaries, some of which are embedded in the basement membrane of capillaries with direct and indirect contact with endothelial cells.^{<xref ref-type="bibr" rid="bib15">15</xref>} Factors produced by injured epithelial cells and other cells, including VEGFs, platelet-derived growth factors (PDGFs), fibroblast growth factors, and transforming growth factor-&#x003b2;, activate pericytes and induce their detachment from capillaries and conversion to MFs.^{<xref ref-type="bibr" rid="bib1">1</xref>} In addition, it is worth noting that pericytes are erythropoietin-producing cells in the kidney, and MFs lose the capacity to produce this hormone.^{<xref ref-type="bibr" rid="bib13">13</xref>}</p><p>Accumulation of inflammatory cells is accompanied by fibrosis. These cells are derived from both resident and recruited cell populations.^{<xref ref-type="bibr" rid="bib16">16</xref>} Among them, monocytes and macrophages have central roles in fibrosis. Resident macrophages maintain tissue homeostasis under physiological conditions and react upon tissue insults. Monocytes are leukocytes in the circulating blood, but they are recruited to damaged tissue, where they differentiate into macrophages. Although it is well known that these monocytes/macrophages have a broad spectrum of functions and are subdivided, e.g., M1 and M2 macrophages, as a whole, they are thought to have a tendency to promote renal fibrosis because their ablation in a typical fibrosis model, unilateral ureteric obstruction (UUO), mitigates fibrosis.^{<xref ref-type="bibr" rid="bib16">16</xref>} These cells produce profibrotic and pro-inflammatory factors, which maintain activation of MFs and their fibrogenesis. Furthermore, these inflammatory cells induce additional epithelial cell injury, creating a positive-feedback loop and developing fibrosis.</p><p>Taken together, fibrosis advances in a manner in which repetitive and unresolved epithelial injury primes pericyte activation, inducing them to become MFs and generate ECM, which is augmented by profibrotic monocytes/macrophages.</p></sec><sec><title>HYPOXIA IN FIBROSIS</title><p>In this section, we will describe the roles of hypoxia, particularly HIFs, in fibrosis, with regard to its effects on epithelial cells, pericytes/fibroblasts, and monocytes/macrophages.</p><sec><title>Epithelial cells</title><p>Increased HIF-&#x003b1; in the renal tubular cells of transgenic mice with conditional von-Hippel-Lindau protein knockout in adulthood enhances VEGF and PDGF-B expression and augments endothelial cell proliferation, increasing their numbers. Although increased production and deposition of ECM were also observed in these transgenic mice compared with control mice, they did not display renal injury or dysfunction^{<xref ref-type="bibr" rid="bib17">17</xref>} These results are consistent with studies showing that conditional knockout of HIF-1&#x003b1; in the proximal tubules lessens fibrosis in mouse UUO.^{<xref ref-type="bibr" rid="bib18">18</xref>} Given that deposition of ECM is a part of repair processes unless it is uncontrolled, it is suggested that HIF activation by hypoxia in tubular cells can mitigate renal damage through the upregulation of angiogenic and fibrogenic factors as adaptive responses. This idea is consistent with the observation that general activation of HIFs mitigates renal injury in a CKD model.^{<xref ref-type="bibr" rid="bib10">10</xref>}</p></sec><sec><title>Pericytes</title><p>Human renal fibroblasts, isolated by morphologic criteria, showed increased expression of collagen I&#x003b1;1 and tissue inhibitor of metalloproteinase-1, when they were exposed to hypoxia.^{<xref ref-type="bibr" rid="bib19">19</xref>} By contrast, in mouse renal interstitial fibroblasts, hypoxia did not induce expression of collagen I, &#x003b1;-smooth muscle actin, or transforming growth factor-&#x003b2;1 <italic>in vitro</italic>.^{<xref ref-type="bibr" rid="bib20">20</xref>} The discrepancy may derive from differences in the origins of the cells and/or methods of culture. For example, in hepatic stellate cells, which are considered as the equivalent of pericytes in the liver, hypoxia increases collagen I and VEGF expression.^{<xref ref-type="bibr" rid="bib21">21</xref>}</p><p>During kidney fibrosis, VEGF and PDGF signaling induces pericyte detachment from capillaries and conversion to MFs. Given that hypoxia promotes the production of both growth factors in epithelial cells as mentioned above, hypoxia may indirectly cause pericyte loss, although its direct effects on pericytes remain to be elucidated.^{<xref ref-type="bibr" rid="bib22">22</xref>}</p><p>It should be noted that even primary pericytes derived from intact tissue tend to be activated and have characteristics of MFs when they are cultured under normal conditions without special stimuli. Therefore, to determine the precise roles of hypoxia and/or HIFs in pericytes, investigation of the effects of specific overexpression and knockout of HIFs in these cells <italic>in vivo</italic> is warranted.</p></sec><sec><title>Monocytes and macrophages</title><p>Lysozyme M (LysM)-Cre is often utilized to control the expression of a gene specifically in monocytes and macrophages. Mice with increased HIF expression in monocytes/macrophages, in which von-Hippel-Lindau protein is knocked out by LysM-Cre, had fewer macrophages, although they exhibited comparable fibrosis and tubular damage in a UUO model.^{<xref ref-type="bibr" rid="bib23">23</xref>} However, specific knockout of both HIF-1&#x003b1; and HIF-2&#x003b1; in macrophages increased the number of macrophages in UUO without affecting fibrosis. This discrepancy between inflammatory cells and fibrosis appears inconsistent with studies, showing that the ablation of macrophages mitigates fibrosis.^{<xref ref-type="bibr" rid="bib16">16</xref>} One possible explanation is that HIF upregulation and HIF downregulation change the characteristics or subpopulations of macrophages, which alters the effects of alterations in macrophage number. This may result from a varied LysM-Cre efficiency in macrophage subtypes because LysM-Cre deletes a floxed gene in no more than 80&#x00025; of macrophages.^{<xref ref-type="bibr" rid="bib24">24</xref>} In addition, LysM is also expressed in neutrophils, and gene knockout can therefore also occur in the neutrophils of mice with a LysM-Cre transgene. Therefore, changes in gene expression in neutrophils may have affected the development of fibrosis.</p><p>Contrasting properties of HIFs in myeloid-lineage cells have been reported in liver fibrosis. In a bile duct ligation model, macrophage-specific HIF-1&#x003b2; knockout in mice using LysM-Cre led to less fibrosis and ameliorated tissue injury. In conditional knockout mice, the numbers of macrophages had a tendency to decrease, but the difference was not significant. Similar results were obtained with HIF-1&#x003b1; knockout. In this study, PDGF-B expression in macrophages was reduced by HIF knockout, which may contribute to the observed decrease in fibrosis.^{<xref ref-type="bibr" rid="bib25">25</xref>}</p></sec></sec><sec><title>PERICYTES AS A TARGET TO TREAT HYPOXIA AND FIBROSIS</title><p>Peritubular capillary rarefaction and fibrosis are causative factors in advancing CKD, as well as common hallmarks of CKD. Therefore, it is important to overcome these two defects in CKD therapy.</p><p>This has been attempted through efforts to augment angiogenesis, and to preserve peritubular capillaries and mitigate hypoxia in tubulointerstitial areas.^{<xref ref-type="bibr" rid="bib26">26</xref>} Considering that the simple invasion of endothelial cells into nascent organs during development provides inductive signals to promote organogenesis even without blood flow,^{<xref ref-type="bibr" rid="bib27">27</xref>} angiogenesis may also promote tissue repair and regeneration through effects beyond simple oxygen and nutrition supply. As such, angiogenesis appears to be a more promising therapy for CKD, but success using this approach has not been achieved. However, angiogenesis occurs through complicated mechanisms including the coordinated actions of angiogenic factors. Among them, VEGF-A (VEGF) has predominant roles and by itself can induce angiogenesis.^{<xref ref-type="bibr" rid="bib28">28</xref>}</p><p>Although treatment with VEGF gives rise to new vessel formation, the new vessels are leaky and unstable. Because leukocytes can be recruited through leaky vessels, they are also pro-inflammatory. This may be related to the fact that proliferation of endothelial cells unexpectedly occurs during the early phase of UUO, when increased VEGF expression and macrophages numbers are also observed, followed by the loss of peritubular capillaries and advanced fibrosis.^{<xref ref-type="bibr" rid="bib29">29</xref>} This suggests that incomplete angiogenesis may leave capillaries unstable with subsequent worsening of inflammation and fibrosis.</p><p>These flaws primarily result from the absence of pericytes, which cover immature vessels composed of endothelial cells, inhibit the recruitment of inflammatory cells, and enable the vessels to function properly. It is natural to speculate that pericytes are converted into MFs, and physiological, or properly functioning, pericytes are scarce in CKD. Therefore, deactivation of MFs with subsequent restoration of pericytes is the cornerstone for ameliorating both hypoxia and fibrosis.</p><p>How can we achieve this goal? First, we should consider why hypoxic tubular cells cannot induce sufficient angiogenesis although hypoxia is one of the most potent angiogenic stimuli. As mentioned above, in CKD, tubular cells are under hypoxic stress and can express angiogenic factors, including VEGF. It is possible that an imbalance and/or a deficiency exists in the ability of some angiogenic factors to form mature and stable vessels by recruiting pericytes. It is known that several growth factor families, such as PDGFs, angiopoietins, and transforming growth factor-&#x003b2;, contribute to the recruitment of pericytes to immature vessels composed of endothelial cells.^{<xref ref-type="bibr" rid="bib28">28</xref>} If tubular cells predominantly express VEGF and insufficient pericyte-recruiting growth factors, the new vessels will be immature and have fewer pericytes. In addition, pericyte-recruiting factors secreted from tubular cells maladaptively induce the detachment of pericytes from capillaries rather than recruitment to capillaries (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Future studies that verify the profiles and actions of angiogenic growth factors may facilitate CKD therapy aimed at proper angiogenesis.</p><p>Given that fibrosis is essentially a protective response to tissue injury and is resolved upon removal of the injury, MFs should be capable of reverting to pericytes. In fact, chronic fibrosis can be reversible in patients when the primary insult is eliminated.^{<xref ref-type="bibr" rid="bib30">30</xref>} However, in advancing fibrosis, pericyte recruitment to newer vessels may be hindered by its fibrogenic phenotype. The exact mechanisms that maintain activated states in MFs and prevent them from reverting to pericytes remain to be elucidated, although many of the growth factors involved in these processes have been identified. Verifying the culprit in intracellular signaling should provide clues to a novel therapeutic method for pathologic fibrosis. Furthermore, restored pericytes should resume the production of erythropoietin,^{<xref ref-type="bibr" rid="bib13">13</xref>} which would ameliorate anemia in CKD. Hence, pericytes mitigate kidney hypoxia not only by increasing vessel stability but also by enhancing the capacity of blood to convey oxygen.</p><p>Macrophages also have roles in both angiogenesis and fibrosis.^{<xref ref-type="bibr" rid="bib16">16</xref>, <xref ref-type="bibr" rid="bib24">24</xref>} They can express VEGFs, PDGFs, fibroblast growth factors, and angiopoietins, sometimes in response to hypoxia. During angiogenesis and/or fibrosis, these factors are secreted from so-called &#x02018;M2' macrophages, which promote tissue repair. However, M2 macrophages are thought to be further subdivided into angiogenic and fibrogenic macrophages, although their relevance <italic>in vivo</italic> remains unclear.^{<xref ref-type="bibr" rid="bib16">16</xref>, <xref ref-type="bibr" rid="bib24">24</xref>} It is also possible that macrophages express a relatively homogenous repertoire of growth factors that is dependent on the surrounding milieu. Regardless, macrophages can affect pericyte activation. Therefore, control of the differentiation of macrophage subtypes and/or growth factor production is another possible method for CKD therapy.</p><p>In addition to determining the roles of particular cell populations, studies have revealed that the general activation of HIFs by PHD inhibition can promote angiogenesis. For example, peptide-mediated inhibition of PHDs generates more mature and less leaky vessels than those generated by growth factors.^{<xref ref-type="bibr" rid="bib31">31</xref>} Knockdown of PHD2 in endothelial cells or pericytes also leads to more stable vessels.^{<xref ref-type="bibr" rid="bib32">32</xref>} These findings indicate the existence of some unidentified factors or mechanisms promoting vessel stabilization through pericyte recruitment. Chemical PHD inhibitors are under development and have therapeutic potential for the treatment of CKD.</p></sec><sec sec-type="conclusions"><title>CONCLUSIONS</title><p>Although CKD and the resultant renal replacement therapy are an increasing burden for patients and society, no decisive therapeutic method has been developed. Fibrosis and hypoxia are major therapeutic targets for treating tubulointerstitial lesions in CKD. During fibrosis, pericytes leave capillaries and become ECM-producing MFs. This process results in capillary fragility and rarefaction, which are central causes of hypoxia. Therefore, deactivating MFs to restore pericytes naturally emerges as a potential CKD therapy. Much still remains to be elucidated, but future investigations may unravel the detailed mechanisms of the fibrotic process and thereby open new avenues for promising therapies to prevent or reverse CKD.</p></sec>

Obesity, oxidative stress, and fibrosis in chronic kidney disease

<p>Obesity in combination with diabetes and hypertension likely is contributing to the increasing incidence of chronic kidney disease (CKD) in the 21st century worldwide and requires novel insights and strategies for treatment. There is an increasing recognition that the kidney has an important role in the complex inter-organ communication that occurs with the development of inflammation and fibrosis with obesity. Inhibition of the adiponectin-AMPK pathway has now become established as a critical pathway regulating both inflammation and pro-fibrotic pathways for both obesity-related kidney disease and diabetic kidney disease. AMPK regulates NF&#x003ba;B activation and is a potent regulator of NADPH oxidases. Nox4 in particular has emerged as a key contribtor to the early inflammation of diabetic kidney disease. AMPK also regulates several transcription factors that contribute to stimulation of the transforming growth factor-beta (TGF-&#x003b2;) system. Another key aspect of AMPK regulation is its control of mammalian target of rapamycin (mTOR) and mitochondrial biogenesis. Inhibition of PGC-1&#x003b1;, the transcriptional co-activator of mitochondrial biogenesis is being recognized as a key pathway that is inhibited in diabetic kidney disease and may be linked to inhibition of mitochondrial function. Translation of this concept is emerging via the field of urine metabolomics, as several metabolites linked to mitochondria are consistently downregulated in human diabetic kidney disease. Further studies to explore the role of AMPK and related energy-sensing pathways will likely lead to a more comprehensive understanding of why the kidney is affected early on and in a progressive manner with obesity and diabetes.</p>

<contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Kumar</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Center for Renal Translational Medicine, Institute for Metabolomic Medicine, Department of Medicine, University of California San Diego/Veterans Affairs San Diego Healthcare System</institution>, La Jolla, California, <country>USA</country></aff>

Kidney International Supplements

<p>The worldwide increase in obesity likely contributes to development and progression of kidney disease with both diabetes and hypertension. The contribution of obesity has been highlighted by a large epidemiologic study, where the body mass index was found to be the second most important contributor to relative risk for developing end-stage renal disease, after proteinuria, among all subjects who were registered as patients and followed for 27&#x02013;36 years.^{<xref ref-type="bibr" rid="bib1">1</xref>} With obesity contributing to multi-organ disease, there is increasing recognition that the kidney is affected at the very stages of obesity and may contribute to systemic inflammation. In this review, studies linking the adiponectin-5&#x02032;-AMP activated protein kinase (AMPK) pathway will be highlighted with respect to development of inflammation and fibrosis with obesity-related and diabetic kidney disease.</p><sec><title>ADIPONECTIN IN OBESITY-RELATED KIDNEY DISEASE</title><p>Adiponectin is a 30-kDa (Acrp30) protein predominantly produced in the adipose tissue and circulates in the plasma as a trimer (low molecular weight), a hexamer generated from two trimers, or as multimers consisting of 12&#x02013;18 hexamers (high molecular weight; HMW). Levels of adiponectin are decreased in obesity, coronary artery disease, and type 2 diabetes mellitus.^{<xref ref-type="bibr" rid="bib2">2</xref>} In our prior studies, adiponectin levels were found to be inversely correlated with low-grade albuminuria in obese African-American subjects.^{<xref ref-type="bibr" rid="bib3">3</xref>} A similar relationship was found in patients with hypertension from Europe and in patients from Japan.^{<xref ref-type="bibr" rid="bib4">4</xref>, <xref ref-type="bibr" rid="bib5">5</xref>} Interestingly, this relationship was primarily found in subjects with elevated body mass index and with low-grade albuminuria. A large study with 440 subjects recently found the same inverse relationship between adiponectin levels and albuminuria in subjects with obesity, even after adjusting for other risk factors.^{<xref ref-type="bibr" rid="bib6">6</xref>} However, in patients with established diabetes or chronic kidney disease (CKD), adiponectin is positively correlated with albuminuria or proteinuria.^{<xref ref-type="bibr" rid="bib7">7</xref>, <xref ref-type="bibr" rid="bib8">8</xref>} It remains controversial whether serum adiponectin levels predict future cardiovascular risk factors in CKD subjects.^{<xref ref-type="bibr" rid="bib9">9</xref>, <xref ref-type="bibr" rid="bib10">10</xref>} A potential role for adiponectin to act on podocytes has been identified based on expression studies of the receptors for adiponectin. Adiponectin receptor 1 and adiponectin receptor 2 are the two major receptors for adiponectin and are described as a new class of heptahelix receptors structurally and functionally distinct from G-protein-coupled receptors.^{<xref ref-type="bibr" rid="bib11">11</xref>} Both receptors signal via the AMPK pathway. Adiponectin receptor 1 gene expression is expressed in the mouse kidney and podocytes to a similar degree as in liver, whereas adiponectin receptor 2 gene expression of kidney and podocyte is much less than liver.^{<xref ref-type="bibr" rid="bib3">3</xref>} Protein studies are difficult to interpret as the commercially available antibodies may not be specific and sensitive. Presently, based on the published epidemiologic studies there is convincing evidence that in subjects who are obese (without diabetes or CKD) there is a relationship between circulating adiponectin levels and low-grade albuminuria, however, the role for adiponectin to mechanistically contribute to albuminuria will be difficult to establish in human studies.</p><p>A cause and effect role for adiponectin in the development of kidney disease has been supported by several independent studies using different mouse models of manipulating adiponectin. Studies in one strain of adiponectin knockout (KO) mice by our group identified that the KO mice have elevated levels of albuminuria but only two- to threefold greater than controls on a C57Bl6 background.^{<xref ref-type="bibr" rid="bib3">3</xref>, <xref ref-type="bibr" rid="bib12">12</xref>} With addition of hyperglycemia, there was a progressive increase in albuminuria in the KO mice, but not in the wild-type diabetic mice. Treatment with exogenous adiponectin was found to attenuate albuminuria and restore podocyte foot process effacement. In a similar vein, 5/6 nephrectomy was found to lead to an accelerated disease in a different adiponectin KO mouse.^{<xref ref-type="bibr" rid="bib13">13</xref>} In a recent study,^{<xref ref-type="bibr" rid="bib14">14</xref>} mice with a genetically engineered inducible podocyte injury have worse disease when lacking adiponectin, and have protection when overexpressing adiponectin. Thus, the available studies are in agreement that, in models of glomerular injury adiponectin has a protective role. However, in contrast there is conflicting data with acute kidney injury. In one study, adiponectin-deficient mice were protected from ischemia-reperfusion injury,^{<xref ref-type="bibr" rid="bib15">15</xref>}whereas an independent study found the reverse, i.e., that adiponectin deficiency exacerbated acute kidney injury after ischemia reperfusion.^{<xref ref-type="bibr" rid="bib16">16</xref>} The role of the specific adiponectin receptors in chronic or acute kidney injury remains to be established.</p></sec><sec><title>AMPK PATHWAY, INFLAMMATION, AND CKD</title><p>The major signaling pathways by which adiponectin appears to confer its effects is via stimulation of AMPK, Akt, Rab5, and phospholipase C.^{<xref ref-type="bibr" rid="bib17">17</xref>} AMPK is a stress-activated kinase that is activated in response to depleting ATP or a relative increase in the intracellular AMP/ATP ratio to preserve cell survival under a low-caloric environment.^{<xref ref-type="bibr" rid="bib18">18</xref>} AMPK was established to have a central role in the effects of adiponectin based on studies in the adiponectin receptor KO mice.^{<xref ref-type="bibr" rid="bib2">2</xref>} As obesity is associated with a reduction in adiponectin and an excess of calories will lead to a reduced AMP/ATP ratio, it would follow that AMPK would be reduced with obesity-related kidney disease.</p><p>We first reported that renal AMPK was reduced in a mouse model of high-fat-induced obesity (diet-induced obesity) within 1 week of the onset of the high-fat diet.^{<xref ref-type="bibr" rid="bib19">19</xref>} Surprisingly, there was evidence of renal inflammation (elevated urine hydrogen peroxide, urine, and glomerular monocyte chemoattractant protein-1 induction) by 1 week of the high-fat diet, and the inflammation preceded any increase in albuminuria. Stimulation of AMPK by 5-aminoimidazole-4-carbox-amide-1-beta-D-ribofuranoside (AICAR) was able to completely suppress the inflammatory markers and reduced mesangial cell production of monocyte chemoattractant protein-1 in response to palmitate. More recently, chronic stimulation of AMPK by AICAR (for 12 weeks) was also successful to reduce renal inflammation, albuminuria, and matrix accumulation with the high-fat diet (Decleves, <italic>Kidney Int</italic> 2014; 85: 611&#x02013;623). Furthermore, AMPK activation was able to completely reduce lipid vacuolization in proximal tubular cells as well. Part of the basis for the latter finding may be due to reduction of HMGCoA reductase activity with AMPK activation and reduced cholesterol production.</p><p>AMPK also seems to have a prominent role in regulating macrophage infiltration and activation. The overall numbers of macrophages infiltrating the kidney with a high-fat diet was completely normalized with AMPK activation. Furthermore, AMPK activation lowered the CD11c/CD11b ratio indicating a reduction in M1 macrophages (Decleves, <italic>Kidney Int</italic> 2014; 85: 611&#x02013;623). A role for AMPK in regulating macrophage activation has been highlighted recently^{<xref ref-type="bibr" rid="bib18">18</xref>} and will be an active area of research in future studies.</p><p>AMPK also appears to have a key role in regulating the NADPH oxidase (Nox) system. Of the major Nox isoforms that have been identified, it appears that Nox1, 2, and 4 may have a role in mediating the oxidative stress involved in CKD. We have previously identified that Nox4 was prominent in podocytes and that high glucose-induced upregulation of Nox4 can be blocked with adiponectin or activation of AMPK.^{<xref ref-type="bibr" rid="bib3">3</xref>} In separate studies, AMPK was found to inhibit Nox2 subunits via upregulating I&#x003ba;B and blocking NF-&#x003ba;B-induced stimulation of Nox subunits (p67, p47) in endothelial cells.^{<xref ref-type="bibr" rid="bib20">20</xref>}</p><p>A role for AMPK regulation of Nox4 was demonstrated in diabetic kidney disease by several groups and there is a growing consensus that Nox4 may be the most critical Nox linked to progression of diabetic kidney disease.^{<xref ref-type="bibr" rid="bib21">21</xref>, <xref ref-type="bibr" rid="bib22">22</xref>} Our group found that mice with Nox2 deficiency have the same degree of hyperglycemia and weight loss with streptozotocin-induced diabetes, however, the degree of diabetic kidney disease was not affected in the Nox2 KO diabetic group.^{<xref ref-type="bibr" rid="bib23">23</xref>} The degree of albuminuria, glomerular matrix expansion, and urine hydrogen peroxide was essentially the same in the wild-type and Nox2 KO diabetic groups. As there was a marked increase in Nox4 in the Nox2 KO diabetic kidney, it is possible that Nox4 may compensate for Nox2 and be sufficient to promote diabetic kidney disease. There are ongoing studies with a combined Nox1/Nox4 inhibitor. In other studies, inhibition of Nox1/Nox4 was found to be protective with liver disease and cardiac disease.^{<xref ref-type="bibr" rid="bib24">24</xref>, <xref ref-type="bibr" rid="bib25">25</xref>} These studies have taken on added importance, as new phase II studies are underway to evaluate the role of Nox inhibition for diabetic kidney disease.</p></sec><sec><title>AMPK, FIBROSIS, AND CKD</title><p>In addition to inflammation, AMPK has also been closely linked to fibrosis promoting pathways. In the high-fat diet model, chronic AMPK activation with AICAR was able to reduce mesangial matrix expansion and reduce urinary levels of TGF-&#x003b2;1 (Decleves, <italic>Kidney Int</italic> 2014; 85: 611&#x02013;623). Recently, we found that AMPK activation also markedly reduced glomerular TGF-&#x003b2;, collagen, and fibronectin accumulation in several mouse models of diabetic kidney disease (Dugan, <italic>J Clin Invest</italic> 2013; 123: 4888&#x02013;4899). Similar findings were also found with the OVE 26 mouse.^{<xref ref-type="bibr" rid="bib22">22</xref>} The mechanistic basis for how AMPK activation inhibits TGF-&#x003b2; is unclear at present. A prior study found that adiponectin and AMPK reduced TGF-&#x003b2;-induced matrix and myofibroblast transformation,^{<xref ref-type="bibr" rid="bib26">26</xref>} however, Smad2/3 phosphorylation was not affected. Recently, we found that a key transcription factor USF1 was translocated to the nucleus with high glucose exposure and completely blocked by AMPK activation.^{<xref ref-type="bibr" rid="bib27">27</xref>} As USF1 has been found to mediate glucose-induced stimulation of the TGF-&#x003b2;1 gene transcription, there could be an important effect of AMPK to regulate USF1-induced TGF-&#x003b2;1 synthesis.^{<xref ref-type="bibr" rid="bib28">28</xref>}</p><p>In addition to downstream effects by AMPK to regulate Nox and TGF-&#x003b2;, there is a well-established pathway by which AMPK inhibits mTOR activity. Several groups have identified that mTOR is activated in diabetic kidney disease.^{<xref ref-type="bibr" rid="bib29">29</xref>} Inhibition of mTOR is protective against diabetic kidney disease.^{<xref ref-type="bibr" rid="bib29">29</xref>} However, deletion of mTOR in podocytes also contributes to disease,^{<xref ref-type="bibr" rid="bib30">30</xref>} and treatment with rapamycin has been found to enhance proteinuria in some patients,^{<xref ref-type="bibr" rid="bib31">31</xref>} thus limiting its utility as a therapeutic for diabetic kidney disease. A recent study found that mTOR inhibition also led to reduced Nox4 levels in podocytes, suggesting that mTOR may have a direct effect to regulate Nox4 independent of AMPK.^{<xref ref-type="bibr" rid="bib32">32</xref>}</p></sec><sec><title>AMPK AND MITOCHONDRIAL FUNCTION</title><p>A key pathway by which AMPK stimulation protects cells in a calorie-deprived state is to stimulate the master regulator of mitochondrial biogenesis, PGC-1&#x003b1;. This transcriptional co-activator is a potent stimulator of many mitochondrial proteins and increases mitochondrial content.^{<xref ref-type="bibr" rid="bib33">33</xref>} In states of reduced AMPK activation, it would be expected that PGC-1&#x003b1; is also reduced. Indeed, PGC-1&#x003b1; was found to be markedly reduced in the muscle of patients with diabetes^{<xref ref-type="bibr" rid="bib34">34</xref>} and may be due partly to epigenetic modification of the PGC-1&#x003b1; promoter. Recently, we found that the diabetic kidney also had reduced PGC-1&#x003b1; levels in association with reduced AMPK, reduced mitochondrial content, and reduced mitochondrial complex activity (Dugan, <italic>J Clin Invest</italic> 2013; 123: 4888&#x02013;4899). This led to the question that, if there is reduced mitochondrial complex activity in the electron transport chain would there be a concomitant change in mitochondrial superoxide production? Indeed, we found that there was reduced superoxide production in the diabetic kidney using a real-time imaging protocol and further verified by <italic>ex vivo</italic> studies with electron paramagnetic resonance measurements. Thus, we found that the diabetic kidney is actually in a state of reduced mitochondrial activity and reduced mitochondrial superoxide production. This is in direct contrast to the prevailing notion that diabetic complications are due to an excess of mitochondrial superoxide!</p><p>We further sought to ask this question in patients with established diabetic kidney disease. To get an index of mitochondrial activity, we performed quantitative measurements of a variety of metabolites linked to various biochemical pathways linked to human disorders (Sharma, <italic>J Am Soc Nephrol</italic> 2013; 24: 1901&#x02013;1912). The predominant signature that was identified was a reduction of metabolites produced by mitochondrial enzymes. Semi-quantitative analysis of mitochondrial complex IV revealed reduction in kidney biopsies from patients with diabetic nephropathy. Furthermore, there was a reduction of gene expression for PGC-1&#x003b1; in diabetic kidney tissues, but not in minimal change disease. These set of studies help to establish a new paradigm for understanding diabetic kidney disease. An early and progressive reduction in mitochondrial content, potentially driven by reduced AMPK/PGC-1&#x003b1;, is linked to early renal inflammation and pro-fibrotic pathways (see <xref ref-type="fig" rid="fig1">Figure 1</xref>). Chronic exposure of cells to caloric excess, from excess glucose and/or high fat, is linked to reduction of AMPK, possibly due to transient or sustained reduction in the AMP/ATP ratio. Persistent reduction of AMPK activity allows for stimulation of inflammatory pathways mediated by NF&#x003ba;B and pro-fibrotic pathways mediated by USF1. Downstream of the transcription factors, Nox and TGF-&#x003b2; are stimulated and directly contributing to inflammation and fibrosis in the kidney and heart. Pathways that mitigate AMPK reduction are the adiponectin-LKB1 pathway as well as direct and indirect activators of AMPK, including AICAR, metformin, weight loss, and exercise. On the basis of several animal studies, stimulation of AMPK may mediate many of the beneficial effects to reduce inflammation and fibrosis.^{<xref ref-type="bibr" rid="bib18">18</xref>, <xref ref-type="bibr" rid="bib35">35</xref>, <xref ref-type="bibr" rid="bib36">36</xref>} Stimulation of AMPK via pharmacologic and non-pharmacologic interventions may well be beneficial in human kidney disease as well. Of note, non-pharmacologic means of increasing AMPK has been identified by exercise and food restriction, and exercise^{<xref ref-type="bibr" rid="bib37">37</xref>} has been shown to reduce diabetic kidney disease independently of weight loss and glucose lowering.^{<xref ref-type="bibr" rid="bib38">38</xref>}</p><p>In conclusion, recent studies in the past 2&#x02013;3 years on the basis of inflammation and fibrosis via the AMPK pathway has led to new insights and paradigms in our understanding of diabetic kidney disease. Additional investigation to understand the mechanistic underpinnings as to how reduced mitochondrial function is linked to inflammation and fibrosis will likely be an exciting and rewarding path to identify new biomarkers and therapeutics for obesity-related and diabetic CKD.</p></sec>

Overview of the cellular and molecular basis of kidney fibrosis

<p>The common pathogenetic pathway of progressive injury in patients with chronic kidney disease (CKD) is epitomized as normal kidney parenchymal destruction due to scarring (fibrosis). Understanding the fundamental pathways that lead to renal fibrosis is essential in order to develop better therapeutic options for human CKD. Although complex, four cellular responses are pivotal. (1) An interstitial inflammatory response that has multiple consequences&#x02014;some harmful and others healing. (2) The appearance of a unique interstitial cell population of myofibroblasts, primarily derived from kidney stromal cells (fibroblasts and pericytes), that are the primary source of the various extracellular matrix proteins that form interstitial scars. (3) Tubular epithelial cells that have variable and time-dependent roles as early responders to injury and later as victims of fibrosis due to the loss of their regenerative abilities. (4) Loss of interstitial capillary integrity that compromises oxygen delivery and leads to a vicious cascade of hypoxia&#x02013;oxidant stress that accentuates injury and fibrosis. In the absence of adequate angiogenic responses, a healthy interstitial capillary network is not maintained. The fibrotic &#x02018;scar' that typifies CKD is an interesting consortium of multifunctional macromolecules that not only change in composition and structure over time, but can be degraded via extracellular and intracellular proteases. Although transforming growth factor beta appears to be the primary driver of kidney fibrosis, a vast array of additional molecules may have modulating roles. The importance of genetic and epigenetic factors is increasingly appreciated. An intriguing but incompletely understood cardiorenal syndrome underlies the high morbidity and mortality rates that develop in association with progressive kidney fibrosis.</p>

<contrib contrib-type="author"><name><surname>Eddy</surname><given-names>Allison A</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Department of Pediatrics, Faculty of Medicine, University of British Columbia</institution>, Vancouver, British Columbia, <country>Canada</country></aff>

Kidney International Supplements

<p>The high prevalence and burden of chronic kidney disease (CKD) is well established. It is generally accepted that all primary causes of CKD share a common pathogenetic pathway of progressive injury due to the destructive consequences of scarring (fibrosis). Despite extensive research efforts to identify the critical cellular and molecular mediators of fibrosis, this rapidly expanding body of new knowledge has yet to be translated into clinical practice. Failure to do so has resulted in an estimated 13&#x02013;16&#x00025; of the adult population living with CKD (defined as individuals with an estimated glomerular filtration rate (eGFR) of &#x0003c;60&#x02009;ml/min per 1.73&#x02009;m²) who face the possible need of renal replacement therapy with dialysis and/or a kidney transplant at some point in the future. Unfortunately, the majority will never reach that point as CKD confers at least a fivefold increased risk of premature death due to accelerated cardiovascular disease. Recent and ongoing advances in basic science research provide the necessary platform for designing and testing novel therapies to change the unfortunate fate of CKD patients. The potential therapeutic repertoire is vast. It is conceivable that the next generation of personalized medicine will make it possible to offer specific therapeutic protocols comprising multiple agents that are selected based on the specific molecular signature of the scar-generating response that uniquely defines the CKD response in an individual. The key participants are now known, and therapeutically targetable steps are rapidly emerging. Four distinct cellular participants are known and the ideal therapeutic protocol should significantly regulate each of them (<xref ref-type="fig" rid="fig1">Figure 1</xref>). <list list-type="order"><list-item><p>Multifunctional inflammatory cells, especially macrophages;</p></list-item><list-item><p>myfibroblasts that determine the accumulation rate and molecular composition of the extracellular matrix proteins that constitute the kidney scars;</p></list-item><list-item><p>microvascular endothelial cells;</p></list-item><list-item><p>tubular epithelial cells.</p></list-item></list></p><p>Transforming growth factor beta (TGF&#x003b2;) is thought to be the grand master that elicits numerous signals that culminate in fibrosis and renal parenchymal loss.</p><sec><title>INFLAMMATORY CELLS</title><p>All CKD is characterized by an interstitial infiltrate of macrophages, the density of which correlates inversely with kidney survival.^{<xref ref-type="bibr" rid="bib1">1</xref>} Depending upon local environmental cues, macrophages can synthesize and secrete a variety of products that influence fibrogenesis. These include growth factors and cytokines (TGF&#x003b2;, platelet-derived growth factor, fibroblast growth factor, tumor necrosis factor alpha, interferon gamma, hepatocyte growth factor), enzymes and their inhibitors (angiotensin-converting enzyme, plasminogen activators, plasminogen activator inhibitor-1, collagenases, tissue inhibitor of metalloproteinases), matrix proteins (collagen, fibronectin, thrombospondin), and many others (complement proteins, coagulation factors, bioactive lipids, reactive oxygen species, nitric oxide, endothelin, etc.)^{<xref ref-type="bibr" rid="bib2">2</xref>} A variety of experimental cell depletion strategies have shown that reducing the number of interstitial macrophages reduces kidney fibrosis. The most recent experiments have taken advantage of genetic engineering strategies to selectively deplete macrophages and have shown remarkable benefit, even in genetic models such as polycystic kidney disease, a common cause of human CKD for which the important pathological interstitial changes have lived in the shadows of cystogenesis until recently.^{<xref ref-type="bibr" rid="bib3">3</xref>}</p><p>Although the multifunctional potential of macrophages associated with tissue injury has long been recognized, important scientific advances over the past decade, derived in large part from studies in mice, have begun to clarify the molecular basis of their functional diversity.^{<xref ref-type="bibr" rid="bib4">4</xref>} Classically activated &#x02018;M1' macrophages and alternatively activated &#x02018;M2' macrophages appear to originate from monocytes that are exposed to distinct and non-overlapping local stimuli. Best characterized are interferon gamma, lipopolysaccharide, tumor necrosis factor and granulocyte&#x02013;macrophage colony&#x02013;stimulating factor for the M1 subset that are primarily associated with tissue injury, whereas interleukin-4, interleukin-13, interleukin-10, corticosteroids, vitamin D, macrophage colony&#x02013;stimulating factor and TGF&#x003b2; preferentially polarize to the M2 subset that are more likely to promote tissue repair and injury resolution. Further work is needed to delineate the differential effector functions between M1 injury induction and M2 injury resolution. Some clues may come from gene-, protein- and metabolic profiling studies. For e.g., mannose receptor I, Yim-1 and arginase 1 appear restricted to M2 cells. The role of macrophages in injury repair was clearly demonstrated in a model of reversible liver injury: macrophage depletion during the phase of injury induction lessened the degree of fibrosis, but when their depletion was delayed until the phase of injury resolution, fibrosis severity was worse.^{<xref ref-type="bibr" rid="bib5">5</xref>} As fibrosis is an integral component of wound healing, further investigation is necessary to understand the differences between M2 responses that lead to &#x02018;adaptive' tissue repair with minimal scarring and restoration of normal parenchyma versus &#x02018;maladaptive' tissue repair with irreversible parenchyma loss that leads to CKD. However, this emerging body of knowledge highlights the therapeutic potential of cell-based therapies using macrophages that are primed <italic>ex vivo</italic> to mediate adaptive repair of injured kidney tissue. Proof of concept experiments using animal models support this hypothetical approach in the future.^{<xref ref-type="bibr" rid="bib6">6</xref>}</p></sec><sec><title>MYOFIBROBLASTS</title><p>Myofibroblasts are a unique population of cells that appear <italic>de novo</italic> in the renal interstitium during fibrosis.^{<xref ref-type="bibr" rid="bib7">7</xref>} The presence of these cells appears essential for scar formation and despite limited human data, their numbers appear to correlate with renal outcomes. Expression of alpha smooth muscle actin (&#x003b1;SMA) by interstitial cells with a characteristic fibroblastic morphology is their defining feature. A variety of <italic>in vitro</italic> studies and mRNA <italic>in situ</italic> hybridization studies in kidney tissues identify myofibroblasts as the primary source of the scar-forming matrix proteins, suggesting that their presence is essential for fibrosis. Given their central importance, the cellular origin of these cells is a critical question that has been extensively investigated in animal models, but conflicting data leave this as a subject of an ongoing debate. Recent lineage-tracing studies have employed unique genetic engineering strategies and a variety of cell-tracing methodologies and, perhaps not surprisingly, contradictory results have been reported.^{<xref ref-type="bibr" rid="bib7">7</xref>, <xref ref-type="bibr" rid="bib8">8</xref>, <xref ref-type="bibr" rid="bib9">9</xref>, <xref ref-type="bibr" rid="bib10">10</xref>} What is clear is that matrix-producing &#x003b1;SMA&#x0002b; interstitial cells have several potential cellular origins. It is likely that each has unique recruitment and activation pathways in addition to TGF&#x003b2;. Furthermore, &#x003b1;SMA expression itself may not be a critical fibrosis-promoting protein, as renal fibrosis was reported to be more severe in mice with genetic &#x003b1;SMA deficiency.^{<xref ref-type="bibr" rid="bib11">11</xref>} Subsets of these cells also express receptors that can internalize and degrade extracellular matrix, as has recently been shown for mannose receptor 2.^{<xref ref-type="bibr" rid="bib12">12</xref>} Whether cellular origin has a role in myofibroblasts functional heterogeneity remains to be determined.</p><p>Although the jury is still deliberating, it appears that the majority of the interstitial myofibroblasts are derived from a pool of endogenous kidney cells that migrate, proliferate, and transform. Resident kidney fibroblasts and microvascular pericytes are the favored leading contenders as the primary myofibroblast source(s).^{<xref ref-type="bibr" rid="bib13">13</xref>} Small numbers of matrix-producing interstitial cells also originate from myeloid lineage bone marrow cells (fibrocytes), whereas in severely damage kidneys, local cells (tubular epithelium, endothelium, and perhaps macrophages) may transdifferentiate into &#x003b1;SMA&#x0002b; matrix-producing cells, but overall their numbers appear to be small and their presence delayed until the advanced stages of CKD.^{<xref ref-type="bibr" rid="bib14">14</xref>}</p></sec><sec><title>TUBULAR EPITHELIA</title><p>During the induction phase of chronic kidney injury, tubular epithelial cells actively participate in injurious pathways through their ability to synthesize products, such as reactive oxygen species, and inflammatory mediators, such as chemokines, that find their way into the interstitium via basolateral secretion or via paracellular pathways by escaping through tight junction barriers. A variety of abnormally filtered urinary proteins derived from the systemic plasma pool or upstream glomerular cells may engage tubular epithelia in these events.^{<xref ref-type="bibr" rid="bib15">15</xref>} Urinary proteins such as members of the complement cascade or cytokines may activate specific cellular responses by binding to their cognate receptors on tubular apical membranes. An alternative activation pathway that is triggered by biochemically modified or conjugated urinary albumin involves proximal tubular megalin receptors that mediate protein endocytosis and activate specific signaling responses in partnership with its co-receptors cubilin and amnionless.^{<xref ref-type="bibr" rid="bib16">16</xref>} This latter pathway has been associated with stimulated synthesis of inflammatory chemokines (monocyte chemoattractant protein-1, regulated on activation normal T-cell expressed and secreted, interleukin-8, fractalkine), profibrotic molecules (TGF&#x003b2;, endothelin), and the transdifferentiation of tubular epithelial into &#x003b1;SMA&#x0002b; cells. Extensively investigated in cell culture systems, the degree to which proteinuria triggers these tubular cell responses <italic>in vivo</italic> is still not clear, but this paradigm is thought to explain in part the undisputed fact that the degree of proteinuria closely correlates with chronic inflammatory and fibrosis pathways that typify CKD.</p><p>As fibrosis severity increases, tubular epithelia that normally have a potent regenerative capacity lose this ability and succumb due to apoptosis or accelerated senescence. The reason for this transition from regenerative to dying cells is not well understood, but appears to involve cell-cycle specific factors, autophagy failure, endoplasmic reticulum stress, oxidative stress, and the loss of unknown &#x02018;regenerative signals'.^{<xref ref-type="bibr" rid="bib17">17</xref>, <xref ref-type="bibr" rid="bib18">18</xref>, <xref ref-type="bibr" rid="bib19">19</xref>} Tubular cell death is a hallmark feature of renal parenchymal damage that leads to serious negative outcomes, as it leaves behind non-functional atubular glomeruli. Histological measures of tubular cell area closely correlate with renal function.^{<xref ref-type="bibr" rid="bib20">20</xref>} An essential ingredient of effective human CKD treatment will be the ability to preserve and/or regenerate functional epithelia to preserve intact and functional nephrons. These regenerative responses are likely to recapitulate normal nephrogenic pathways of kidney development. Important clues to new candidate regenerative pathways are likely to emerge now that next-generation sequencing technologies are delineating genetic mutations that cause human renal hypodysplasia, such as wingless-type MMTV integration family, member 4.^{<xref ref-type="bibr" rid="bib21">21</xref>}</p></sec><sec><title>INTERSTITIAL CAPILLARIES</title><p>Given the high oxygen needs to support the metabolic activities of the kidney, an analogy has been made between CKD due to progressive fibrosis and suffocation.^{<xref ref-type="bibr" rid="bib22">22</xref>} One of the early events in chronic kidney injury is an increase in the permeability of the interstitial microvasculature.^{<xref ref-type="bibr" rid="bib23">23</xref>} As a result, many normally excluded plasma proteins such as fibrinogen and albumin conjugates leak into the interstitium (the kidney capillary leak syndrome) and trigger an inflammatory and potentially profibrotic response. The critical plasma proteins have not been identified in CKD but one of them appears to be fibrinogen (ogen), as genetic fibrinogen deficiency reduces the number of &#x003b1;SMA&#x0002b; interstitial cells in experimental CKD.^{<xref ref-type="bibr" rid="bib24">24</xref>} Although many chronic pathological disorders are characterized by excessive angiogenesis, CKD suffers from the opposite problem&#x02014;failure of reparative angiogenesis and a progressive decline in the surface area of interstitial capillaries. This has led investigators to consider the possibility of pro-angiogenic factors as therapy for CKD or, alternatively, blockade of anti-angiogenic factors that are presumed to be unregulated and harmful during the course of progressive kidney scarring.^{<xref ref-type="bibr" rid="bib25">25</xref>}</p><p>The hypoxia&#x02013;oxidant stress connection is thought to be closely coupled with the damaging consequences of kidney fibrosis. Evidence of significant tubular cell oxidant stress is a universal feature of chronically damaged kidneys, which is likely a consequence of both the excessive generation of reactive oxygen species and inadequate antioxidant defenses. Much remains to be learned about the identity and effects of the specific molecular targets of reactive oxygen species that promote kidney fibrosis, something we are likely to learn more about in the near future, as metabolomic studies are defining the specific profile of sugars, nucleotides, amino acids, and lipids in normal and fibrotic kidneys. A role for oxygen species as cell signaling molecules has been recognized and may also prove relevant to fibrogenic pathways.^{<xref ref-type="bibr" rid="bib26">26</xref>} With the growing application of high-throughput screening technologies to screen large drug libraries, the possibility that drugs with the ability to alter the redox potential in damaged kidneys should be therapeutically beneficial. For e.g., our group has recently shown that the drug cysteamine (approved for human use to prevent nephropathic cystinosis) also significantly reduces kidney fibrosis in non-cystinotic experimental kidney disease models, an effect that is associated with a reduction in oxidant generation and a reduction in protein oxidation.^{<xref ref-type="bibr" rid="bib27">27</xref>}</p></sec><sec><title>MODELING AND REMODELING OF INTERSTITIAL KIDNEY SCARS</title><p>Far from being a boring conglomerate of collagen, the fibrotic &#x02018;scar' that typifies CKD is an interesting consortium of multifunctional macromolecules that change in composition and structure over time. Although the fibrillar collagens I and III often predominate, additional members include other collagens, traditional basement membrane proteins (collagen IV, laminin, nidogen, heparin sulfate proteoglycan), large proteoglycans (aggrecan, versican), small proteoglycans (decorin, biglycan, fibromodulin), glycoprotein (fibronectin, tenascin), and many others (secreted protein acidic and rich in cysteine, which is also known as SPARC or osteonectin, thrombospondin, vitronectin, hensin, etc). The area of the fibrotic interstitium is the best negative histological correlate of renal function and long-term renal prognosis. Within this destructive maze of molecules are some that serve beneficial roles. Examples include decorin that can block the activity of TGF&#x003b2; and biglycan, which inhibits the conversion of fibroblasts into myofibroblasts.^{<xref ref-type="bibr" rid="bib28">28</xref>, <xref ref-type="bibr" rid="bib29">29</xref>} A variety of cellular receptors engage specific extracellular matrix proteins to activate intracellular signaling pathways and cellular responses. Although collagen deposition has become synonymous with fibrosis, it is not yet known whether it is the key determinant of parenchymal destruction associated with progressive fibrosis, or whether other molecules serve an essential role. This is an important consideration, as many antifibrotic therapies have been designed with the goal of reducing collagen synthesis or enhancing its turnover.</p><p>Assuming the collagen is the primary culprit of fibrosis-induced cellular loss, it is remarkable that the <italic>in vivo</italic> key enzymatic pathways that remodel and degrade collagen are still unclear. Despite high synthesis rates, collagen does not accumulate in normal kidneys. For e.g., in normal mouse kidneys, &#x0223c;20&#x00025; of the total collagen content is newly synthesized every 2 weeks. Five enzyme groups have identified collagenase activities: certain matrix metalloproteinases, serine proteases, the ADAMTS family and lysosomal enzymes (cysteine and aspartic proteases). It was widely assumed that specific extracellular enzymes of the matrix metalloproteinase family were primarily responsible for degrading collagen molecules during wound healing to prevent excessive scarring. But remarkably, none of the specific matrix metalloproteinases that have been tested in knockout mice have been shown to reduce fibrosis severity, whereas some (matrix metalloproteinase-2, -7, -9) actually worsen fibrosis. These adverse outcomes are likely a consequence of their pleitropic effects that include proteolytic activation of several latent growth factors and cell receptor&#x02013;dependent activities. It has only been in recent years that the importance of cell-dependent pathways of collagen remodeling have been recognized. In a sequence of steps that involve collagen endocytosis, lysosomal transport and cathepsin-medicated proteolysis, extracellular collagen molecules can be degraded. In experimental models of kidney fibrosis, the mannose receptor 2 (Mrc2) (also known as urokinase receptor&#x02013;associated protein and Endo180), which is expressed by ^15&#x00025; of interstitial myofibroblasts and macrophages, significantly reduces fibrosis severity compared with mice with genetic Mrc2 deficiency.^{<xref ref-type="bibr" rid="bib12">12</xref>} The appearance of these Mrc2&#x0002b; cells is unique to damaged kidneys, and the specific inducers of Mrc2 expression are still under investigation. Other receptors known to endocytose and degrade collagen include mannose receptor 1, &#x003b1;1&#x003b2;1 and &#x003b1;2&#x003b2;1 integrins and milk fat globule epidermal growth factor 8.^{<xref ref-type="bibr" rid="bib30">30</xref>} The recent recognition that lysosomal enzymes of the cathepsin family appear to serve an important antifibrotic role suggests another way that specifically engineered cellular therapies might find a role in the clinical arena in the future.</p></sec><sec><title>MOLECULAR DRIVES OF FIBROSIS: DOES EVERYONE NEED TGF&#x003b2;?</title><p>TGF&#x003b2; is the prototypic fibrogenic growth factor. Engagement of its cognate receptors (II and I) has the potential to activate a variety of canonical and non-canonical intracellular signaling and regulating pathways that have been extensively investigated and numerous fibrosis-promoting target genes have been identified.^{<xref ref-type="bibr" rid="bib31">31</xref>} Both tubular and interstitial cells can synthesize TGF&#x003b2; and, although not the only cellular targets, fibroblasts and myofibroblasts are particularly responsive to TGF&#x003b2; stimulation. Although TGF&#x003b2; is theoretically the ideal molecular target for antifibrotic therapies, it has multiple cellular functions including essential immunoregulatory actions. At least in mice, genetic TGF&#x003b2;1 deficiency is not compatible with life. An ever-growing panel of molecules are known to elicit fibrosis-promoting effects and, for the majority, effects on the TGF&#x003b2; pathway are directly or indirectly involved. For most of them, it remains unknown whether fibrosis could develop in the complete absence of TGF&#x003b2;. Some of the best known fibrogenic molecules are members of the rein-angiogenesis system, reactive oxygen species, other growth factors (connective tissue growth factor, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, tumor necrosis factor alpha), plasminogen activator ihibtor-1, endothelin-1, a variety of proteases, protease inhibitors, chemokines, adhesion molecules (especially certain integrins), specific matrix molecules, cluster of differentiation 36 (a class B scavenger receptor), the Wnt1/&#x003b2; catenin pathway, Notch, hedgehog legends, K_Ca3.1 channel, lysophosphatidic acid, homeodomain-interacting protein kinase 2, the myeloid differentiation primary response gene 88 pathway, and parathyroid hormone&#x02013;related protein. Similarly, the extent to which TGF&#x003b2; is an essential target of the various antifibrotic molecules is unclear. Some of better known antifibrotic molecules that have been considered candidates for the treatment of human CKD are hepatocyte growth factor, bone morphogenic protein-7, bradykinin, relaxin, heme oxygenase, interferon gamma, vitamin D, adiponectin, adenosine A2A, klotho, kielin/chordin-like, rapamycin pathway, transient receptor potential cation channel subfamily V member 1, Wnt7 and lipoxins.</p></sec><sec><title>GENETIC AND EPIGENETICS REGULATION OF THE FIBROTIC RESPONSE</title><p>The high degree of variability in the outcome and long-term prognosis among groups of patients with the same primary kidney disease is well recognized. The quest to understand this variability is a fundamental step if personalized medicine is ever to become a reality for CKD patients. There is no question that genetics plays a significant role, perhaps best illustrated by ethnicity-dependent variations in kidney outcomes. The apolipoprotein L1 genotype (G1 and G2 risk alleles) influences prognosis in African Americans is a recent example of genetic determinants of fibrosis severity.^{<xref ref-type="bibr" rid="bib32">32</xref>} New insights have emerged recently through the use of genome-wide association studies to establish significant associations with the risk and severity of CKD, although these associations do not establish causality and gene polymorphisms do not necessarily result in changes in protein function. Nonetheless, genome-wide association studies in human CKD represent an unbiased approach to identify new candidates that deserve further investigation for their potential role in the fibrogenic responses that led to CKD. One such example is uromodulin (UMOD), identified in several recent human genome-wide association studies as a significant determinant of CKD risk and severity (reviewed in ref. <xref ref-type="bibr" rid="bib33">33</xref>). <italic>UMOD</italic> encodes a protein that is uniquely expressed in the thick ascending limb of the loop of Henle and the early distal tubule. Although the primary function of UMOD remains unknown, it is the most abundant protein found in normal human urine, and protein-encoding genetic mutations are a known cause of familial CKD characterized as chronic tubulointerstitial nephritis. With the rapidly declining cost and analysis time for human genetic studies, it is anticipated that significant new insights into the genetic basis of CKD risk and prognosis will soon emerge.</p><p>Other heritable factors may also influence fibrogenic responses without altering the protein-coding gene sequences. Specific mechanisms may include DNA methylation, histone modification, and microRNA activities. Each of these processes has the potential to modify renal fibrotic responses and specific examples have recently been published, with many more likely to emerge. For e.g., in experimental models of kidney fibrosis, the number of interstitial myofibroblasts was significant reduced by a demethylating agent, a histone deacetylase inhibitor, and anti-microRNA-21 therapy, noting that microRNA-21 unregulation has been reported in several animal and human CKD tissue samples.^{<xref ref-type="bibr" rid="bib34">34</xref>, <xref ref-type="bibr" rid="bib35">35</xref>, <xref ref-type="bibr" rid="bib36">36</xref>, <xref ref-type="bibr" rid="bib37">37</xref>} Epigenetic regulation of fibrotic responses provides a new way to investigate environmental influences on genetically regulated pathways, such as kidney fibrosis.</p></sec><sec><title>CARDIORENAL SYNDROME</title><p>Ending where this overview began, it is important to remember that most CKD patients will never reach the point of needing renal replacement therapy to sustain life; they are more likely to die prematurely due to accelerated cardiovascular diseases. The CKD risk rises almost exponentially as renal function declines. In a study by DuBose <italic>et al.</italic>,^{<xref ref-type="bibr" rid="bib38">38</xref>} the age-specific risk of cardiovascular events was 17-fold higher in individuals with an eGFR&#x0003c;15&#x02009;ml/min per 1.73&#x02009;m² compared with these with an eGFR&#x0003e;60&#x02009;ml/min per 173&#x02009;m². Several factors are thought to contribute to the pathogenesis of this cardiorenal syndrome, including many that are injurious for both organs and cause endothelial cell dysfunction, inflammation, smooth muscle cell proliferation, oxidative stress, and vascular calcification. Therapeutic interventions that attenuate kidney fibrosis, hypertension, and/or proteinuria should also diminish the incidence and severity of cardiovascular disease and improve patient survival.</p><p>Over the past two decades, basic science research has greatly advanced our understanding of the cellular and molecular pathways that transform a kidney with normal structure and function into one of compromised function that is likely to worsen over time due to a relentless process of fibrosis. Although many questions remain to be answered, we must now do a better job of translating this knowledge into new and effective strategies to prevent, treat and perhaps even cure human CKD.</p></sec>

Terlipressin-Induced Ischemic Skin Necrosis: A Rare Association

<p><bold>Patient: Male, 65</bold></p><p><bold>Final Diagnosis: Drug-iduced skin necrosis</bold></p><p><bold>Symptoms: &#x02014;</bold></p><p><bold>Medication: &#x02014;</bold></p><p><bold>Clinical Procedure: Skin graft</bold></p><p><bold>Specialty: Surgery</bold></p><sec><title>Objective:</title><p><bold>Adverse events of drug therapy</bold></p></sec><sec><title>Background:</title><p>Terlipressin is a synthetic vasopressin analogue that is used in the treatment of bleeding esophageal varices and hepatorenal syndrome in patients with cirrhosis. Serious ischemic adverse events, such as skin necrosis involving the extremities, scrotum, trunk, and abdominal skin, are rarely observed. In the literature to date, 20 cases that developed ischemic skin necrosis due to terlipressin usage have been reported.</p></sec><sec><title>Case Report:</title><p>We report a patient with extensive skin necrosis on the infusion site of the right forearm and hand, which developed after the use terlipressin used to treat bleeding oesophageal varices in a 65-year-old man with cirrhosis.</p></sec><sec><title>Conclusions:</title><p>Although rare, ischemic complications of terlipressin do occur.</p></sec>

<contrib contrib-type="author"><name><surname>Coskun</surname><given-names>Banu Demet Ozel</given-names></name><xref ref-type="author-notes" rid="fn1-amjcaserep-15-476">^A</xref><xref ref-type="author-notes" rid="fn2-amjcaserep-15-476">^B</xref><xref ref-type="author-notes" rid="fn5-amjcaserep-15-476">^E</xref><xref ref-type="aff" rid="af1-amjcaserep-15-476">¹</xref><xref ref-type="corresp" rid="c1-amjcaserep-15-476"/></contrib><contrib contrib-type="author"><name><surname>Karaman</surname><given-names>Ahmet</given-names></name><xref ref-type="author-notes" rid="fn2-amjcaserep-15-476">^B</xref><xref ref-type="author-notes" rid="fn6-amjcaserep-15-476">^F</xref><xref ref-type="aff" rid="af1-amjcaserep-15-476">¹</xref></contrib><contrib contrib-type="author"><name><surname>Gorkem</surname><given-names>Hasan</given-names></name><xref ref-type="author-notes" rid="fn2-amjcaserep-15-476">^B</xref><xref ref-type="author-notes" rid="fn4-amjcaserep-15-476">^D</xref><xref ref-type="aff" rid="af1-amjcaserep-15-476">¹</xref></contrib><contrib contrib-type="author"><name><surname>Bu&#x0011f;day</surname><given-names>Irfan</given-names></name><xref ref-type="author-notes" rid="fn4-amjcaserep-15-476">^D</xref><xref ref-type="author-notes" rid="fn6-amjcaserep-15-476">^F</xref><xref ref-type="aff" rid="af1-amjcaserep-15-476">¹</xref></contrib><contrib contrib-type="author"><name><surname>Poyrazo&#x0011f;lu</surname><given-names>Orhan Kursad</given-names></name><xref ref-type="author-notes" rid="fn2-amjcaserep-15-476">^B</xref><xref ref-type="author-notes" rid="fn6-amjcaserep-15-476">^F</xref><xref ref-type="aff" rid="af1-amjcaserep-15-476">¹</xref></contrib><contrib contrib-type="author"><name><surname>Senel</surname><given-names>Fatma</given-names></name><xref ref-type="author-notes" rid="fn7-amjcaserep-15-476">^G</xref><xref ref-type="aff" rid="af2-amjcaserep-15-476">²</xref></contrib>

The American Journal of Case Reports

<sec><title>Background</title><p>Terlipressin is a synthetic long-acting analogue of vasopressin, which is widely used in the treatment of cirrhotic patients with variceal bleeding and hepatorenal syndrome. It is a vasoconstrictor that acts preferentially on the splanchnic circulation, lowering portal venous pressure [<xref rid="b1-amjcaserep-15-476" ref-type="bibr">1</xref>]. In the management of bleeding esophageal varices, terlipressin was shown to decrease mortality, reduce the failure rate of initial hemostasis, and reduce the number of emergency procedures to stop uncontrolled bleeding or rebleeding. The overall efficacy of terlipressin in controlling variceal bleeding is approximately 75&#x02013;80%, and is especially effective when administered early [<xref rid="b2-amjcaserep-15-476" ref-type="bibr">2</xref>]. The safety profile of terlipressin is better than vasopressin. However, vasoconstrictor effects on the systemic circulation result in ischemic complications in &#x0003c;5% of cases [<xref rid="b3-amjcaserep-15-476" ref-type="bibr">3</xref>,<xref rid="b4-amjcaserep-15-476" ref-type="bibr">4</xref>]. In the literature to date, 20 patients who developed ischemic skin necrosis due to terlipressin usage have been reported. Here, we report a patient with extensive skin necrosis on the infusion site of the right forearm and hand, which developed after the use terlipressin used to treat bleeding oesophageal varices in a 65-year-old man with cirrhosis.</p></sec><sec><title>Case Report</title><p>A 65-year-old man presented with variceal hemorrhage secondary to non-alcoholic fatty liver disease (NAFLD) cirrhosis. His medical history included hypertension and ischemic heart disease. His body mass index (BMI) was 28 kg/m². He had no bleeding episode and laboratory test results were: hemoglobin (Hgb): 10.3 g/dl, mean corpuscular volume (MCV): 86 fL, platelets (PLT): 110.000/mm³, prothrombin time (PT): 16-s international normalized ratio (INR): 1.4, alanine aminotransferase (ALT): 45 U/L, aspartate aminotransferase (AST): 65 U/L, gamma-glutamyl transferase (GGT): 87 IU/L, albumin: 2.8 g/dl, creatinine: 1.0 mg/dl, sodium (Na): 131 mmol/L, and potassium (K): 4.02 mmol/L. His model for end-stage liver disease (MELD) score on presentation was 10. Ultrasound (US) imaging showed hepatomegaly with Grade 2&#x02013;3 hepatosteatosis and splenomegaly. There was no history of alcohol use. His viral serologic test results and test results for autoimmune hepatitis did not reveal any abnormality. The bleeding oesophageal varices were controlled with endoscopic banding and the addition of terlipressin and intravenous antibiotics. Terlipressin was used at a dose of 1 mg intravenously every 4 h. Following 2 days of treatment, the patient developed large areas of erythema, as well as swelling and bruising on the skin of the extensor side of the right forearm and hand but sparing the fingers (<xref ref-type="fig" rid="f1-amjcaserep-15-476">Figure 1</xref>). We attempted to diagnose the causes of the skin lesion by repeated blood culture, lesion and skin biopsy, and Doppler US. Cultures from the lesion and blood cultures were sterile. Doppler US demonstrated normal arterial blood flow in the major arteries (ulnar, radial, and brachial). Cutaneous biopsy revealed non-specific inflammation extending from the dermis into the subcutaneous fatty tissue, and vascular congestion in the upper dermis. There was no evidence of thrombi or vasculitic signs in the dermal vessels (<xref ref-type="fig" rid="f2-amjcaserep-15-476">Figure 2</xref>). From these results and physical examination, we suspected a terlipressin-induced ischemic skin complication. The terlipressin injection was immediately discontinued and the wound on the right forearm was dressed with hydrocolloid solution. Despite cessation of terlipressin, his skin condition worsened, with development of extensive areas of necrosis. Finally, necrotic tissue was extracted and a skin graft was done.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>Terlipressin is a prohormone of lysine-vasopressin (VP) (3 glycyl residues and lysine-VP). Following intravenous administration, the glycyl residues are cleaved from the prohormone by endothelial peptidases, allowing prolonged release of lysine-VP. This mechanism prolongs the half-life of terlipressin, enabling administration of undivided doses without the need for an infusion as with vasopressin [<xref rid="b1-amjcaserep-15-476" ref-type="bibr">1</xref>]. Terlipressin administration, after 30 min, has been shown to significantly increase mean arterial pressure and systemic vascular resistance, while heart rate, cardiac output, hepatic venous portal pressure gradient, and portal venous blood flow decrease significantly [<xref rid="b5-amjcaserep-15-476" ref-type="bibr">5</xref>,<xref rid="b6-amjcaserep-15-476" ref-type="bibr">6</xref>]. It has been found to be superior to placebo in the control of variceal bleeding and to decrease renal vasoconstrictor system activity and improve renal function in patients with hepatorenal syndrome [<xref rid="b1-amjcaserep-15-476" ref-type="bibr">1</xref>,<xref rid="b3-amjcaserep-15-476" ref-type="bibr">3</xref>].</p><p>Terlipressin is the agent most frequently recommended for the control of acute bleeding. It is administered in a dose of 2 mg every 4 h for 2&#x02013;5 days. After bleeding is controlled, it may be administered at a lower dose of 1 mg ever 4 h for up to 5 days [<xref rid="b7-amjcaserep-15-476" ref-type="bibr">7</xref>]. Adverse effects of terlipressin are usually mild and include skin pallor, headache, abdominal pain, bradycardia, hypertension and hyponatremia. It can exert vasoconstrictor effects on the systemic circulation; serious ischemic complications are rare and include myocardial infarction, ischemic colitis, and skin necrosis [<xref rid="b8-amjcaserep-15-476" ref-type="bibr">8</xref>&#x02013;<xref rid="b10-amjcaserep-15-476" ref-type="bibr">10</xref>]. In the English literature to date, terlipressin-induced skin necrosis have been reported in 20 cases, located on the abdominal wall, thigh, leg, calves, fingers, toes, tongue, scalp, scrotum, breast, and esophagus [<xref rid="b11-amjcaserep-15-476" ref-type="bibr">11</xref>&#x02013;<xref rid="b14-amjcaserep-15-476" ref-type="bibr">14</xref>].</p><p>Because our patient had ischemic heart disease, he was closely monitored for myocardial infarction. On the second day of the treatment, a red-purple color change was observed, covering the patient&#x02019;s right forearm. Despite the discontinuation of terlipressin treatment, the lesions progressed for 1 week. To the best of our knowledge, this is the first report of skin necrosis on the forearm due to terlipressin.</p><p>In all cases, including ours, skin manifestations evolved after 2&#x02013;3 days of treatment, suggesting a dose-related effect. Potential predisposing factors of ischemic skin complications are hypovolemia, concomitantly administered pressor drugs, and patients with ischemic disease, obesity, venous insufficiency, or spontaneous bacterial peritonitis [<xref rid="b12-amjcaserep-15-476" ref-type="bibr">12</xref>,<xref rid="b15-amjcaserep-15-476" ref-type="bibr">15</xref>,<xref rid="b16-amjcaserep-15-476" ref-type="bibr">16</xref>]. Some experts have suggested that continuous intravenous infusion of terlipressin was the possible risk factor of ischemic adverse events [<xref rid="b17-amjcaserep-15-476" ref-type="bibr">17</xref>,<xref rid="b18-amjcaserep-15-476" ref-type="bibr">18</xref>]. In our case, terlipressin was administered as an intravenous bolus and no other vasoactive drug was concomitantly administered. In the present case, history of atherosclerotic change of upper extremity vessels, hypovolemia, and overweight were possible risk factors for the peripheral gangrene and necrosis.</p><p>When we examined the cases of skin necrosis due to terlipressin, the reasons of liver failure were reported as alcoholic cirrhosis in 10 cases, obesity and NAFLD in 5 cases, metastatic carcinoma in 2 cases, viral hepatitis in 2 cases, and autoimmune hepatitis in 1 case. Therefore, we conclude that alcohol use should be considered as a potential risk factor for terlipressin-related skin necrosis.</p><p>The development of skin necrosis is related to the particular distribution of the target receptor of terlipressin &#x02013; the vasopressin receptor type 1 (V1 receptor) &#x02013; which is located in smooth muscles of the blood vessels, mainly in the area of the splanchnic circulation, kidney, myometrium, bladder, adipocytes, and skin circulation. Thus, the damaged areas in our case reinforce the probability of terlipressin as the cause [<xref rid="b17-amjcaserep-15-476" ref-type="bibr">17</xref>].</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>In conclusion, we report a patient with extensive skin necrosis in an unusual location, secondary to terlipressin therapy for management of variceal bleeding. Although rare, clinicians must bear in mind the possibility of ischemic complications caused by terlipressin. In addition to the obesity, NAFLD, and ischemic heart disease, it was important to think that alcohol use was a possible risk factor for progressive ischemic necrosis due to terlipressin.</p></sec>

Comparison of the antibacterial activity of essential oils and extracts of medicinal and culinary herbs to investigate potential new treatments for irritable bowel syndrome

<sec><title>Background</title><p>Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, which may result from alteration of the gastrointestinal microbiota following gastrointestinal infection, or with intestinal dysbiosis or small intestinal bacterial overgrowth. This may be treated with antibiotics, but there is concern that widespread antibiotic use might lead to antibiotic resistance. Some herbal medicines have been shown to be beneficial, but their mechanism(s) of action remain incompletely understood. To try to understand whether antibacterial properties might be involved in the efficacy of these herbal medicines, and to investigate potential new treatments for IBS, we have conducted a preliminary study <italic>in vitro</italic> to compare the antibacterial activity of the essential oils of culinary and medicinal herbs against the bacterium, <italic>Esherichia coli</italic>.</p></sec><sec><title>Methods</title><p>Essential oils were tested for their ability to inhibit <italic>E. coli</italic> growth in disc diffusion assays and in liquid culture, and to kill <italic>E. coli</italic> in a zone of clearance assay. Extracts of coriander, lemon balm and spearmint leaves were tested for their antibacterial activity in the disc diffusion assay. Disc diffusion and zone of clearance assays were analysed by two-tailed t tests whereas ANOVA was performed for the turbidometric assays.</p></sec><sec><title>Results</title><p>Most of the oils exhibited antibacterial activity in all three assays, however peppermint, lemon balm and coriander seed oils were most potent, with peppermint and coriander seed oils being more potent than the antibiotic rifaximin in the disc diffusion assay. The compounds present in these oils were identified by gas chromatography mass spectrometry. Finally, extracts were made of spearmint, lemon balm and coriander leaves with various solvents and these were tested for their antibacterial activity against <italic>E. coli</italic> in the disc diffusion assay<italic>.</italic> In each case, extracts made with ethanol and methanol exhibited potent antibacterial activity.</p></sec><sec><title>Conclusions</title><p>Many of the essential oils had antibacterial activity in the three assays, suggesting that they would be good candidates for testing in clinical trials. The observed antibacterial activity of ethanolic extracts of coriander, lemon balm and spearmint leaves suggests a mechanistic explanation for the efficacy of a mixture of coriander, lemon balm and mint extracts against IBS in a published clinical trial.</p></sec>

<contrib contrib-type="author" equal-contrib="yes" id="A1"><name><surname>Thompson</surname><given-names>Aiysha</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>486599@swansea.ac.uk</email></contrib><contrib contrib-type="author" equal-contrib="yes" id="A2"><name><surname>Meah</surname><given-names>Dilruba</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>meahd@cardiff.ac.uk</email></contrib><contrib contrib-type="author" equal-contrib="yes" id="A3"><name><surname>Ahmed</surname><given-names>Nadia</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Nadiahmed6@yahoo.com</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Conniff-Jenkins</surname><given-names>Rebecca</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Beckij219@msn.com</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Chileshe</surname><given-names>Emma</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Emma.chileshe@gmail.com</email></contrib><contrib contrib-type="author" id="A6"><name><surname>Phillips</surname><given-names>Chris O</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>C.O.Phillips@swansea.ac.uk</email></contrib><contrib contrib-type="author" id="A7"><name><surname>Claypole</surname><given-names>Tim C</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>T.C.Claypole@swansea.ac.uk</email></contrib><contrib contrib-type="author" id="A8"><name><surname>Forman</surname><given-names>Dan W</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>D.W.Forman@swansea.ac.uk</email></contrib><contrib contrib-type="author" corresp="yes" id="A9"><name><surname>Row</surname><given-names>Paula E</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>paula.row@cantab.net</email></contrib>

BMC Complementary and Alternative Medicine

<sec><title>Background</title><p>Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder, which affects 10 &#x02013; 22% of the UK population and is responsible for 20 &#x02013; 50% of the workload of gastroenterology departments [<xref ref-type="bibr" rid="B1">1</xref>]. IBS is characterised by altered bowel habit, pain, and wind or bloating, which all severely affect quality of life and may last for decades. IBS can be classified into subgroups depending on the predominant bowel symptom, namely constipation-predominant (IBS-C), diarrhoea-predominant (IBS-D) or alternating between the two (IBS-A) [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B3">3</xref>]. Post-infectious (PI) IBS may arise after an episode of acute gastroenteritis [<xref ref-type="bibr" rid="B4">4</xref>].</p><p>The causes of IBS remain poorly understood, and include altered gut motor function, visceral hypersensitivity, abnormal gas handling, alterations in the central nervous system, mild inflammation, disturbances in serotonin handling and genetic factors (reviewed in [<xref ref-type="bibr" rid="B5">5</xref>]). Evidence has been mounting in the past decade that alterations in the gastrointestinal microbiota may also play a role in IBS. An early study showed that patients with IBS-D excreted abnormally high levels of hydrogen compared to healthy individuals, suggesting that there was abnormal bacterial fermentation of ingested foods [<xref ref-type="bibr" rid="B6">6</xref>]. An exclusion diet reduced this. More recently the gastrointestinal microbiota of IBS patients have been compared with those from healthy subjects by bacterial culture or molecular approaches [reviewed in [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. Many studies have found alterations in the numbers of bacterial species present, or in the abundance of particular bacterial species. The findings vary, but a common feature seems to be a reduction in the number of Bifidobacteria and increased numbers of Firmicutes and Enterobacteriaceae in samples from IBS patients compared to control samples.</p><p>IBS has also been linked to small intestinal bacterial overgrowth, [reviewed in [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>], although this is currently somewhat controversial [<xref ref-type="bibr" rid="B8">8</xref>]. In SIBO, bacterial numbers can rise from normal levels of 10^0-4 colony forming units (cfu) of bacteria/ml in the terminal jejunum, 10^0-5&#x000a0;cfu/ml in the proximal ileum and 10^5-8&#x000a0;cfu/ml in the terminal ileum [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>] to 10¹¹&#x000a0;cfu/ml [<xref ref-type="bibr" rid="B9">9</xref>]. SIBO is currently defined as the presence of &#x02265;&#x02009;1 &#x000d7; 10⁵&#x000a0;cfu/ml of colonic bacteria in the jejunum [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>]. In contrast to the normal situation in which digestion and absorption of food is complete before the bolus reaches bacteria in the colon, in patients with SIBO, ingested food comes into contact with bacteria in the jejunum and ileum, and is fermented to produce gas, which has been visualised in the small intestine by abdominal radiography [<xref ref-type="bibr" rid="B15">15</xref>]. SIBO is typically diagnosed by giving patients a drink containing a poorly digestible sugar such as lactulose, and analysing their breath gases at intervals (reviewed in [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>]). Glucose can also be used. The main gases excreted are hydrogen, which is associated with IBS-D, and methane, which is associated with IBS-C [<xref ref-type="bibr" rid="B18">18</xref>-<xref ref-type="bibr" rid="B21">21</xref>]. In one study, 78% of IBS patients tested positive for SIBO using the lactulose hydrogen breath test (LHBT) [<xref ref-type="bibr" rid="B22">22</xref>].</p><p>There is currently some controversy surrounding the use of lactulose hydrogen breath tests to diagnose SIBO and, consequently, the role of SIBO in IBS. Early breath tests diagnosed SIBO if hydrogen was excreted in two bursts, corresponding to bacterial fermentation in the small intestine, and in the colon. Later, hydrogen excretion within 90&#x000a0;minutes of lactulose ingestion was taken as diagnostic of SIBO. However, a recent study has combined LHBTs with scintigraphy to follow the movement of a radiolabelled tracer through the gastrointestinal tract [<xref ref-type="bibr" rid="B23">23</xref>]. Hydrogen excretion within 90&#x000a0;minutes of lactulose ingestion was found to correlate with fast oro-caecal transit rather than SIBO [<xref ref-type="bibr" rid="B23">23</xref>], and it has been suggested that glucose would be a better fermentation substrate since it is absorbed before it reaches the colon [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. Using the ability to culture 1 &#x000d7; 10⁵ colonic bacteria from duodenal or jejunal aspirates as the gold standard for SIBO diagnosis, SIBO was diagnosed in 4% of IBS patients and healthy control subjects in one study [<xref ref-type="bibr" rid="B14">14</xref>] and in 10.9% of IBS patients in another [<xref ref-type="bibr" rid="B25">25</xref>]. Modestly raised bacterial counts (&#x02265; 1 &#x000d7; 10³&#x000a0;cfu/ml) were found in 43% of IBS patients compared to 12% of healthy controls in the first study [<xref ref-type="bibr" rid="B14">14</xref>] and 37% of IBS patients in the second [<xref ref-type="bibr" rid="B25">25</xref>], suggesting that perhaps a more modest overgrowth of bacteria than previously thought might contribute to IBS. Of note, lipopolysaccharide from Gram-negative bacteria speeds up gastrointestinal transit [<xref ref-type="bibr" rid="B26">26</xref>], raising the possibility that modest numbers of bacteria in the small intestine could speed up transit of lactulose (and chyme) to the colon, and result in a positive LHBT (and IBS) without fermenting the lactulose themselves.</p><p>One Gram-negative bacterium that has been implicated in IBS and SIBO is <italic>Escherichia coli.</italic> An early study found <italic>E. coli</italic> throughout the gastrointestinal tract of patients with SIBO [<xref ref-type="bibr" rid="B27">27</xref>]. <italic>E. coli</italic> was present in jejunal and duodenal aspirates from patients with IBS and SIBO [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B25">25</xref>], in addition to <italic>Klebsiella</italic> and <italic>Enterococcus</italic> species. <italic>E. coli</italic> was found to be more prevalent in the mucosal microbiota of IBS patients than those of healthy control subjects when biopsy specimens were labelled with fluorescent in situ hybridisation probes [<xref ref-type="bibr" rid="B28">28</xref>], and enteroaggregative strains of <italic>E. coli</italic> were present in higher numbers in faecal samples from IBS patients than those from healthy individuals [<xref ref-type="bibr" rid="B29">29</xref>].</p><p>There are currently various medicines available to treat IBS, including fiber, antispasmodic agents, and antidepressants to modulate pain perception [<xref ref-type="bibr" rid="B30">30</xref>]. Tricyclic antidepressants block diarrhoea whereas serotonin reuptake inhibitors can benefit IBS-C patients by stimulating gastrointestinal motility [<xref ref-type="bibr" rid="B30">30</xref>]. In agreement with the idea that alterations in the gastrointestinal microbiota are involved in IBS, there has been considerable success with treatments that reverse this. Clinical trials have shown that antibiotics can be effective in treating IBS [<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. Successful eradication of SIBO, and reversal of the symptoms of IBS have been achieved by treating IBS patients with antibiotics such as metronidazole [<xref ref-type="bibr" rid="B33">33</xref>], neomycin [<xref ref-type="bibr" rid="B18">18</xref>], the non-absorbable antibiotic rifaximin (reviewed in [<xref ref-type="bibr" rid="B34">34</xref>]), a combination of rifaximin and ciprofloxacin [<xref ref-type="bibr" rid="B35">35</xref>], or a combination of rifaximin and neomycin for IBS-C patients who produce methane [<xref ref-type="bibr" rid="B36">36</xref>]. Reduction of hydrogen or methane excretion was linked to improvement in gastrointestinal symptoms and rifaximin was particularly effective in treating wind and bloating. Currently, NICE guidelines do not mention the use of antibiotics for the treatment of IBS in the UK [<xref ref-type="bibr" rid="B37">37</xref>]. SIBO can return after treatment, however [<xref ref-type="bibr" rid="B38">38</xref>] and there is concern that widespread and prolonged treatment with antibiotics could lead to the emergence of antibiotic resistant bacterial strains [<xref ref-type="bibr" rid="B39">39</xref>]. Indeed rifaximin can be used to treat infections with <italic>Clostridium difficile</italic>[<xref ref-type="bibr" rid="B40">40</xref>], but rifaximin-resistant strains have been isolated [<xref ref-type="bibr" rid="B41">41</xref>], and the fact that some IBS patients are carriers of <italic>C. difficile</italic>[<xref ref-type="bibr" rid="B42">42</xref>] raises the possibility that widespread treatment of IBS patients with rifaximin could lead to the production of more rifaximin-resistant strains of <italic>C. difficile.</italic></p><p>In contrast, the use of probiotics aims to increase the number of beneficial bacteria. Probiotics are live microorganisms that, when administered in sufficient numbers, reduce visceral hypersensitivity, improve gastrointestinal dysmotility and epithelial integrity, improve immune function, and modulate the gut microbiota [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. Two recent systematic reviews showed that probiotics had a moderate therapeutic benefit in improving IBS symptoms [<xref ref-type="bibr" rid="B43">43</xref>,<xref ref-type="bibr" rid="B44">44</xref>]. Probiotics reduced the pain of IBS and this was statistically significant; there was also a significant reduction in flatulence and a trend towards reduction in bloating [<xref ref-type="bibr" rid="B43">43</xref>]. In particular, lactic acid bacteria were shown to be useful, although the therapeutic benefit stopped when administration of the probiotics was terminated [<xref ref-type="bibr" rid="B44">44</xref>]. Related to this, prebiotics have also been shown to be of modest benefit in treating IBS &#x02013; these are nondigestible dietary supplements that increase the growth of beneficial bacteria. One clinical trial showed that ingestion of galactooligosaccharide stimulated the growth of bifidobacteria and alleviated IBS symptoms [<xref ref-type="bibr" rid="B45">45</xref>]. Finally, faecal transplantation has been successfully used to modulate the gastrointestinal microbiota in a small number of IBS patients (reviewed in [<xref ref-type="bibr" rid="B46">46</xref>]). This has the advantage that the entire community of colonic bacteria is transplanted, rather than the one or two bacterial species that might be present in a probiotic preparation.</p><p>An alternative approach to the treatment of IBS is the use of dietary modifications to reduce the amount of fermentable substrates available to the gastrointestinal microbiota. Early work pioneered the use of an exclusion diet consisting of one meat, one fruit and distilled or spring water for a week. If the patient&#x02019;s IBS symptoms resolved, foods were reintroduced one at a time and any resulting IBS symptoms noted [<xref ref-type="bibr" rid="B47">47</xref>]. Two thirds of the patients reported resolution of their symptoms on the exclusion diet, and wheat, corn, dairy products, coffee, tea and citrus fruits were found to provoke IBS symptoms, even in a double blind food challenge. A more relaxed exclusion diet consisting of fish and meat (apart from beef) and rice, and lacking dairy products, citrus fruits, yeast, tap water and caffeinated drinks, reduced hydrogen excretion by IBS patients in addition to resolving their symptoms [<xref ref-type="bibr" rid="B6">6</xref>]. This exclusion diet has been described in more detail [<xref ref-type="bibr" rid="B48">48</xref>]. These and other exclusion diets have been found to be useful under medical supervision by NICE [<xref ref-type="bibr" rid="B37">37</xref>], which also recommended that it may be helpful for IBS patients to limit both their fibre intake, and to eat no more than three portions of fruit per day. A recent approach to dietary manipulation has rationalised knowledge of the foods that commonly provoke IBS symptoms with an understanding of their chemical composition, namely the adoption of a diet low in FODMAPs (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) [<xref ref-type="bibr" rid="B49">49</xref>]. This involves avoiding 1) fruits that are high in fructose, 2) dairy products apart from butter and hard cheese, because they contain lactose, 3) vegetables, legumes and cereals (including wheat) that contain the oligosaccharides fructans or galactans and 4) fruits, vegetables and artificial sweeteners that contain polyols [<xref ref-type="bibr" rid="B49">49</xref>]. Restriction of fructose and fructans led to an improvement in gastrointestinal symptoms in three out of four IBS patients with fructose malabsorption [<xref ref-type="bibr" rid="B50">50</xref>]. When other patients who had attained remission of their IBS symptoms for 3 &#x02013; 36&#x000a0;months on a low FODMAPs diet were rechallenged with fructose or fructans in a placebo-controlled, double blind clinical trial, their symptoms returned, indicating that the fructose and fructans could provoke IBS symptoms [<xref ref-type="bibr" rid="B51">51</xref>]. In another double blind trial by the same group (in Australia), IBS patients on a high FODMAPs diet excreted more hydrogen and experienced more gastrointestinal symptoms than patients on a low FODMAPs diet [<xref ref-type="bibr" rid="B52">52</xref>]. More recently, a British group has compared the efficacy of a low FODMAPs diet in reducing IBS symptoms with the standard dietary guidelines recommended by NICE: more of the patients on the low FODMAPs diet reported an improvement in their symptoms than the patients following the NICE dietary guidelines [<xref ref-type="bibr" rid="B53">53</xref>], this was especially true for wind and bloating.</p><p>Herbal medicines are also used to treat IBS, both in mainstream medicine, and in complementary and alternative medicines from different traditions; these have been discussed in several excellent reviews [<xref ref-type="bibr" rid="B54">54</xref>-<xref ref-type="bibr" rid="B56">56</xref>]. Peppermint (<italic>Mentha piperita</italic>) oil has been recommended for the treatment of IBS by NICE guidelines [<xref ref-type="bibr" rid="B37">37</xref>] and is widely prescribed in the form of enteric-coated peppermint oil tablets; it has antispasmodic activity [<xref ref-type="bibr" rid="B57">57</xref>,<xref ref-type="bibr" rid="B58">58</xref>]. Grigoleit and Grigoleit performed a meta-analysis of sixteen placebo-controlled clinical trials studying the use of enteric-coated peppermint oil capsules to treat IBS, and found that the overall success rate of peppermint oil capsules was 58% compared to 29% for placebo [<xref ref-type="bibr" rid="B59">59</xref>]. They concluded that &#x0201c;peppermint oil&#x02026;may the drug of first choice in IBS patients with non-serious constipation or diarrhoea to alleviate general symptoms and to improve quality of life e.g. pain or bloating&#x0201d;. A recent meta-analysis of four clinical trials confirmed this view [<xref ref-type="bibr" rid="B60">60</xref>]. In fact peppermint oil, in the form of a preparation called Peppermint water BP1973, has long been used to treat dyspepsia, flatulence and stomach cramps, and has just been awarded a traditional herbal registration certificate [<xref ref-type="bibr" rid="B61">61</xref>]. Another herbal preparation that has shown promise in the treatment of IBS is Iberogast&#x000ae; (also called STW-5), a liquid formulation of nine different herbs, namely bitter candytuft, or clown&#x02019;s mustard plant (<italic>Iberis amara</italic>), German chamomile <italic>(Matricaria recutita</italic>) flowers, angelica (<italic>Angelica archangelica</italic>) root, caraway (<italic>Carum carvi</italic>) fruit, lemon balm (<italic>Melissa officinalis</italic>) leaves, greater celandine (<italic>Chelidonium majus</italic>) aerial parts, liquorice (<italic>Glycyrrhiza glabra</italic>) root, milk thistle <italic>(Silybum marianum</italic> L) and peppermint oil [<xref ref-type="bibr" rid="B62">62</xref>]. In a randomised, double-blind, placebo-controlled trial, STW 5 (Iberogast&#x000ae;) reduced abdominal pain, problems with bowel habit and flatulence in IBS patients [<xref ref-type="bibr" rid="B63">63</xref>]. Iberogast&#x000ae; is also effective against functional dyspepsia [<xref ref-type="bibr" rid="B64">64</xref>] and is widely prescribed in Germany with more than a million prescriptions being written for it in Germany in 2002 [<xref ref-type="bibr" rid="B65">65</xref>]. Iberogast&#x000ae; has been shown to have antispasmodic, anti-inflammatory and antioxidant activity, and also acts as a secretagogue (reviewed in [<xref ref-type="bibr" rid="B55">55</xref>,<xref ref-type="bibr" rid="B62">62</xref>]). A similar mixed herbal extract, called carmint, which consists of herbal extracts of lemon balm, spearmint (<italic>Mentha spicata</italic>) and coriander (<italic>Coriandrum sativum</italic>), reduced abdominal pain and bloating in IBS patients in a clinical trial [<xref ref-type="bibr" rid="B66">66</xref>]. IBS-D patients were prescribed loperamide, and IBS-C patients were prescribed psyllium, plus either carmint or placebo; there was a significant reduction in symptoms in the IBS patients taking carmint compared to those taking the placebo after eight weeks.</p><p>Herbs have traditionally been used to treat bacterial infections [<xref ref-type="bibr" rid="B67">67</xref>], for instance lavender (<italic>Lavandula angustifolia</italic>) oil and tincture were used to treat wounds before the First World War. Many culinary herbs have been reported to possess antibacterial properties [<xref ref-type="bibr" rid="B67">67</xref>,<xref ref-type="bibr" rid="B68">68</xref>], as have the essential oils of these herbs [<xref ref-type="bibr" rid="B69">69</xref>]. Many herbs, for instance fennel (<italic>Foeniculum vulgare</italic>), lavender, peppermint, rosemary (<italic>Rosmarinus officinalis</italic>) and sage (<italic>Salvia officinalis</italic>), have been used traditionally as digestives, aiding digestion or reducing flatulence [<xref ref-type="bibr" rid="B67">67</xref>,<xref ref-type="bibr" rid="B68">68</xref>,<xref ref-type="bibr" rid="B70">70</xref>]. Our hypothesis is that the digestive properties of a particular herb may be linked, at least in part, to the herb&#x02019;s antibacterial action. Essential oils and herbal extracts have an advantage over conventional antibiotics since they may contain several antibacterial compounds that act in different ways, so that it would be more difficult for bacteria to develop resistance. For instance lemon grass (<italic>Cymbopogon citratus</italic>) essential oil contains at least sixteen compounds [<xref ref-type="bibr" rid="B71">71</xref>] and successfully inhibited the growth of <italic>Helicobacter pylori</italic> over many bacterial generations, whereas antibiotic resistant <italic>H. pylori</italic> emerged after ten passages on plates containing only the antibiotic clarithromycin [<xref ref-type="bibr" rid="B72">72</xref>].</p><p>NICE guidelines recommend that further research should be conducted to study the possibility of using herbal medicines to treat IBS. With this in mind, in order to increase our understanding of the mechanism of action of herbal medicines that have been shown to be beneficial in treating IBS, and to identify other essential oils or extracts that would be useful candidates for clinical trials, we have conducted a preliminary study <italic>in vitro</italic> using a non-pathological strain of <italic>E. coli.</italic> We have compared the antibacterial activity of essential oils of a range of herbs that have been used traditionally as digestives, in three separate assays. We found that the essential oils with the most potent antibacterial activity in the three assays were those of coriander seed, lemon balm and peppermint. Interestingly, essential oils or extracts of coriander, lemon balm and peppermint are all present in herbal medicines that have been validated for use in the treatment of IBS by at least one clinical trial (without their mechanism of action being ascribed to an antibacterial effect). We identified the compounds present in the coriander seed, lemon balm and peppermint essential oils that we had used, by thermal desorption gas chromatography mass spectrometry. Finally we tested the antibacterial activity of extracts of coriander, lemon balm and spearmint leaves that had been made with various solvents to determine whether ethanolic tinctures of these herbs (which have been used instead of essential oils in some of the IBS medicines) would have antibacterial activity. This has allowed us to propose a new mechanism of action for these herbal medicines, and suggest some other herbs/essential oils that could be tested in further clinical trials.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Materials</title><p><italic>E. coli</italic> strain DH5&#x003b1;, was obtained from Dr. Geertje van Keulen (Swansea University). All essential oils (listed in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>) were obtained from Amphora Aromatics, Bristol, UK. Coriander plants were obtained from Tesco Supermarket, Swansea, lemon balm plants were from the Swansea City Council Botanic Gardens shop and spearmint plants were obtained from the Homebase superstore in Swansea. All chemicals were obtained from Sigma Chemical Company (Poole, Dorset) unless otherwise stated.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Summary of results of antibacterial activity of essential oils</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Vernacular name</bold></th><th align="left"><bold>Botanical name</bold></th><th align="left"><bold>Activity in disc diffusion assay</bold></th><th align="left"><bold>Activity in turbido-metric assay</bold></th><th align="left"><bold>Activity in zone of clearance assay</bold></th></tr></thead><tbody valign="top"><tr><td rowspan="2" align="left" valign="bottom">Coriander seed<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Coriandrum sativum</italic><hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+++<hr/></td></tr><tr><td align="left" valign="bottom">(0.0053)<hr/></td><td align="left" valign="bottom">(5.82 &#x000d7; 10^-5)<hr/></td><td align="left" valign="bottom">(0.0002)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Fennel<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Foeniculum vulgare</italic><hr/></td><td align="left" valign="bottom">+<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0003)<hr/></td><td align="left" valign="bottom">(8.01 &#x000d7; 10^-6)<hr/></td><td align="left" valign="bottom">(0.0027)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Grapeseed<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Vitis vinifera</italic><hr/></td><td align="left" valign="bottom">-<hr/></td><td align="left" valign="bottom">-<hr/></td><td align="left" valign="bottom">-<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">(0.3216)<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Lavender<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Lavendula angustifolia</italic><hr/></td><td align="left" valign="bottom">+<hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0039)<hr/></td><td align="left" valign="bottom">(0.0031)<hr/></td><td align="left" valign="bottom">(0.0011)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Lemon balm<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Melissa officinalis</italic><hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+++<hr/></td></tr><tr><td align="left" valign="bottom">(0.0041)<hr/></td><td align="left" valign="bottom">(0.0008)<hr/></td><td align="left" valign="bottom">(0.0014)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Lemon grass<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Cymbopogon citratus</italic><hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0004)<hr/></td><td align="left" valign="bottom">(0.0030)<hr/></td><td align="left" valign="bottom">(0.0003)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Mandarin<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Citrus reticulata</italic><hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0092)<hr/></td><td align="left" valign="bottom">(3.39 &#x000d7; 10^-5)<hr/></td><td align="left" valign="bottom">(0.0484)<hr/></td></tr><tr><td align="left" valign="bottom">Neem<hr/></td><td align="left" valign="bottom"><italic>Azadirachta indica</italic><hr/></td><td align="left" valign="bottom">-<hr/></td><td align="left" valign="bottom">ND<hr/></td><td align="left" valign="bottom">-<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Peppermint<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Mentha piperita</italic><hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+++<hr/></td></tr><tr><td align="left" valign="bottom">(0.0390)<hr/></td><td align="left" valign="bottom">(3.14 &#x000d7; 10^-5)<hr/></td><td align="left" valign="bottom">(0.0000)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Pine<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Pinus sylvestris</italic><hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">++<hr/></td></tr><tr><td align="left" valign="bottom">(0.0027)<hr/></td><td align="left" valign="bottom">(0.0003)<hr/></td><td align="left" valign="bottom">(0.0001)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Rosemary<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Rosmarinus officinalis</italic><hr/></td><td align="left" valign="bottom">+<hr/></td><td align="left" valign="bottom">++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0000)<hr/></td><td align="left" valign="bottom">(0.0085)<hr/></td><td align="left" valign="bottom">(0.0036)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Sage<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Salvia lavendulifolia</italic><hr/></td><td align="left" valign="bottom">+<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0022)<hr/></td><td align="left" valign="bottom">(3.39 &#x000d7; 10^-6)<hr/></td><td align="left" valign="bottom">(0.0021)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Tea tree<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Melaleuca alternifolia</italic><hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+++<hr/></td></tr><tr><td align="left" valign="bottom">(0.0079)<hr/></td><td align="left" valign="bottom">(5.83 &#x000d7; 10^-6)<hr/></td><td align="left" valign="bottom">(0.0000)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Thyme<hr/></td><td rowspan="2" align="left" valign="bottom"><italic>Thymus vulgaris</italic><hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+++<hr/></td><td align="left" valign="bottom">+<hr/></td></tr><tr><td align="left" valign="bottom">(0.0020)<hr/></td><td align="left" valign="bottom">(5.84 &#x000d7; 10^-5)<hr/></td><td align="left" valign="bottom">(0.0193)<hr/></td></tr><tr><td rowspan="2" align="left">Ylang ylang</td><td align="left" valign="bottom"><italic>Cananga odorata</italic> var. <italic>genuina</italic><hr/></td><td align="left" valign="bottom">+/-<hr/></td><td align="left" valign="bottom">+<hr/></td><td align="left" valign="bottom">-<hr/></td></tr><tr><td align="left">&#x000a0;</td><td align="left">(0.0075)</td><td align="left">(0.0477)</td><td align="left">&#x000a0;</td></tr></tbody></table><table-wrap-foot><p>Crosses denote the magnitude of the antibacterial activity; the numbers in brackets are the P values obtained from t tests comparing the size of the halos produced with the essential oils with those produced by grapeseed oil for the disc diffusion and zone of clearance assays, and from comparing the A₆₀₀ of the turbidometric assay in the presence of essential oil after 6&#x000a0;hours with that lacking essential oil.</p></table-wrap-foot></table-wrap></sec><sec><title>Disc diffusion assay: growth inhibition</title><p>A modification of the Kirby-Bauer disc diffusion assay was used [<xref ref-type="bibr" rid="B73">73</xref>]. <italic>E. coli</italic> bacteria were grown overnight in Luria Bertani medium [<xref ref-type="bibr" rid="B74">74</xref>] at 37&#x000b0;C with shaking at 150&#x000a0;rpm for 16&#x000a0;h and 100&#x000a0;&#x003bc;l of the resulting culture were spread onto LB agar plates [<xref ref-type="bibr" rid="B74">74</xref>] as a lawn. Sterile 7&#x000a0;mm glass fibre discs (Whatmann GF-C, Sigma, Poole, Dorset, UK) were immediately placed on the surface of the bacterial plates and essential oil (10&#x000a0;&#x003bc;l) was added to each disc. Grapeseed (<italic>Vitis vinifera</italic> seed) oil was included as a negative control, since it was not expected to exhibit any antibacterial activity [<xref ref-type="bibr" rid="B75">75</xref>]. Following growth at 37&#x000b0;C for 24&#x000a0;h, the zones of inhibition (halos) were measured with a ruler to an accuracy of 0.5&#x000a0;mm. An absence of antibacterial activity would produce a halo of 7&#x000a0;mm diameter, the size of the glass fibre discs. Disc diffusion assays were also carried out using 10&#x000a0;&#x003bc;l of the following antibiotic solutions in order to compare the antibacterial activities of some essential oils with those of the antibiotics: ampicillin (100&#x000a0;mg/ml in water), neomycin (10&#x000a0;mg/ml in water), and rifaximin (100&#x000a0;mg/ml in methanol), with disc diffusion assays also being carried out with water or methanol for comparison.</p></sec><sec><title>Disc diffusion assay: zone of clearance</title><p>This assay was adapted from that of Bexfield <italic>et al.</italic>[<xref ref-type="bibr" rid="B76">76</xref>]. <italic>E. coli</italic> bacterial overnight culture (100&#x000a0;&#x003bc;l) was spread aseptically onto LB agar plates, and grown overnight at 37&#x000b0;C for 16&#x000a0;h. Sterile 7&#x000a0;mm glass fibre discs containing 10&#x000a0;&#x003bc;l essential oil were added to each disc. Grapeseed oil was used as a control. The plates were grown at 37&#x000b0;C for a further 2&#x000a0;days and the diameters of the zones of clearance were measured with a ruler to an accuracy of 0.5&#x000a0;mm.</p></sec><sec><title>Turbidometric assay</title><p><italic>E. coli</italic> DH5&#x003b1; were grown in LB medium for 16&#x000a0;h overnight and the concentration of bacteria in the overnight culture was determined by measuring A₆₀₀[<xref ref-type="bibr" rid="B77">77</xref>]. Essential oil (100&#x000a0;&#x003bc;l) was added to 102&#x000a0;ml LB medium in a 500&#x000a0;ml conical flask. The flask was shaken to mix the contents and 2&#x000a0;ml of the liquid were removed to cuvettes for use as blanks. Bacterial overnight culture containing 1 &#x000d7; 10⁸<italic>E. coli</italic> cfu was added to the flask, which was shaken to mix the contents. A sample was removed to a cuvette and the A₆₀₀ was measured against the blank. The bacteria were grown at 37&#x000b0;C with shaking at 150&#x000a0;rpm and the A₆₀₀ was measured after 1&#x000a0;h and every 20&#x000a0;min thereafter for a further 5&#x000a0;h by sampling from the flask. Controls were performed, either lacking essential oil, or containing 100&#x000a0;&#x003bc;l grapeseed oil.</p></sec><sec><title>GC/MS of essential oils</title><p>The chemical composition of the oils was evaluated by injecting samples of dilute oil onto sorbent tubes, then extracting with thermal desorption for analysis using gas chromatography with mass spectrometry (GC/MS).</p><p>Oils were diluted to 1 part in 1000 in HPLC grade methanol and injected in 10&#x000a0;&#x003bc;L quantities into sorbent tubes (6.4&#x000a0;mm diameter stainless steel containing Tenax TA&#x000ae; sorbent). A stream of helium was used to remove the methanol over 3&#x000a0;minutes at a flow rate of 30&#x000a0;ml/min and at room temperature. Sample tubes containing the oils were loaded into an Ultra unit (Markes International, Llantrisant, UK) for automated processing via a Unity thermal desorption unit and fed with inert helium at 10&#x000a0;psi to desorb the tubes in a 30&#x000a0;ml/min stream of inert helium at 300&#x000b0;C for 5&#x000a0;minutes. To further concentrate, the flow was driven onto a cold trap (U-T11GPC, general purpose graphitized carbon C4/5-C30/32) set at -10&#x000b0;C. The trap was then desorbed at 300&#x000b0;C for 3&#x000a0;minutes with 50&#x000a0;ml of the sample stream vented and the remainder injected into the GC (Agilent Technologies 6890&#x000a0;N gas chromatograph). The VOCs were separated using a capillary column (30&#x000a0;m &#x000d7; 0.25&#x000a0;mm id, HP-5MS film thickness 0.25&#x000a0;&#x003bc;m). The column temperature was initially set at 40&#x000b0;C and then increased steadily to 200&#x000b0;C at a rate of 5&#x000b0;C/min. Mass spectrometry (Agilent Technologies 5973 network mass selective detector) was used in electronic ionisation mode and mass spectral data obtained in the SCAN mode with a mass range m/z 40 - 550. Each tube was run twice to prevent carry-over of volatiles onto the cold trap in subsequent samples and to check for VOCs inherent in the system. Three samples were taken for each type of oil.</p><p>Automated peak detection and baseline correction was used to calculate peak area and retention time (RT) for each compound. Using an automated library search function in the Chemstation GC/MS software (Agilent Technologies), VOCs were tentatively identified using the NIST 98 mass spectral library (The National Institute of Standards and Technology) at the apex of each peak and utilising probability based matching (PBM). The list of compounds generated was re-checked using forward-search matching and compared with the literature. The consistency of the mass response across the retention time range was checked by injecting aliquots of toluene, xylene and dodecane directly into sorbent tubes (1 part in 10,000 in methanol), and measuring the area under the curve. These compounds gave very similar mass responses for given concentrations, indicating that the area under the curve for each compound could be used to approximately indicate the abundance level.</p><p>The compounds that make up essential oils are often present in a range of optical and structural isomers with similar mass spectra and sometimes differing retention times. It is therefore difficult to precisely identify all compounds [<xref ref-type="bibr" rid="B78">78</xref>]. The separation and analysis methods employed do not allow identification of optical isomers, or all structural isomers, so while the compounds present will probably be specific optical and structural isomers, compound identification has been limited to generic, rather than specific, isomeric structures.</p></sec><sec><title>Preparation of plant extracts</title><p>Extracts were made from freeze dried coriander, lemon balm and spearmint leaves with various solvents using a modification of the methods of Wong and Kitts [<xref ref-type="bibr" rid="B79">79</xref>] for coriander and lemon balm or Lopez <italic>et al.</italic>[<xref ref-type="bibr" rid="B80">80</xref>] for mint. In each case, freeze dried leaf material was ground or chopped with a razor blade, resuspended in 10&#x000a0;ml solvent and left to macerate at room temperature for 18 or 24&#x000a0;h. The extracts were filtered through filter paper (Whatmann No. 1, Fisher Scientific. Loughborough, UK) and either used neat, or dried in a rotary evaporator set to 50&#x000b0;C and resuspended in the appropriate solvent. Extracts were stored at &#x02013;20&#x000b0;C and equilibrated to room temperature before use. The extracts were tested in disc diffusion assays. Negative controls consisted of the solvent that had been used to prepare the extract, in each case, whereas the appropriate essential oil was used as the positive control.</p></sec><sec><title>Statistics</title><p>The null hypothesis states that there is no difference between the diameter of the halos in the disc diffusion assays or the A₆₀₀ values in the turbidometric assays, and those of the controls. Two-tailed independent samples t tests were carried out to determine the probability that the diameter of the halos in the disc diffusion asays differed from those of the controls by chance. The results of the turbidometric assays were analysed by a one-way ANOVA and post-hoc test using SPSS.</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>Disc diffusion assays to examine the antibacterial activity of essential oils against <italic>E. coli</italic></title><p>Plant essential oils were tested for their antibacterial activity against <italic>E. coli</italic> DH5&#x003b1; by disc diffusion assays (see Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref>). The largest halo diameters were produced by pine (<italic>Pinus sylvestris</italic>), thyme (<italic>Thymus officinalis</italic>) and tea tree (<italic>Melaleuca alternifolia</italic>) oils, with mean halo diameters of 38.7 +/- 3.3&#x000a0;mm for thyme, 31.3 +/- 3.7&#x000a0;mm for pine and 27 +/- 1.5&#x000a0;mm for tea tree, against a mean halo diameter of 7 +/- 0&#x000a0;mm for the grapeseed oil negative control. The results were highly significantly different from the control in each case (P&#x02009;&#x0003c;&#x02009;0.01). This demonstrates that these oils have strong antibacterial activity towards <italic>E. coli</italic>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Antibacterial activity of essential oils in disc diffusion assays. A)</bold> Example of disc diffusion assay plate showing the halos in the bacterial lawn resulting from the antibacterial activity of lemon balm and coriander oils against <italic>E. coli</italic> DH5&#x003b1;. <bold>B)</bold> Graph of mean halo diameters from three disc diffusion assays showing the effect of essential oils on the growth of <italic>E. coli,</italic> +/- SEM. <bold>C)</bold> Graph of mean halo diameters from four replicates of a single disc diffusion assay showing the effect of essential oils or antibiotics on the growth of <italic>E. coli,</italic> +/- SEM. t tests were carried out to determine whether the halos produced by rifaximin were significantly different from the solvents, essential oils or other antibiotics. Significance levels obtained from two-tailed t tests are denoted by stars: *&#x02009;=&#x02009;significant (P&#x02009;&#x0003c;&#x02009;0.05); **&#x02009;=&#x02009;highly significant (P&#x02009;&#x0003c;&#x02009;0.01); ***&#x02009;=&#x02009;very highly significant (P&#x02009;&#x0003c;&#x02009;0.001).</p></caption><graphic xlink:href="1472-6882-13-338-1"/></fig><p>Antibacterial activity against <italic>E. coli</italic> was recorded at a more moderate, but still statistically significant level with coriander seed (mean halo diameter of 18 +/- 2.4&#x000a0;mm), lemon grass (18 +/- 1&#x000a0;mm), peppermint (17.7 +/- 3.5&#x000a0;mm), lemon balm (16 +/- 1.5&#x000a0;mm), fennel (14 +/- 0.6&#x000a0;mm), sage (14 +/- 2&#x000a0;mm), rosemary (13.3 +/- 0.3&#x000a0;mm), lavender (13 +/- 1&#x000a0;mm) and mandarin (<italic>Citrus reticulata</italic>) (12.7 +/- 1.2&#x000a0;mm). The antibacterial activity of peppermint essential oil was statistically significantly different from that of the grapeseed oil control whereas the activities of coriander seed, lemon balm, sage, lavender and mandarin essential oils were highly significantly different and those of lemon grass, fennel and rosemary very highly significantly different from that of the control. Ylang ylang (<italic>Cananga odorata</italic> var. <italic>genuina</italic>) demonstrated only a very weak antibacterial activity against <italic>E. coli</italic> (mean halo diameter&#x02009;=&#x02009;8.7 +/- 0.3&#x000a0;mm), which was, however, highly statistically significant. Like grapeseed oil, neem (<italic>Azadirachta indica</italic>) oil did not exhibit any antibacterial effect in this assay.</p><p>Disc diffusion assays were also conducted to compare the diameter of the halos produced by the antibiotic rifaximin (which has been shown to reverse SIBO in clinical trials) with halos produced by two other antibiotics: neomycin (used to treat SIBO and IBS clinically) and ampicillin (used to inhibit the growth of <italic>E. coli</italic> DH5a in the laboratory), and essential oils of peppermint, coriander seed and spearmint. The results are shown in Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref>C. As expected, the mean diameter of the halos produced by rifaximin in the disc diffusion assays was larger and very highly significantly different from that produced by the methanol control, demonstrating that the rifaximin solution had antibacterial activity against <italic>E. coli</italic> DH5&#x003b1; which was not due to the methanol that was used to dissolve the rifaximin. The halos produced by neomycin and ampicillin were larger than those produced by rifaximin, showing that these antibiotics are more potent at killing/inhibiting the growth of <italic>E. coli</italic> in the disc diffusion assay than rifaximin, and the results were very highly significant. Interestingly, there was no significant difference between the diameter of the halos produced by rifaximin with those produced by spearmint essential oil, suggesting that, at least in this assay, spearmint essential oil is just as potent as rifaximin. Moreover, the halos produced by peppermint and coriander seed essential oils were larger and either significantly different, or very highly significantly different from the rifaximin in each case, demonstrating that peppermint and coriander seed essential oils have a larger antibacterial activity against <italic>E. coli</italic> DH5&#x003b1; in this assay than rifaximin.</p></sec><sec><title>Turbidometric assays to examine the antibacterial activity of essential oils against <italic>E. coli</italic></title><p>The antibacterial activities of the various essential oils against <italic>E. coli</italic> were also examined using turbidometric assays since this method is said to be more sensitive than the disc diffusion assay [<xref ref-type="bibr" rid="B81">81</xref>]. It can also reveal whether the oil slows down the growth of bacteria, or inhibits growth altogether. The results are shown in Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>. The Absorbance₆₀₀ values obtained after 360&#x000a0;minutes, corresponding to bacterial growth were analysed by one way ANOVA. There were significant differences in variance detected between the mean absorbance values at t&#x02009;=&#x02009;360 mins, F_{14, 30}&#x02009;=&#x02009;3253.911, p&#x02009;&#x0003c;&#x02009;0.0001. Post hoc extraction (Least Significant Difference method) revealed that there were significant differences between the standard and the samples containing the essential oils, with the exception of grapeseed oil (p&#x02009;&#x0003c;&#x02009;0.05), i.e. all of the essential oils inhibited the growth of <italic>E. coli</italic> DH5&#x003b1; in the turbidometric assay, except for grapeseed oil.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>Antibacterial activity of essential oils in turbidometric assays.</bold> Graphs showing the results of turbidometric assays to examine the effect of plant essential oils on the growth of <italic>E. coli</italic> in liquid culture. Data are the mean of three experiments +/- SEM.</p></caption><graphic xlink:href="1472-6882-13-338-2"/></fig><p>In fact grapeseed oil produced a small and reproducible stimulation of the growth of <italic>E. coli</italic> compared to the standard lacking essential oil in this assay (Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>), suggesting that it is acting as a prebiotic. The other oils all inhibited the growth of <italic>E. coli</italic>. Ylang ylang oil inhibited <italic>E. coli</italic> growth by a small amount whereas rosemary, lavender, lemon balm, lemon grass, fennel and pine inhibited <italic>E. coli</italic> growth more strongly. The curves for samples containing these oils were exponential, indicating that there was some bacterial growth, albeit less than in the control. Mandarin, peppermint, sage, tea tree, thyme and coriander seed essential oils almost completely inhibited the growth of <italic>E. coli</italic> in the turbidometric assay, the growth curves were flat.</p><p>Samples containing lemon balm, lemon grass or mandarin essential oils had a high OD₆₀₀, even at the start of the bacterial incubation, although the absorbances did not rise very steeply after that and, in the case of mandarin oil, fell. This should not have happened and cannot be attributed to light absorbance at 600&#x000a0;nm by coloured compounds in the oils, since the spectrophotometer was zeroed against LB blanks containing the appropriate oil. The effect was reproducible with certain oils, but the reason for it is not clear &#x02013; perhaps some of the oils formed light scattering micelles.</p></sec><sec><title>Zone of clearance assay</title><p>In order to investigate whether the essential oils could lyse <italic>E. coli</italic> that were already present rather than inhibiting their growth, zone of clearance assays were conducted, ie glass fibre discs containing essential oils were placed on pre-existing <italic>E. coli</italic> lawns, and halos were allowed to develop. This was important because treatments to target SIBO would need to kill bacteria that were already present in the small intestine, rather than simply inhibiting their growth.</p><p>Coriander seed, lemon balm, peppermint, pine and tea tree oils exhibited strong antibacterial activity in the zone of clearance assay (see Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>), with mean halo diameters of 28.0 +/- 1.4&#x000a0;mm for tea tree, 25.8 +/- 1.8&#x000a0;mm for peppermint, 24.1 +/- 3.1&#x000a0;mm for lemon balm, 22.8 +/- 2.0&#x000a0;mm for coriander seed and 19.1&#x000a0;mm +/- 1.4&#x000a0;mm for pine essential oils, compared to a mean halo diameter of 7 +/- 0&#x000a0;mm for grapeseed oil. t tests revealed that the halo diameters for coriander seed, peppermint, pine and tea tree were very highly significantly different from those of the grapeseed oil control (P&#x02009;&#x0003c;&#x02009;0.001) whereas the halo diameters for lemon balm were highly significantly different from those of the control (p&#x02009;&#x0003c;&#x02009;0.01). Many of the other oils exhibited a small effect in the zone of clearance assay, namely fennel (mean halo diameter: 11.6 +/- 0.9), rosemary (11.4 +/- 0.9&#x000a0;mm), thyme (10.6 +/- 1.1&#x000a0;mm), lavender (10.4 +/- 0.6&#x000a0;mm), mandarin (10.3 +/- 1.3&#x000a0;mm), sage (10.3 +/- 0.6&#x000a0;mm) and lemon grass (10.1 +/- 0.4&#x000a0;mm). The halos for mandarin and thyme were significantly different from those of the grapeseed oil control whereas those for fennel, lavender, rosemary and sage were highly significantly different and the halos for lemon grass were very highly significantly different from the halos produced by the grapeseed oil control.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Antibacterial activity of essential oils in zone of clearance assays.</bold> Graph showing mean halo diameters +/- SEM obtained from four separate zone of clearance assays to determine the ability of plant essential oils to lyse <italic>E. coli.</italic> Significance levels obtained from two-tailed t tests are denoted by stars: *&#x02009;=&#x02009;Significant, P&#x02009;&#x0003c;&#x02009;0.05; **&#x02009;=&#x02009;highly significant, P&#x02009;&#x0003c;&#x02009;0.01; ***&#x02009;=&#x02009;very highly significant, P&#x02009;&#x0003c;&#x02009;0.001.</p></caption><graphic xlink:href="1472-6882-13-338-3"/></fig><p>The results of the disc diffusion assays, the turbidometric assays and the zone of clearance assays have been summarised in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>. It can be seen that coriander seed, lemon balm, peppermint, pine and tea tree essential oils performed well in all three assays.</p></sec><sec><title>Mass spectrometry of coriander seed, lemon balm and peppermint essential oils</title><p>Since coriander, lemon balm and peppermint are all culinary herbs and their essential oils are all on the list of food additives that are generally recognised as safe to ingest [<xref ref-type="bibr" rid="B82">82</xref>], thermal desorption gas chromatography mass spectrometry (GC/MS) was conducted on the coriander seed, lemon balm and peppermint essential oils used in this study to identify the compounds present, and to assess whether the oils were representative of their respective types.</p><p>Sample chromatograms are shown in Figure&#x000a0;<xref ref-type="fig" rid="F4">4</xref>. Lists of the main compounds detected by GC/MS are tabulated for lemon balm, peppermint and coriander seed oils in Tables&#x000a0;<xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref> and <xref ref-type="table" rid="T4">4</xref> respectively. The approximate concentration is calculated as the mean of three samples of each essential oil. Compound identification is tentative and based on correlation with a mass spectra database. The compounds shown are those present in approximate concentrations of 1% or more and were primarily in the form of terpenes or terpinoids.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Chromatograms of lemon balm, peppermint and coriander oils.</p></caption><graphic xlink:href="1472-6882-13-338-4"/></fig><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Major compounds detected in lemon balm oil</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Compound (Tentative identification)</bold></th><th align="left"><bold>Approximate percentage mass</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Citronellal<hr/></td><td align="left" valign="bottom">29.79<hr/></td></tr><tr><td align="left" valign="bottom">Geraniol/Nerol<hr/></td><td align="left" valign="bottom">19.85<hr/></td></tr><tr><td align="left" valign="bottom">Citronellol<hr/></td><td align="left" valign="bottom">11.20<hr/></td></tr><tr><td align="left" valign="bottom">Citronellol acetate<hr/></td><td align="left" valign="bottom">4.07<hr/></td></tr><tr><td align="left" valign="bottom">Isopulegol<hr/></td><td align="left" valign="bottom">3.59<hr/></td></tr><tr><td align="left" valign="bottom">Limonene<hr/></td><td align="left" valign="bottom">3.23<hr/></td></tr><tr><td align="left" valign="bottom">Linalyl acetate<hr/></td><td align="left" valign="bottom">2.80<hr/></td></tr><tr><td align="left" valign="bottom">Unknown<hr/></td><td align="left" valign="bottom">2.62<hr/></td></tr><tr><td align="left" valign="bottom">Unknown<hr/></td><td align="left" valign="bottom">2.55<hr/></td></tr><tr><td align="left" valign="bottom">Unknown<hr/></td><td align="left" valign="bottom">1.87<hr/></td></tr><tr><td align="left" valign="bottom">Linalool<hr/></td><td align="left" valign="bottom">1.65<hr/></td></tr><tr><td align="left" valign="bottom">Camphene<hr/></td><td align="left" valign="bottom">1.44<hr/></td></tr><tr><td align="left" valign="bottom">Citral<hr/></td><td align="left" valign="bottom">1.42<hr/></td></tr><tr><td align="left" valign="bottom">Caryophyllene<hr/></td><td align="left" valign="bottom">1.35<hr/></td></tr><tr><td align="left">Unknown</td><td align="left">1.05</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Major compounds detected in peppermint oil</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Compound (Tentative identification)</bold></th><th align="left"><bold>Approximate percentage mass</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Menthol<hr/></td><td align="left" valign="bottom">52.33<hr/></td></tr><tr><td align="left" valign="bottom">Menthone<hr/></td><td align="left" valign="bottom">29.96<hr/></td></tr><tr><td align="left" valign="bottom">1,8-cineole (eucalyptol)<hr/></td><td align="left" valign="bottom">3.70<hr/></td></tr><tr><td align="left" valign="bottom">Menthyl acetate<hr/></td><td align="left" valign="bottom">2.96<hr/></td></tr><tr><td align="left" valign="bottom">Unknown<hr/></td><td align="left" valign="bottom">1.80<hr/></td></tr><tr><td align="left">Caryophyllene</td><td align="left">1.24</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Major compounds detected in coriander seed oil</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Compound (Tentative identification)</bold></th><th align="left"><bold>Approximate percentage mass</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Linalool<hr/></td><td align="left" valign="bottom">72.16<hr/></td></tr><tr><td align="left" valign="bottom">Cymene<hr/></td><td align="left" valign="bottom">5.58<hr/></td></tr><tr><td align="left" valign="bottom">Camphor<hr/></td><td align="left" valign="bottom">5.58<hr/></td></tr><tr><td align="left" valign="bottom">Pinene<hr/></td><td align="left" valign="bottom">3.97<hr/></td></tr><tr><td align="left" valign="bottom">Linalyl acetate<hr/></td><td align="left" valign="bottom">3.15<hr/></td></tr><tr><td align="left" valign="bottom">Limonene<hr/></td><td align="left" valign="bottom">2.12<hr/></td></tr><tr><td align="left">Terpinene</td><td align="left">1.86</td></tr></tbody></table></table-wrap><p>The lemon balm oil primarily consisted of citronellal, geraniol/nerol (alcohol form of citral) and citronellol, with smaller amounts of citronellol acetate, isopulegol and limonene (see Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>). It was unusual in containing only a low level of citral, which consists of a mixture of the two isomeric compounds geranial and neral, and is usually reported to be a major constituent of lemon balm oil [<xref ref-type="bibr" rid="B83">83</xref>-<xref ref-type="bibr" rid="B85">85</xref>], although lemon balm oils with only slightly higher levels of citral than ours have been documented [<xref ref-type="bibr" rid="B86">86</xref>]. Some of the lower concentration compounds gave similar library matches to one another despite being detected at different retention times. This suggests similar structures, which could not be adequately distinguished by the techniques used in the study, and these compounds have been labelled as &#x0201c;unknown&#x0201d;. The peppermint oil consisted primarily of menthol and menthone, with smaller amounts of eucalyptol (1,8-cineole), menthyl acetate and caryophyllene (see Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>), which is in agreement with other published analyses of peppermint oil [<xref ref-type="bibr" rid="B87">87</xref>-<xref ref-type="bibr" rid="B89">89</xref>]. The coriander seed oil consisted primarily of linalool, with smaller amounts of cymene, camphor, pinene, linalyl acetate, limonene and terpinene (see Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>). The compounds were similar to those reported elsewhere for coriander seed [<xref ref-type="bibr" rid="B90">90</xref>-<xref ref-type="bibr" rid="B93">93</xref>]. Again this suggests that the oil used in this study was representative of its type. Our analysis could not differentiate between isomers and many compounds present at lower concentrations were not adequately identified, and require further investigation.</p></sec><sec><title>Disc diffusion assays to examine the effect of plant extracts on the growth of <italic>E. coli</italic></title><p>The experiments in the earlier sections showed that the essential oils of coriander seed, lemon balm and peppermint exhibited antibacterial activity against <italic>E. coli</italic> in three separate assays, suggesting that they might reduce numbers of <italic>E. coli</italic> in the gastrointestinal tract, and thus be able to reverse intestinal dysbiosis and SIBO, and resolve the symptoms of IBS. In fact, a clinical trial has shown that a mixed herbal extract of coriander, lemon balm and spearmint (called carmint) reduced the symptoms of IBS after eight weeks [<xref ref-type="bibr" rid="B66">66</xref>]. Presumably carmint (said to contain &#x0201c;total extracts&#x0201d; of the three herbs [<xref ref-type="bibr" rid="B66">66</xref>]) is an ethanolic extract of coriander, lemon balm and spearmint leaves (and possibly stems), although this is not explicitly stated. The mechanism of action underlying carmint&#x02019;s efficacy in treating IBS has not been described. In order to determine whether ethanolic extracts of coriander, lemon balm and spearmint leaves would have antibacterial activity, which could explain carmint&#x02019;s efficacy, we made extracts of coriander, lemon balm and spearmint leaves using ethanol or various other solvents of differing polarity, and carried out disc diffusion assays. It should be noted that even if the extracts made with solvents other than ethanol had antibacterial activity, they would be useless therapeutically because the solvents would be toxic. The solvents were used as negative controls, since some would exhibit antibacterial activity in their own right, and the appropriate essential oil was used as the positive control.</p></sec><sec><title>Coriander leaf extracts</title><p>The results for disc diffusion assays with coriander leaf extracts are shown in Figure&#x000a0;<xref ref-type="fig" rid="F5">5</xref>. The mean halo diameter produced by the coriander extract that was made with chloroform is no larger than those produced by the chloroform control (8.7 +/- 0.3&#x000a0;mm versus 7.7 +/- 0.3&#x000a0;mm), thus the chloroform extract of coriander does not exhibit any antibacterial effects against <italic>E. coli.</italic> The coriander extract made with DMSO seemed to exhibit antibacterial activity, with a mean halo diameter of 16 +/- 3&#x000a0;mm, compared to 7.7 +/- 0.3&#x000a0;mm for the DMSO control, although the results fell short of significance. The ethanolic extract of coriander exhibited potent antibacterial activity, with a mean halo diameter of 22.7 +/- 1.2&#x000a0;mm versus 10.7 +/- 0.9&#x000a0;mm for the ethanol control, with the results being highly significant. The coriander extract made with ethyl acetate was not significantly different from the ethyl acetate control (mean halo diameters of 14.3 +/- 2.9&#x000a0;mm versus 9.7 +/- 0.9&#x000a0;mm). Finally, the methanolic extract of coriander also exhibited strong antibacterial activity, with a mean halo diameter of 25 +/- 3.5&#x000a0;mm versus 13 +/- 3.6&#x000a0;mm for the methanol control, with the results being statistically significant. Thus the ethanolic and methanolic extracts of coriander were the most potent at inhibiting the growth of <italic>E. coli</italic> in the disc diffusion assay, suggesting that the antibacterial compounds have been extracted into these polar solvents.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>Antibacterial activity of coriander extracts in disc diffusion assays.</bold> Graph showing mean halo diameters +/- SEM obtained from three separate disc diffusion assays to determine the effect of coriander extracts made with various solvents on the growth of <italic>E. coli.</italic> Significance levels obtained from two-tailed t tests are denoted by stars: *&#x02009;=&#x02009;Significant, P&#x02009;&#x0003c;&#x02009;0.05; **&#x02009;=&#x02009;highly significant, P&#x02009;&#x0003c;&#x02009;0.01; ***&#x02009;=&#x02009;very highly significant, P&#x02009;&#x0003c;&#x02009;0.001. Significance levels for coriander essential oil were not determined.</p></caption><graphic xlink:href="1472-6882-13-338-5"/></fig></sec><sec><title>Lemon balm extracts</title><p>The results for disc diffusion assays with lemon balm extracts are shown in Figure&#x000a0;<xref ref-type="fig" rid="F6">6</xref>. Lemon balm extract made with chloroform did not exhibit any antibacterial activity against <italic>E. coli</italic>, since the mean halo diameter was 7.7 +/- 0.3&#x000a0;mm for both the chloroform control and the chloroform extract. Extracts of lemon balm made with DMSO exhibited moderate antibacterial activity against <italic>E. coli,</italic> with a mean halo diameter of 12.3 +/- 1.2&#x000a0;mm versus 7 +/- 0&#x000a0;mm, with the results being statistically significant. More potent antibacterial activity was observed with lemon balm extracts made with ethanol, ethyl acetate and methanol, with mean halo diameters of 18 +/- 2.5&#x000a0;mm, 20.7 +/- 3&#x000a0;mm and 19 +/- 2&#x000a0;mm respectively versus 9 +/- 1.5, 10.3 +/- 1.8 and 8 +/- 0&#x000a0;mm respectively, for the solvent controls. The results for the extracts made with ethanol and ethyl acetate were statistically significant, and those for the methanolic extract were highly statistically significant. This suggests that the antibacterial compounds in lemon balm have been extracted into ethyl acetate, methanol and ethanol.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold>Antibacterial activity of lemon balm extracts.</bold> Graph showing mean halo diameters +/- SEM obtained from three separate disc diffusion experiments to determine the effect of lemon balm extracts made with various solvents on the growth of <italic>E. coli.</italic> Significance levels obtained from two-tailed t tests are denoted by stars: *&#x02009;=&#x02009;Significant, P&#x02009;&#x0003c;&#x02009;0.05; **&#x02009;=&#x02009;highly significant, P&#x02009;&#x0003c;&#x02009;0.01; ***&#x02009;=&#x02009;very highly significant, P&#x02009;&#x0003c;&#x02009;0.001. Significance levels for disc diffusion assays with lemon balm essential oil were not determined.</p></caption><graphic xlink:href="1472-6882-13-338-6"/></fig></sec><sec><title>Spearmint extracts</title><p>The results for disc diffusion assays with spearmint extracts are shown in Figure&#x000a0;<xref ref-type="fig" rid="F7">7</xref>. Spearmint extracts made with dichloromethane showed very little antibacterial effect against <italic>E. coli,</italic> producing halos with an average diameter of 8.8 +/- 0.2&#x000a0;mm compared to 8.0 +/- 0.0&#x000a0;mm for the dichloromethane control. The results were highly statistically significant, however. Spearmint extracts made with ethanol inhibited the growth of <italic>E. coli</italic>, producing halos of average diameter 16.2 +/- 1.2&#x000a0;mm versus 8.8 +/- 0.2&#x000a0;mm for the ethanol control, and the results were highly significant. Spearmint extracts made with ethyl acetate displayed antibacterial activity, producing halos of average diameter 14.3 +/- 0.7&#x000a0;mm versus average halo diameters of 11.0 +/- 0.6&#x000a0;mm for the solvent control; the results were statistically significant. Spearmint extracts prepared with methanol displayed a similar level of antibacterial activity, producing halos with a mean diameter of 13.2&#x02009;+&#x02009;0.4&#x000a0;mm versus 8.8 +/- 0.4&#x000a0;mm for the methanol control. The results were highly significant. This suggests that the antibacterial compounds in spearmint have been extracted into ethanol, ethyl acetate and methanol.</p><fig id="F7" position="float"><label>Figure 7</label><caption><p><bold>Antibacterial activity of spearmint extracts.</bold> Graph showing mean halo diameters +/- SEM from three disc diffusion assays to determine the effect of spearmint extracts made with various solvents on the growth of <italic>E. coli.</italic> Significance levels obtained from two-tailed t tests are denoted by stars: *&#x02009;=&#x02009;Significant, P&#x02009;&#x0003c;&#x02009;0.05; **&#x02009;=&#x02009;highly significant, P&#x02009;&#x0003c;&#x02009;0.01; ***&#x02009;=&#x02009;very highly significant, P&#x02009;&#x0003c;&#x02009;0.001. Significance levels for disc diffusion assays with peppermint essential oil were not determined.</p></caption><graphic xlink:href="1472-6882-13-338-7"/></fig><p>The results show that ethanolic extracts of coriander, lemon balm and spearmint leaves have antibacterial activity, and thus that the mixed herbal extract carmint (which contains extracts of coriander, lemon balm and spearmint) would be expected to have antibacterial activity, which would probably be responsible for its efficacy as a treatment for IBS.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>The data above show that pine, tea tree and thyme oils all exhibited strong antibacterial activity against <italic>E. coli</italic> in the disc diffusion assay, with coriander seed, lemon balm, lemon grass, mandarin and peppermint essential oils having more moderate antibacterial activity. In each case the results were statistically significant. This agrees with reports in the literature since inhibition of the growth of different strains of <italic>E. coli</italic> has been demonstrated by coriander [<xref ref-type="bibr" rid="B94">94</xref>,<xref ref-type="bibr" rid="B95">95</xref>], fennel [<xref ref-type="bibr" rid="B94">94</xref>-<xref ref-type="bibr" rid="B97">97</xref>], lavender [<xref ref-type="bibr" rid="B94">94</xref>,<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B99">99</xref>], lemon balm [<xref ref-type="bibr" rid="B98">98</xref>], lemon grass [<xref ref-type="bibr" rid="B94">94</xref>,<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B101">101</xref>], mandarin [<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B102">102</xref>], peppermint [<xref ref-type="bibr" rid="B94">94</xref>,<xref ref-type="bibr" rid="B96">96</xref>,<xref ref-type="bibr" rid="B97">97</xref>,<xref ref-type="bibr" rid="B99">99</xref>,<xref ref-type="bibr" rid="B103">103</xref>], pine [<xref ref-type="bibr" rid="B104">104</xref>], rosemary [<xref ref-type="bibr" rid="B96">96</xref>,<xref ref-type="bibr" rid="B97">97</xref>,<xref ref-type="bibr" rid="B105">105</xref>,<xref ref-type="bibr" rid="B106">106</xref>], sage [<xref ref-type="bibr" rid="B96">96</xref>,<xref ref-type="bibr" rid="B106">106</xref>], tea tree [<xref ref-type="bibr" rid="B94">94</xref>,<xref ref-type="bibr" rid="B107">107</xref>] thyme [<xref ref-type="bibr" rid="B96">96</xref>,<xref ref-type="bibr" rid="B107">107</xref>,<xref ref-type="bibr" rid="B108">108</xref>], and ylang ylang [<xref ref-type="bibr" rid="B94">94</xref>]. Most of these reports used disc diffusion assays or growth inhibition assays to determine the minimum inhibitory concentration of essential oils that would inhibit the growth of <italic>E. coli</italic>. In this present study, coriander seed, fennel, mandarin, peppermint, sage, tea tree and thyme essential oils all blocked the growth of <italic>E. coli</italic> in a turbidometric assay as well, whereas lavender, lemon balm, lemon grass, pine and rosemary oils exhibited strong antibacterial activity in this assay, at least for six hours, the approximate length of time that would elapse between doses of a theoretical IBS medicine, which would be taken before breakfast, lunch, dinner, and possibly bed time. Any one of these essential oils, or a mixture of them, would be a potential candidate for the reversal of SIBO and treatment of IBS. The advantages of using a mixture of essential oils rather than a single oil are that the larger number of compounds would act on a wider range of bacteria, the oils might act synergistically to have a stronger effect on any given bacterium (as has been documented for thyme and anise (<italic>Pimpinella anisum</italic>) oils [<xref ref-type="bibr" rid="B109">109</xref>]), and there would be a reduced chance of producing resistant bacteria.</p><p>Coriander seed, lemon balm, peppermint and tea tree essential oils demonstrated strong antibacterial effects against <italic>E. coli</italic> in a zone of clearance assay (and pine had a more moderate effect), indicating that they are bacteriocidal towards <italic>E. coli</italic>. Of these, coriander seed, lemon balm and peppermint essential oils are on the FDA list of food additives that are generally recognised as safe to ingest [<xref ref-type="bibr" rid="B82">82</xref>]. Indeed peppermint and coriander seed essential oils displayed higher antibacterial activity in a disc diffusion assay with <italic>E. coli</italic> DH5&#x003b1; than rifaximin, considered to be the antibiotic of choice for treating SIBO [<xref ref-type="bibr" rid="B34">34</xref>]. Thus coriander seed, lemon balm and peppermint essential oils would be very good candidates for the treatment of IBS, either singly or in combination, since they would be able to kill bacteria that were already present in the small intestine as SIBO, rather than just inhibiting their growth.</p><p>The compounds present in these three essential oils were identified by thermal desorption gas chromatography mass spectrometry; many have been reported to have antibacterial activity. Antibacterial activity against <italic>E. coli</italic> has been documented for linalool [<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B110">110</xref>-<xref ref-type="bibr" rid="B113">113</xref>], cymene [<xref ref-type="bibr" rid="B114">114</xref>], camphor [<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B110">110</xref>] and pinene [<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B110">110</xref>], which are all present in coriander seed oil; citronellal [<xref ref-type="bibr" rid="B112">112</xref>], geraniol/nerol [<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B111">111</xref>,<xref ref-type="bibr" rid="B112">112</xref>,<xref ref-type="bibr" rid="B115">115</xref>,<xref ref-type="bibr" rid="B116">116</xref>], citronellol [<xref ref-type="bibr" rid="B111">111</xref>], and isopulegol [<xref ref-type="bibr" rid="B117">117</xref>], which are present in lemon balm oil; limonene [<xref ref-type="bibr" rid="B102">102</xref>,<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B110">110</xref>,<xref ref-type="bibr" rid="B113">113</xref>,<xref ref-type="bibr" rid="B118">118</xref>], which is present in both coriander seed and lemon balm oil; as well as caryophyllene [<xref ref-type="bibr" rid="B113">113</xref>], which is present in lemon balm and peppermint oil; and eucalyptol [<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B110">110</xref>,<xref ref-type="bibr" rid="B111">111</xref>,<xref ref-type="bibr" rid="B119">119</xref>], menthol [<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B110">110</xref>] and menthone [<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B118">118</xref>], which are all present in peppermint oil. Citral, which is usually reported to be a major constituent of lemon balm oil, but was present only at a low level in the lemon balm oil that we used, has also been shown to have antibacterial activity against <italic>E. coli</italic>[<xref ref-type="bibr" rid="B98">98</xref>,<xref ref-type="bibr" rid="B100">100</xref>,<xref ref-type="bibr" rid="B108">108</xref>,<xref ref-type="bibr" rid="B112">112</xref>,<xref ref-type="bibr" rid="B115">115</xref>].</p><p>Lemon balm essential oil varies enormously in its chemical composition. This can be due to the use of different plant lines or cultivars [<xref ref-type="bibr" rid="B83">83</xref>-<xref ref-type="bibr" rid="B85">85</xref>], variation in plant growth conditions such as climate/soil [<xref ref-type="bibr" rid="B83">83</xref>] or salinity [<xref ref-type="bibr" rid="B86">86</xref>], harvesting at different points in the growing season, or in different years [<xref ref-type="bibr" rid="B84">84</xref>], different harvest cut height [<xref ref-type="bibr" rid="B120">120</xref>], use of either fresh or dried plant material to produce the essential oil [<xref ref-type="bibr" rid="B84">84</xref>] and storage of the essential oil, since citral levels decrease with storage time [<xref ref-type="bibr" rid="B84">84</xref>]. Citral levels have been shown to range between 3.1 +/- 0.35% and 5.4 +/- 0.12%, in essential oils from plants that were grown in the presence of salt and harvested at the full bloom stage [<xref ref-type="bibr" rid="B86">86</xref>], to between 10.86% and 64.56% in essential oils from plants that were not flowering [<xref ref-type="bibr" rid="B84">84</xref>], with Cosge <italic>et al.</italic>[<xref ref-type="bibr" rid="B85">85</xref>] reporting intermediate citral levels between 10.1% and 17.43% and higher citronellal levels between 36.62% and 43.78%. Patora <italic>et al</italic>. hypothesised that citral is converted to citronellal as the plant ages [<xref ref-type="bibr" rid="B84">84</xref>]. This highlights the need to conduct rigorous quality control analysis, if lemon balm (or any herbal product) is to be used medicinally. Further work is ongoing in our laboratory to investigate the variation in the chemical composition and antibacterial activity of different batches of lemon balm oil (and other essential oils) obtained from different companies, and to analyse the antibacterial activity of the individual compounds in the oils, with the aim of informing eventual quality control analysis of essential oils for use as medicines. We have several batches of lemon balm oil from the same company as the one that we used in this study, with very similar chemical composition, and several batches from another company with higher concentrations of citral.</p><p>For more in depth evaluation of essential oils, the use of more appropriate separation technologies such as chiral columns would aid the identification and separation of the various isomeric forms common in essential oils. Furthermore, the use of systems such as Kovats retention index would produce data independent of the chromatographic setting, which could be readily compared with the literature and hence aid identification [<xref ref-type="bibr" rid="B78">78</xref>].</p><p>Carmint (a mixture of coriander, lemon balm and spearmint extracts) has been demonstrated to reduce IBS symptoms [<xref ref-type="bibr" rid="B66">66</xref>]. Our findings with the essential oils of coriander seed, lemon balm and peppermint suggested that carmint&#x02019;s efficacy may be due to antibacterial activity, and the ability to reverse gastrointestinal dysbiosis or SIBO. To investigate this further, extracts were made of coriander, lemon balm and spearmint leaves using a variety of solvents, and tested for their antibacterial activity. In each case, extracts made with ethanol or methanol displayed the most potent antibacterial activity, and since carmint is assumed to contain ethanolic extracts of the herbs, this suggests that carmint is effective against IBS because it has antibacterial activity. Other properties may also contribute to carmint&#x02019;s mechanism of action, however, since both peppermint and lemon balm essential oils and extracts have been shown to have antispasmodic activity [<xref ref-type="bibr" rid="B57">57</xref>,<xref ref-type="bibr" rid="B58">58</xref>], peppermint has also been shown to act on serotonin receptors [<xref ref-type="bibr" rid="B121">121</xref>] and lemon balm reduces stress [<xref ref-type="bibr" rid="B122">122</xref>] and could therefore modulate the gut-brain axis.</p><p>Peppermint and lemon balm extracts are also present in the mixed herbal extract, Iberogast&#x000ae;, or STW-5, which has also been shown to be effective against IBS in a clinical trial [<xref ref-type="bibr" rid="B63">63</xref>]. This suggests that antibacterial activity may also underlie Iberogast&#x000ae;&#x02019;s mechanism of action, in addition to its documented antispasmodic, anti-inflammatory, antioxidant and prosecretory effects [<xref ref-type="bibr" rid="B55">55</xref>,<xref ref-type="bibr" rid="B62">62</xref>]. This hypothesis is further strengthened by the fact that Iberogast&#x000ae; has been shown in clinical trials to be effective in treating functional dyspepsia [<xref ref-type="bibr" rid="B64">64</xref>], which has been linked to infection with <italic>Helicobacter pylori</italic> and can be treated with antibiotics [<xref ref-type="bibr" rid="B123">123</xref>].</p><p>There has been one case study that suggested that enteric-coated peppermint oil tablets could reduce SIBO. Treatment of a patient with IBS and SIBO with enteric-coated peppermint oil tablets for 20&#x000a0;days resulted in a 32% reduction in hydrogen and methane excretion at the 60&#x000a0;minute time-point of a lactulose breath test (six days after the end of treatment), which correlated with an improvement in the IBS symptoms of pain, bloating, eructation and altered bowel habit [<xref ref-type="bibr" rid="B124">124</xref>]. The was ascribed to an antibacterial effect of the peppermint oil in the capsules reducing bacterial numbers in the small intestine, although it has been suggested that the treatment may instead have altered gastrointestinal transit time [<xref ref-type="bibr" rid="B125">125</xref>]. It is debatable whether enteric-coated peppermint oil tablets would be able to reverse SIBO in all patients, however, because they are designed to uncoat in the lower intestine in order to exert an antispasmodic effect on the colon, and thus may traverse much of the small intestine intact. The type of enteric-coated peppermint oil capsules that were used in the study was not identified, which is relevant because different types vary in their pharmacokinetics. Mintec&#x000ae; tablets uncoat earlier with a lag time of 0.5&#x000a0;h and a time to peak release of 2.8&#x000a0;h, and display an intense release of peppermint oil, whereas Colpermin&#x000ae; tablets uncoat later with a lag time of 1.07&#x000a0;h and a time to peak release of 5&#x000a0;h and have a more steady release profile [<xref ref-type="bibr" rid="B126">126</xref>,<xref ref-type="bibr" rid="B127">127</xref>]. Thus Mintec&#x000ae; tablets would be more likely to be able to address SIBO. It could be argued that peppermint water [<xref ref-type="bibr" rid="B61">61</xref>] would be better still, although it would be important not to have too high a dose, since it could cause heartburn due to relaxation of the oesophageal sphincter [<xref ref-type="bibr" rid="B128">128</xref>], and also tachycardia [<xref ref-type="bibr" rid="B129">129</xref>]. Whether the patient whose case study was reported [<xref ref-type="bibr" rid="B124">124</xref>], had achlorhydria, which might have caused the enteric-coated peppermint oil tablets to uncoat early [<xref ref-type="bibr" rid="B130">130</xref>], was not addressed.</p><p>Some of the other herbs whose essential oils have been shown to have antibacterial properties in this current study are also present in herbal medicines that have been proposed to be useful for treating IBS, or used to treat digestive disorders in the past. Mandarin is present in the Chinese herbal medicine Tong Xie Yao Fang (TXYF), and a modified version TXYFa, which has been shown by a systematic review to be potentially effective in the treatment of IBS, since it reduced abdominal pain, distension, flatulence and diarrhoea for up to 6&#x000a0;months after treatment [<xref ref-type="bibr" rid="B131">131</xref>]. Absinthe contains fennel (shown in this study and others to have antibacterial properties) as well as wormwood (<italic>Artemisia absinthium</italic> L.), anise and often lemon balm, Roman wormwood (<italic>Artemisia pontica</italic> L.) and hyssop (<italic>Hyssopus officinalis</italic> L.) [<xref ref-type="bibr" rid="B132">132</xref>]. Although absinthe is now thought of as an alcoholic beverage, with unhappy connotations due to its ability to cause absinthism when drunk to excess, it was originally used in the 1780s to cure colic (aka IBS) and fight dysentery, which would correlate with its probable antibacterial activity.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>New treatments are needed for IBS. Since IBS has been linked to intestinal dysbiosis or small intestinal bacterial overgrowth, we have investigated the antibacterial activity of the essential oils of a range of culinary and medicinal herbs, many of which have traditionally been used as digestives or are present in herbal medicines that have been shown to be effective in treating IBS in clinical trials, in three assays: the disc diffusion assay, the turbidometric assay and the zone of clearance assay. Of the essential oils that are on the FDA list of essential oils that are generally recognised as safe to ingest, coriander seed, lemon balm and peppermint essential oils exhibited strong antibacterial activity in all three assays, suggesting that the reason why carmint is effective for treating IBS is because it has antibacterial activity. The fact that ethanolic extracts of coriander, lemon balm and spearmint leaves all exhibited antibacterial activity, provided more evidence to support this hypothesis. Since other herbal medicines that have been shown/proposed to be useful in treating IBS and other digestive disorders, contain essential oils or extracts with antibacterial activity, we hypothesise that antibacterial properties are an important mechanism of action for herbal IBS medicines. We consider that essential oils of coriander seed, lemon balm and peppermint and other antibacterial essential oils on the FDA GRAS list, as well as carmint and Iberogast&#x000ae; all warrant further investigation in mechanistic studies and randomised, double-blind, placebo controlled clinical trials to assess their ability to act on intestinal dysbiosis or SIBO and resolve the symptoms of IBS.</p></sec><sec><title>Abbreviations</title><p>ANOVA: Analysis of variance; cfu: Colony forming units; DMSO: Dimethyl sulphoxide; FODMAPs: Fermentable oligosaccharides, disaccharides, monosaccharides and polyols; GC/MS: Gas chromatography with mass spectrometry; HPLC: High Purification Liquid Chromatography; IBS: Irritable bowel syndrome; LHBT: Lactulose hydrogen breath test; NICE: National Institute for Health and Clinical Excellence; NIST: The National Institute of Standards and Technology; PBM: Probability based matching; SIBO: Small intestinal bacterial overgrowth; VOC: Volatile organic compound.</p></sec><sec><title>Competing interests</title><p>We have no competing interests to declare. Aiysha Thompson, Dilruba Meah, Nadia Ahmed, Rebecca-Conniff-Jenkins and Emma Chileshe were all undergraduate students at Swansea University while this work was carried out. Dr. Chris Phillips, Professor Tim Claypole, Dr. Dan Forman and Dr. Paula Row are all employed by Swansea University and received no funding for this work. Paula Row has since been the recipient of funding for summer vacation studentships from the British Society for Antimicrobial Chemotherapy and The Biochemical Society, respectively, for two other students to continue this work, and (together with Tim Claypole, Chris Phillips and Dr. Nidhika Berry, the director of the Public Health Laboratories in Singleton Hospital, Swansea) has also been awarded a &#x0201c;Bridging the Gaps&#x0201d; Grant from Funds awarded by EPSRC to Swansea University, to continue this work.</p></sec><sec><title>Authors&#x02019; contributions</title><p>AT, DM and NA are all joint first authors. AT helped to design and carried out the turbidometric assays, designed and carried out the experiments with peppermint extracts, performed corroborative disc diffusion assays and zone of clearance assays, performed some of the statistical analysis, helped to prepare the figures, helped to write the manuscript, and critically reviewed the manuscript. DM helped to design and carried out the disc diffusion assays, designed the zone of clearance assays, performed the experiments with coriander and lemon balm extracts, performed corroborative turbidometric assays and zone of clearance assays, performed some of the statistical analysis, helped to write the manuscript, and critically reviewed the manuscript. NA carried out the zone of clearance assays, performed corroborative disc diffusion assays and turbidometric assays, performed some of the statistical analysis, helped to write the manuscript, and critically reviewed the manuscript. RC-J performed corroborative disc diffusion assays and turbidometric assays, helped to write the manuscript, and critically reviewed the manuscript. EC performed corroborative disc diffusion assays and turbidometric assays, helped to write the manuscript, and critically reviewed the manuscript. COP and TCC performed the GC/MS analysis, analysed the GC/MS data and helped to write the manuscript, and critically reviewed the manuscript. DWF helped carry out statisctical analysis of the data and provided critical intellectual input into the study and the manuscript, helped to write the manuscript and critically reviewed it. PER conceived and designed the study, helped to design the experiments, supervised AT, DM, NA, RCC and EC in carrying out the experiments, helped carry out statistical analysis of the data, helped to prepare the figures, took the lead in writing the manuscript, amalgamating the other authors&#x02019; contributions into it, and critically reviewed the manuscript. All authors read and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6882/13/338/prepub">http://www.biomedcentral.com/1472-6882/13/338/prepub</ext-link></p></sec>

Usage patterns and adverse experiences in traditional Korean medicine: results of a survey in South Korea

<sec><title>Background</title><p>Although traditional medicine (TM) in South Korea is included in the national health care system, it is considered complementary and alternative medicine (CAM), and not mainstream medicine. Therefore, the lack of statistical data regarding the usage and adverse experiences of traditional Korean medicine (TKM) makes difficult to understand the current status of TM. In this study, we aimed to report usage patterns and adverse experiences on TKM targeting consumers in South Korea.</p></sec><sec><title>Methods</title><p>A total of 2000 consumers participated in the survey on usage and adverse experiences in 2008. Among the 2,000 participants, 915 (45.8%) had taken herbal medicine or received traditional medicinal therapies; these individuals were further surveyed on the internet or in an interview.</p></sec><sec><title>Results</title><p>The usage rate was higher among women and among patients in their 30s. Of the total TKM usage, acupuncture accounted for 36.7%, and herbal medicine accounted for 13.4%. Regarding the frequency of use of TKM, 73.8% of patients reported using TM less than 5 times in 1 year. Of the 915 respondents, 8.2% of individuals had some type of adverse experience resulting from TKM. Adverse experiences were primarily caused by acupuncture and herbal medicines, and they primarily involved diseases of the digestive system and skin. The incidence of adverse experiences was less than 3.7% for acupuncture and 3.8% for herbal medicine. Overall, the incidence rate of adverse experiences for TKM for the entire population was 0.04 per 10,000 individuals.</p></sec><sec><title>Conclusions</title><p>The medical usage and occurrence of adverse events on TKM should be surveyed periodically, and the statistical trends should be analysed. The disparity between the survey results for traditional herbal medicines and medical practices, and those for the national pharmacovigilance system or academic reports of adverse experiences should be examined. The national pharmacovigilance system must be improved to compensate for the disparities. Policies and regulations are required to enhance the reporting of adverse experiences not only for herbal medicines but also for traditional medicinal therapies.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Shin</surname><given-names>Hyeun-Kyoo</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>hkshin@kiom.re.kr</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Jeong</surname><given-names>Soo-Jin</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>sjijeong@kiom.re.kr</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Huang</surname><given-names>Dae Sun</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>the7star@hanmail.net</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Kang</surname><given-names>Byoung-Kab</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>bkkang@kiom.re.kr</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Lee</surname><given-names>Myeong Soo</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>drmslee@gmail.com</email></contrib>

BMC Complementary and Alternative Medicine

<sec><title>Background</title><p>Recently, interest in complementary and alternative medicine (CAM) and traditional medicine (TM) has increased in the clinical settings of many countries, and herbal medicines and acupuncture have become globally popular therapies [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>]. Herbal medicines have been used in the clinics of China, Korea and Japan for thousands of years, and the toxicity and efficacy of herbal therapies have been well established in the literature. These studies have become important sources of information and techniques for treating intractable diseases [<xref ref-type="bibr" rid="B3">3</xref>]. However, the largest problems regarding CAM/TM are the lack of a sufficient scientific basis for establishing efficacy and the questions that have arisen regarding the safety of increasing the use of CAM/TM.</p><p>Traditional Korean medicine (TKM), which is one part of CAM, faces similar problems. In 2008, 14,818 TKM doctors in South Korea treated a total of 12,128,657 patients with TKM that were reimbursable by the national health insurance system. These therapies included 56 types of herbal formulations and 68 types of single herbal crude extracts as well as traditional therapies, such as acupuncture, moxibustion, cupping therapy [<xref ref-type="bibr" rid="B4">4</xref>]. Since TKM was first included in the national insurance system, its usage rate has increased, whereas the number of reports of adverse events remains low.</p><p>The first case involving the adverse events of herbal medicine was reported at a conference in South Korea in 1979 [<xref ref-type="bibr" rid="B5">5</xref>]. The reporting of adverse events caused by all drugs and treatments for human diseases is an important topic for which the government has set specific regulations. The Korean government first established regulations of pharmacovigilance in the national public healthcare system in 1988, which required that all adverse drug events be reported according to the regulations [<xref ref-type="bibr" rid="B6">6</xref>]. In 2007 and 2008, the number of reported adverse events in South Korea was 3,750 and 7,210, respectively. Among these cases, only 8 cases were reported on herbal medicines in both 2007 and 2008 [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. Adverse events of chiropractic therapy and acupuncture were first reported in 1988 and 2006, respectively [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. Since then, reports of adverse events of TM therapy have been rare.</p><p>The current pharmacovigilance system does include a method for the spontaneous reporting of adverse events of herbal medicines; however, there are no systems in place for monitoring and reporting adverse events of other traditional therapy methods. Any adverse event that occurs can only be determined from publications reported by relevant experts. Thus, there are currently no methods for assessing the number of adverse events arising from herbal medicines or other traditional therapies in South Korea.</p><p>To recognise the increasing interest in traditional therapies among the public, this survey aims to assess the usage patterns and adverse experiences of TKM affecting consumers in South Korea.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Study design</title><p>This study is a survey of Koreans on usage patterns and adverse experiences in Korean medicine. We collected the participants for the survey by sex, age, and residence and designed a statistically number of samples.</p></sec><sec><title>Setting</title><p>A total of 2,000 South Koreans (1,000 each, male and female) over the age of 20 were surveyed at the NI-Korea company between December 1 and December 17, 2008. We promoted the study to the public on the internet and interviewed individuals with trained interviewers from a professional survey research company. We randomly advertised the survey and sent a questionnaire by email to the study participants.</p></sec><sec><title>Participants</title><p>We clearly determine the number of survey population (n&#x02009;=&#x02009;2,000) using sample size calculation and stratified them by sex, age and residence. Then, we randomly sampled from the sampling frame provided by the Statistics Korea based on Neyman&#x02019;s allocation method. Among the 2,000 individuals by sample size, 1,771 were surveyed online, while 229 individuals were interviewed face-to-face. If there is survey nonresponse, we randomly chose and added new sample from the sampling frame.</p></sec><sec><title>Variables</title><p>The survey questions used are as follows:</p><p>&#x02013; Experience and number of TKM usage events</p><p>&#x02013; Usage patterns of TKM treatment (e.g., acupuncture, moxibustion, cupping therapy, physical therapy, and chiropractic) and herbal medicine</p><p>&#x02013; Number of adverse experiences and the targeted organ of TKM</p></sec><sec><title>Data sources/measurement</title><p>The symptoms of adverse experience were classified into five groups as follows: diseases of the digestive system, diseases of the skin, diseases of the nervous system, disorders of the kidneys, and other disorders.</p></sec><sec><title>Bias</title><p>This study was a retrospective survey. Recall bias and selection bias may have existed in this study.</p></sec><sec><title>Study size</title><p>A weighted application three-society stratified sample (sex, age, and area of administration) method was used for analysis by proportionate probability sampling. The stratified random sampling targeted a sampling error of &#x000b1;2.2 with a confidence interval of 95%.</p><p>The total sampling number:</p><p><disp-formula><mml:math id="M1" name="1472-6882-13-340-i1" overflow="scroll"><mml:mrow><mml:mi mathvariant="normal">n</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:msup><mml:mfenced open="(" close=")"><mml:mrow><mml:mstyle displaystyle="true"><mml:mi mathsize="big">&#x02211;</mml:mi></mml:mstyle><mml:mi mathvariant="italic">N&#x003c3;</mml:mi></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mrow><mml:msup><mml:mfenced open="(" close=")"><mml:mrow><mml:mfrac><mml:mi>&#x003f5;</mml:mi><mml:msub><mml:mi>t</mml:mi><mml:mi>&#x003b1;</mml:mi></mml:msub></mml:mfrac><mml:mo>&#x000d7;</mml:mo><mml:mstyle displaystyle="true"><mml:mi mathsize="big">&#x02211;</mml:mi></mml:mstyle><mml:mi>X</mml:mi></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:mstyle displaystyle="true"><mml:mi mathsize="big">&#x02211;</mml:mi></mml:mstyle><mml:mi>N</mml:mi><mml:msup><mml:mi>&#x003c3;</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p>Sampling number in each class:</p><p><disp-formula><mml:math id="M2" name="1472-6882-13-340-i2" overflow="scroll"><mml:mrow><mml:msub><mml:mi>n</mml:mi><mml:mi>s</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi mathvariant="normal">n</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mfrac><mml:mi mathvariant="italic">N&#x003c3;</mml:mi><mml:mrow><mml:mstyle displaystyle="true"><mml:mi mathsize="big">&#x02211;</mml:mi></mml:mstyle><mml:mi mathvariant="italic">N&#x003c3;</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p><italic>n</italic>: national sample population over the age of 20</p><p><italic>n</italic>_{<italic>s</italic>}: sample population in each class</p><p><italic>N</italic>: national parent population over the age of 20</p><p><italic>X</italic>: population in each class</p><p><italic>&#x003c3;</italic>: standard deviation of population in each class</p><p><italic>&#x003c3;</italic>^{<italic>2</italic>}: variance of population in each class</p><p><disp-formula><mml:math id="M3" name="1472-6882-13-340-i3" overflow="scroll"><mml:mfrac><mml:mi>&#x003f5;</mml:mi><mml:msub><mml:mi>t</mml:mi><mml:mi>&#x003b1;</mml:mi></mml:msub></mml:mfrac></mml:math></disp-formula></p><p>: expecting precision (&#x003f5;&#x02009;=&#x02009;tolerance error, <italic>t</italic>_{<italic>&#x003b1;</italic>}&#x02009;=&#x02009;confidence coefficient).</p><p>The total of 2,000 (1,000 each, male and female) people over the age of 20 was calculated using Neyman&#x02019;s sample number decision formula and the optimal allocation method. The 1,085 individuals who did not have any experience with TKM were excluded. The remaining 915 individuals who had experience with TKM were surveyed for the development of any adverse experience of TKM therapies. Thus, the survey was analyzed for 915 respondents.</p></sec><sec><title>Statistical analysis</title><p>Frequency analysis was conducted for the questions. The survey results analyses were performed using SPSS WIN 12.0K (SPSS Inc., Chicago, IL, USA).</p></sec><sec><title>Ethical considerations</title><p>The ethical review committee of the Korea Institute of Oriental Medicine waived the need for formal ethical approval and informed consent to be obtained from participants due to the nature of the study. The survey was already conducted on a voluntary basis with agreement from the participants for the use of the collected data for scientific purposes.</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>Usage patterns of traditional Korean medicine</title><p>Of the initial 2,000 individuals surveyed, only 45.8% had received TKM, including herbal medicines, acupuncture, moxibustion, cupping therapy, physical therapy, or chiropractic therapy, during the previous year. The rates of usage for each TKM in proportion to the total 2000 individuals surveyed were as follows: acupuncture, 36.7%; herbal medicine, 13.4%; physical therapy, 10.0%; moxibustion, 9.0%; cupping therapy, 5.7%; chiropractic therapy, 1.5%; and other therapies, 0.5%.</p><p>Of the 915 individuals who had received TKM, more females (53.6%) used these TKMs than males (46.4%). Among the different age groups, TKM usage varied as follows: 26.7% of those in their 30s used TKM, 25.0% of individuals in their 40s used TKM, 21.2% of individuals in their 20s used TKM, 17.3% of individuals in their 50s used TKM and 9.8% of individuals in their 60s used TKM. In terms of the frequency of usage, 73.8% used TKM fewer than 5 times per year, 13.1% used TKM 6 to 10 times per year and 13.1% used TKM more than 11 times per year.</p><p>Moreover, 915 people who used TKM received combination therapies. Acupuncture was the most commonly repeated therapy at 80.1%, followed by herbal medicines (29.2%), physical therapy (21.9%), moxibustion (19.6%), cupping therapy (12.4%), chiropractic therapy (3.3%) and other therapies (1.1%).</p><p>Of the 425 males, 80.2% received acupuncture, 24.5% took herbal medicines, 21.6% received physical therapy, 19.2% received moxibustion, 13.8% received cupping therapy and 3.9% received chiropractic therapy. Of the 490 females, 80.1% received acupuncture, 33.2% took herbal medicines, 22.2% received physical therapy, 19.9% received moxibustion, 11.1% received cupping therapy and 2.7% received chiropractic therapy. The gender distribution for the use of each therapy was as follows: females comprised 53.5% of the total acupuncture users, 61.0% of the herbal medicine users, 54.0% of the physical therapy patients and 54.2% of the moxibustion users, all of which represented higher usage rates compared with the males. However, males represented 52.2% of the cupping therapy users and 56.7% of the chiropractic therapy users compared with women.</p><p>Regarding the age group distribution, 78.1% of the 194 individuals in their 20s received acupuncture and 30.9% took herbal medicines. Regarding the 244 individuals in their 30s who were surveyed, 77.2% received acupuncture and 31.8% took herbal medicines. Of the 90 seniors in their 60s, 90.0% received acupuncture and 45.3% took herbal medicines; both rates were higher than those of any other age group.</p><p>Regarding the type of therapy, of the 733 total acupuncture cases, the predominant recipients were people in their 30s (25.7%) and 40s (24.8%). Of the 267 individuals who used herbal medicine, 29.2% were people in their 30s. Regarding the 200 cases of physical therapy, 28.0% represented people in their 30s. Regarding the 179 cases of moxibustion, 26.8% were people in their 40s. Regarding the 113 cases of cupping therapy, 35.4% were individuals in their 30s. Regarding the 30 cases of chiropractic therapy, 30.0% were people in their 40s, who were the most frequent recipients of this type of therapy.</p><p>Of the total cases for each therapy, the percentage of individuals receiving 5 or fewer treatments of TKM were 71.6% for acupuncture, 73.8% for herbal medicines, 75.5% for physical therapy, 68.7% for moxibustion, 68.1% for cupping therapy and 70.0% for chiropractic therapy (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Usage of traditional Korean medicine (n&#x02009;=&#x02009;915)</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th rowspan="2" align="center" valign="top"><bold>Demographic characteristics</bold></th><th rowspan="2" align="center" valign="top"><bold>No. of respondents</bold></th><th colspan="7" align="center" valign="bottom"><bold>Types of traditional Korean medicine</bold><hr/></th></tr><tr><th align="center"><bold>Acupuncture</bold></th><th align="center"><bold>Herbal medicine</bold></th><th align="center"><bold>Physical therapy</bold></th><th align="center"><bold>Moxibustion</bold></th><th align="center"><bold>Cupping</bold></th><th align="center"><bold>Chiropractic</bold></th><th align="center"><bold>Others</bold></th></tr></thead><tbody valign="top"><tr><td align="center" valign="bottom">All respondents<hr/></td><td align="center" valign="bottom">915<hr/></td><td align="center" valign="bottom">733(80.1)<hr/></td><td align="center" valign="bottom">267(29.2)<hr/></td><td align="center" valign="bottom">200(21.9)<hr/></td><td align="center" valign="bottom">179(19.6)<hr/></td><td align="center" valign="bottom">113(12.4)<hr/></td><td align="center" valign="bottom">30(3.3)<hr/></td><td align="center" valign="bottom">10(1.1)<hr/></td></tr><tr><td align="center" valign="bottom">Gender<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;Male<hr/></td><td align="center" valign="bottom">425<hr/></td><td align="center" valign="bottom">341(80.2)<hr/></td><td align="center" valign="bottom">104(24.5)<hr/></td><td align="center" valign="bottom">92(21.6)<hr/></td><td align="center" valign="bottom">82(19.2)<hr/></td><td align="center" valign="bottom">59(13.8)<hr/></td><td align="center" valign="bottom">17(3.9)<hr/></td><td align="center" valign="bottom">3(0.7)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;Female<hr/></td><td align="center" valign="bottom">490<hr/></td><td align="center" valign="bottom">392(80.1)<hr/></td><td align="center" valign="bottom">163(33.2)<hr/></td><td align="center" valign="bottom">108(22.2)<hr/></td><td align="center" valign="bottom">97(19.9)<hr/></td><td align="center" valign="bottom">54(11.1)<hr/></td><td align="center" valign="bottom">13(2.7)<hr/></td><td align="center" valign="bottom">7(1.4)<hr/></td></tr><tr><td align="center" valign="bottom">Age<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;20-29<hr/></td><td align="center" valign="bottom">194<hr/></td><td align="center" valign="bottom">152(78.1)<hr/></td><td align="center" valign="bottom">60(30.9)<hr/></td><td align="center" valign="bottom">46(23.6)<hr/></td><td align="center" valign="bottom">41(20.6)<hr/></td><td align="center" valign="bottom">14(7.0)<hr/></td><td align="center" valign="bottom">4(2.2)<hr/></td><td align="center" valign="bottom">1(0.4)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;30-39<hr/></td><td align="center" valign="bottom">244<hr/></td><td align="center" valign="bottom">188(77.2)<hr/></td><td align="center" valign="bottom">78(31.8)<hr/></td><td align="center" valign="bottom">56(22.9)<hr/></td><td align="center" valign="bottom">37(15.2)<hr/></td><td align="center" valign="bottom">40(16.6)<hr/></td><td align="center" valign="bottom">7(2.8)<hr/></td><td align="center" valign="bottom">3(1.2)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;40-49<hr/></td><td align="center" valign="bottom">229<hr/></td><td align="center" valign="bottom">182(79.6)<hr/></td><td align="center" valign="bottom">53(23.3)<hr/></td><td align="center" valign="bottom">40(17.7)<hr/></td><td align="center" valign="bottom">48(21.1)<hr/></td><td align="center" valign="bottom">36(15.7)<hr/></td><td align="center" valign="bottom">9(4.0)<hr/></td><td align="center" valign="bottom">5(2.0)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;50-59<hr/></td><td align="center" valign="bottom">158<hr/></td><td align="center" valign="bottom">130(82.3)<hr/></td><td align="center" valign="bottom">35(22.4)<hr/></td><td align="center" valign="bottom">34(22.2)<hr/></td><td align="center" valign="bottom">39(24.9)<hr/></td><td align="center" valign="bottom">10(6.5)<hr/></td><td align="center" valign="bottom">8(4.9)<hr/></td><td align="center" valign="bottom">1(1.0)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;60-69<hr/></td><td align="center" valign="bottom">90<hr/></td><td align="center" valign="bottom">81(90.0)<hr/></td><td align="center" valign="bottom">41(45.3)<hr/></td><td align="center" valign="bottom">24(26.2)<hr/></td><td align="center" valign="bottom">14(16.0)<hr/></td><td align="center" valign="bottom">13(14.2)<hr/></td><td align="center" valign="bottom">2(2.3)<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="center" valign="bottom">N. of utilizations<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;&#x02264;5<hr/></td><td align="center" valign="bottom">675<hr/></td><td align="center" valign="bottom">525(77.9)<hr/></td><td align="center" valign="bottom">197(29.2)<hr/></td><td align="center" valign="bottom">151(22.5)<hr/></td><td align="center" valign="bottom">123(18.3)<hr/></td><td align="center" valign="bottom">77(11.4)<hr/></td><td align="center" valign="bottom">21(3.2)<hr/></td><td align="center" valign="bottom">7(1.0)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;6-10<hr/></td><td align="center" valign="bottom">120<hr/></td><td align="center" valign="bottom">104(86.4)<hr/></td><td align="center" valign="bottom">35(29.4)<hr/></td><td align="center" valign="bottom">29(24.4)<hr/></td><td align="center" valign="bottom">27(22.2)<hr/></td><td align="center" valign="bottom">17(14.3)<hr/></td><td align="center" valign="bottom">3(2.1)<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="center">&#x02003;&#x000a0;&#x02265;11</td><td align="center">120</td><td align="center">104(86.4)</td><td align="center">35(29.0)</td><td align="center">20(16.6)</td><td align="center">29(24.1)</td><td align="center">19(15.8)</td><td align="center">6(4.6)</td><td align="center">3(2.9)</td></tr></tbody></table><table-wrap-foot><p>All data are in n (%).</p></table-wrap-foot></table-wrap></sec><sec><title>Adverse experience from traditional medicinal therapies and herbal medicines</title><p>The 915 individuals included in this study received a total of 1,532 TKM treatments. When asked about adverse experiences, 7.2% of the individuals reported adverse experiences, while 91.8% individuals reported no adverse experiences. The types of adverse experiences included diseases of the digestive system (40.0%), skin (30.6%), nervous system (14.6%), kidney (6.6%) and others (7.9%).</p><p>Of the 425 male and 490 female participants, 8.7% and 7.9%, respectively, reported adverse experiences. In terms of the type of adverse experiences, diseases of the digestive system (43.2%) and skin (37.8%) were predominant in males. For the females, diseases of the digestive system (36.8%), skin (23.7%) and nervous system (23.7%) were the most common.</p><p>Respondents in their 20s had the highest rate of adverse experiences at 10.3%, while sexagenarians had the lowest rate of the experiences at 4.4%. Age distribution regarding adverse experiences on TKM was as follows: 20s (26.7%), 30s (28.8%), 40s (20.0%), 50s (20.0%) and 60s (5.3%). The most common type of adverse experience for each group was as follows: diseases of the digestive system for individuals in their 20s (60.0%), diseases of the skin for both individuals in their 30s (38.0%) and those in their 40s (40.1%), diseases of the digestive system for individuals in their 50s (60.2%) and diseases of the skin for individuals in their 60s (76.0%).</p><p>The increased use of TM therapy also led to an increase in adverse experiences, as the percentage was 7.7% in those receiving 5 or fewer treatments, 8.3% in those receiving 6 to 10 treatments and 10.8% in those receiving 11 or more treatments. The most common type of adverse experience for each group was as follows: diseases of the digestive system (44.2%) for individuals receiving 5 or fewer treatments, diseases of the skin (60.0%) for those receiving 6 to 10 treatments and diseases of the digestive system (38.4%) for those receiving 11 or more treatments (Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>).</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Adverse experiences on traditional Korean medicinal therapies and the number of disease incidents</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th rowspan="2" align="center" valign="top"><bold>Demographic characteristics</bold></th><th rowspan="2" align="center" valign="top"><bold>No. of respondents</bold></th><th colspan="2" align="center" valign="bottom"><bold>Adverse experience</bold><hr/></th><th colspan="5" align="center" valign="bottom"><bold>Diseases related to adverse experiences</bold><hr/></th></tr><tr><th align="center"><bold>No</bold></th><th align="center"><bold>Yes</bold></th><th align="center"><bold>Digestive system</bold></th><th align="center"><bold>Skin</bold></th><th align="center"><bold>Nervous system</bold></th><th align="center"><bold>Kidney</bold></th><th align="center"><bold>Others</bold></th></tr></thead><tbody valign="top"><tr><td align="center" valign="bottom">All respondents<hr/></td><td align="center" valign="bottom">915<hr/></td><td align="center" valign="bottom">840(91.8)<hr/></td><td align="center" valign="bottom">75(8.2)<hr/></td><td align="center" valign="bottom">30(40.0)<hr/></td><td align="center" valign="bottom">23(30.6)<hr/></td><td align="center" valign="bottom">11(14.6)<hr/></td><td align="center" valign="bottom">5(6.6)<hr/></td><td align="center" valign="bottom">6(8.0)<hr/></td></tr><tr><td align="center" valign="bottom">Gender<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;Male<hr/></td><td align="center" valign="bottom">425<hr/></td><td align="center" valign="bottom">388(91.3)<hr/></td><td align="center" valign="bottom">37(8.7)<hr/></td><td align="center" valign="bottom">16(43.2)<hr/></td><td align="center" valign="bottom">14(37.8)<hr/></td><td align="center" valign="bottom">2(5.4)<hr/></td><td align="center" valign="bottom">3(8.1)<hr/></td><td align="center" valign="bottom">2(5.4)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;Female<hr/></td><td align="center" valign="bottom">490<hr/></td><td align="center" valign="bottom">452(92.1)<hr/></td><td align="center" valign="bottom">38(7.9)<hr/></td><td align="center" valign="bottom">14(36.8)<hr/></td><td align="center" valign="bottom">9(23.7)<hr/></td><td align="center" valign="bottom">9(23.7)<hr/></td><td align="center" valign="bottom">2(5.3)<hr/></td><td align="center" valign="bottom">4(10.5)<hr/></td></tr><tr><td align="center" valign="bottom">Age<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;20-29<hr/></td><td align="center" valign="bottom">194<hr/></td><td align="center" valign="bottom">174(89.7)<hr/></td><td align="center" valign="bottom">20(10.3)<hr/></td><td align="center" valign="bottom">12(60.0)<hr/></td><td align="center" valign="bottom">5(25.0)<hr/></td><td align="center" valign="bottom">1(5.0)<hr/></td><td align="center" valign="bottom">1(5.0)<hr/></td><td align="center" valign="bottom">1(5.0)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;30-39<hr/></td><td align="center" valign="bottom">244<hr/></td><td align="center" valign="bottom">223(91.4)<hr/></td><td align="center" valign="bottom">21(8.6)<hr/></td><td align="center" valign="bottom">6(28.6)<hr/></td><td align="center" valign="bottom">8(38.0)<hr/></td><td align="center" valign="bottom">5(23.8)<hr/></td><td align="center" valign="bottom">1(4.8)<hr/></td><td align="center" valign="bottom">1(4.8)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;40-49<hr/></td><td align="center" valign="bottom">229<hr/></td><td align="center" valign="bottom">214(93.4)<hr/></td><td align="center" valign="bottom">15(6.6)<hr/></td><td align="center" valign="bottom">3(20.0)<hr/></td><td align="center" valign="bottom">6(40.1)<hr/></td><td align="center" valign="bottom">2(13.3)<hr/></td><td align="center" valign="bottom">2(13.3)<hr/></td><td align="center" valign="bottom">2(13.3)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;50-59<hr/></td><td align="center" valign="bottom">158<hr/></td><td align="center" valign="bottom">143(90.5)<hr/></td><td align="center" valign="bottom">15(9.5)<hr/></td><td align="center" valign="bottom">9(60.2)<hr/></td><td align="center" valign="bottom">1(6.6)<hr/></td><td align="center" valign="bottom">2(13.3)<hr/></td><td align="center" valign="bottom">1(6.6)<hr/></td><td align="center" valign="bottom">2(13.3)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;60-69<hr/></td><td align="center" valign="bottom">90<hr/></td><td align="center" valign="bottom">86(95.6)<hr/></td><td align="center" valign="bottom">4(4.4)<hr/></td><td align="center" valign="bottom">-<hr/></td><td align="center" valign="bottom">3(76.0)<hr/></td><td align="center" valign="bottom">1(24.0)<hr/></td><td align="center" valign="bottom">-<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="center" valign="bottom">N. of utilizations<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;&#x02264;5<hr/></td><td align="center" valign="bottom">675<hr/></td><td align="center" valign="bottom">623(92.3)<hr/></td><td align="center" valign="bottom">52(7.7)<hr/></td><td align="center" valign="bottom">23(44.2)<hr/></td><td align="center" valign="bottom">16(30.8)<hr/></td><td align="center" valign="bottom">6(13.5)<hr/></td><td align="center" valign="bottom">2(3.8)<hr/></td><td align="center" valign="bottom">4(7.7)<hr/></td></tr><tr><td align="center" valign="bottom">&#x02003;&#x000a0;6-10<hr/></td><td align="center" valign="bottom">120<hr/></td><td align="center" valign="bottom">110(91.7)<hr/></td><td align="center" valign="bottom">10(8.3)<hr/></td><td align="center" valign="bottom">2(20.0)<hr/></td><td align="center" valign="bottom">6(60.0)<hr/></td><td align="center" valign="bottom">1(10.0)<hr/></td><td align="center" valign="bottom">-<hr/></td><td align="center" valign="bottom">1(10.0)<hr/></td></tr><tr><td align="center">&#x02003;&#x000a0;&#x02265;11</td><td align="center">120</td><td align="center">107(89.2)</td><td align="center">13(10.8)</td><td align="center">5(38.4)</td><td align="center">1(7.7)</td><td align="center">3(23.1)</td><td align="center">3(23.1)</td><td align="center">1(7.7)</td></tr></tbody></table><table-wrap-foot><p>All data are in n (%).</p></table-wrap-foot></table-wrap></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>This study was conducted to report usage patterns and adverse experiences on TKM targeting consumers in South Korea. We found that of the 2,000 respondents, only 45.8% had used TKM in 2008. Among countries with percentages of TM usage in Northeast Asia in 2001, China, Japan and South Korea had usage percentages of 90.0%, 49.0% and 69.0%, respectively [<xref ref-type="bibr" rid="B9">9</xref>]. In contrast, our survey results revealed a decrease in the TM usage rate in South Korea. For individuals over 20 years of age, the usage of TKM was 45.8% compared with the usage by individuals 0-18 years of age at 11.5% [<xref ref-type="bibr" rid="B10">10</xref>]. Additionally, the rate of acupuncture use in South Korea was 36.7%, which was much higher than rates in the UK (1.6%), the US (1.1%), and Japan (6.1%) [<xref ref-type="bibr" rid="B11">11</xref>]. However, approximately 70% of the individuals used TKM 5 or fewer times.</p><p>Among the participants over 20 years of age included in this study, 8.2% reported adverse experiences on TKM. That rate obtained in the present study is higher than the rate of adverse experience of 0.27% reported for CAM in Korean children aged 0 to 18 years [<xref ref-type="bibr" rid="B10">10</xref>].</p><p>TKM therapies function by stimulating or applying pressure to the skin, muscles, or skeleton of the human body and can cause adverse experiences, such as diseases of the skin and nervous system. The representative adverse experiences of these therapies included pain or itching, burning, bleeding or hematoma and paralysis. Regarding the oral administration of traditional herbal medicines, the digestion, absorption, metabolism and excretion of the medicine can cause adverse events on the digestive and kidney systems, including stomachache, jaundice, liver failure, and oedema of the face, hands and feet. In this survey, acupuncture and herbal medicines were considered the main causes of the adverse experiences observed.</p><p>The adverse experiences of acupuncture were primarily diseases of both the skin and nervous system. When assessed using the incidence frequency of the two diseases, the rate of adverse experiences of acupuncture was 3.7%, which is lower than the rates reported in any other country. According to a review of the reported results in databases, the literatures, and randomised clinical trials from countries that are comparable to South Korea, the rates for adverse experiences were 11.8% in Canadian children, 8.6% in Switzerland, and ranged from 6.71% to 15% in China [<xref ref-type="bibr" rid="B12">12</xref>-<xref ref-type="bibr" rid="B14">14</xref>]. Additionally, when the total incidence rate for adverse experiences among the general population of South Korea was calculated based on the rate of 3.7% found in this study, the rate was 0.04 per 10,000, which is lower than the rate of 0.55 per 10,000 reported in the US [<xref ref-type="bibr" rid="B15">15</xref>].</p><p>Studies in China have reported that the rate of adverse experiences from acupuncture depends on age and gender, with older age groups and males having higher rates than females [<xref ref-type="bibr" rid="B16">16</xref>]. In Japan, younger age groups and females were more sensitive to needle stimulation, while elderly individuals were better able to tolerate pain [<xref ref-type="bibr" rid="B11">11</xref>]. In the current study in South Korea, the rate of adverse experiences did not correlate with age; however, males displayed a higher incidence of diseases of the skin, while females had a higher incidence of diseases of the nervous system.</p><p>Regarding the relationship between the frequency and adverse experiences of acupuncture, adverse events occurred more often during the early stage of the treatment than during the late stages. We can speculate that this difference occurs because people may discontinue treatment if they experience adverse events during the beginning stages.</p><p>In this study, the main adverse experiences due to acupuncture were itchiness, bleeding or hematoma and paralysis; these results are similar to those reported in other studies. The adverse events reported for Canadian children included pain, bruising, bleeding and a worsening of symptoms; Americans experienced sedation (30.98%), needle pain (25.44%), and neuropathy/nervous system-related issues (15.42%); and Swiss patients experienced bleeding or hematoma (6.1%), pain (1.7%) and vegetative symptoms (0.7%) [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. However, a Chinese review article found that the adverse events reported in China included symptoms that were more serious, such as pneumothorax, fainting, subarachnoid haemorrhage and infection [<xref ref-type="bibr" rid="B14">14</xref>], thereby showing the disparities in the severity of adverse events between countries.</p><p>Following acupuncture, the next most common cause of adverse experience was herbal medicine, i.e., Chinese herbs. In Korea, significant adverse effects such as hepatitis and neuropathy were first reported at conferences in 1999 and 2000, respectively [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>].</p><p>The main adverse events were diseases of the digestive system and kidney. The rate of adverse experiences based on the incidence of these two types of diseases was 3.8% or less, and the estimated risk of serious adverse experiences in the overall population was as low as 0.04/10,000 patients in Korea. There were no differences relative to gender, although individuals in their 20s who took herbal medicines more frequently showed diseases of the digestive system as an adverse experience. This is contrary to the case of acupuncture, in which more frequent use resulted in less adverse events. These findings indicate that the adverse experiences of with herbal medicines are more serious. Acupuncture is associated with minor and simple adverse experiences, whereas herbal medicines can be metabolised and cause serious damage to the organs.</p><p>In modern society, which requires state-of-the art science in medicine, CAMs have gained popularity. However, tragic adverse events, such as Chinese herbal neuropathy, have already been reported in studies or case reports [<xref ref-type="bibr" rid="B20">20</xref>]. In China, there have been frequent reports of adverse events on TM in recent years, but the causes of these events are complex. TMs are thought to damage the liver and kidney by mechanisms similar to those observed for Western medicines [<xref ref-type="bibr" rid="B21">21</xref>]. The representative symptoms of herbal medicine adverse experiences found in this study included stomachache, diarrhoea, jaundice, liver malfunction, and oedema of the face, hands and feet.</p><p>This study was a survey of the general public and the follow-up of consumers&#x02019; adverse experiences was limited; we were unable to determine the root causes and effects of the adverse events. We conducted a survey of the general public regarding TM treatments, the adverse experiences and disease development to assess the types of adverse events. This type of survey method is not as accurate or detailed as research on adverse experiences. However, given the few reported cases of adverse experiences resulting from herbal medicines as well as the absence of reporting systems for other traditional therapy methods, this study was meaningful in that it allowed for the assessment of the current status of the adverse experiences of herbal medicines in the general population of Korea.</p></sec><sec sec-type="conclusions"><title>Conclusion</title><p>Medical usage and the occurrence of adverse events on TKM should be surveyed periodically and their statistical trends should be analysed. It is necessary to improve the national pharmacovigilance system for spontaneous adverse event reports by reviewing the disparity between the occurrence of herbal medicine reports and the national pharmacovigilance reports in survey data. Policies and regulations are required to enhance the reporting of adverse experiences not only for herbal medicines but also for TKM therapies.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interest.</p></sec><sec><title>Authors&#x02019; contributions</title><p>HS, SJJ, DSH, BKK and MSL designed the study, performed search, extracted data, carried out analyses and interpretations of the data, and drafted this report. All authors read and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6882/13/340/prepub">http://www.biomedcentral.com/1472-6882/13/340/prepub</ext-link></p></sec>

Profiling of phenolic compounds and their antioxidant and anticancer activities in pandan (<italic>Pandanus amaryllifolius</italic> Roxb.) extracts from different locations of Malaysia

<sec><title>Background</title><p>Phytochemicals and antioxidants from plant sources are of increasing interest to consumers because of their roles in the maintenance of human health. Most of the secondary metabolites of herbs are used in a number of pharmaceutical products.</p></sec><sec><title>Methods</title><p>Secondary metabolites composition and content of five flavonoids and three phenolic acids were evaluated and determined in <italic>Pandanus amaryllifolius</italic> extracts from three different locations of Malaysia by RP-HPLC; Total phenolic and total flavonoid content were determined using Folin-Ciocalteau and aluminum chloride colorimetric assay; The antioxidant activity of the extracts was determined by the ferric reducing antioxidant potential (FRAP) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay was employed to screen anticancer activity of extracts against MCF-7 cancer cell line.</p></sec><sec><title>Results</title><p>Highest value of total flavonoids (TF) and total phenolics (TP) was observed in pandan extract from Bachok locattion (1.87 mg/g DW and 6.72 mg/g DW) followed by Klang (1.32 mg/g DW; 5.07 mg/g DW) and Pontian (1.12 mg/g DW; 4.88 mg/g DW). Rutin just detected from Bachok location with value of 0.082 mg/g DW. High content of epicatechin (0.035 mg/g DW) and naringin (0.325 mg/g DW) were observed from Bachok location while, highest content of catechin (0.613 mg/g DW) and kaempferol (0.278 mg/g DW) was observed in pandan extract from Klang location. The extract of pandan from Bachok exhibited highest value of gallic acid (0.423 mg/g DW) and cinnamic acid (0.084 mg/g DW). Ferrulic acid just detected from pandan extract of Bachok location with concentration of 0.281mg/g DW. Between studied locations Bachok exhibited highest value of DPPH (64.27%) and FRAP (517.2 &#x003bc;m of Fe (II)/g) activity followed by Klang (52.16%; 448.6 &#x003bc;m of Fe (II)/g) and Pontian (50.10%; 314.8 &#x003bc;m of Fe (II)/g). The preliminary screening showed pandan extracts from 3 locations possessed anticancer promoting activity against MCF-7 cell line, with 78.3%, 70.5% and 67.4% inhibition rate, respectively. Maximum MCF-7cell line inhibition was observed in pandan extract from Bachok location.</p></sec><sec><title>Conclusions</title><p>The samples collected from the North (Bachok) exhibited the highest TP, TF antioxidant and anticancer activity while those from the Southern portion (Pontian) appeared to have the lowest content of TP, TF and antioxidant activity.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Ghasemzadeh</surname><given-names>Ali</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>alighasemzadeh@upm.edu.my</email></contrib><contrib contrib-type="author" corresp="yes" id="A2"><name><surname>Jaafar</surname><given-names>Hawa ZE</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Hawazej@upm.edu.my</email></contrib>

BMC Complementary and Alternative Medicine

<sec><title>Background</title><p>Most plants are major sources of natural products used in pharmaceuticals, agrochemicals, flavour and fragrance ingredients, food additives, and pesticides. Secondary compounds are unique to a species or group, and they are important for defense, protection and competition [<xref ref-type="bibr" rid="B1">1</xref>]. Most of these compounds are commonly used as flavourings, medicines, or recreational drugs. Secondary chemicals are important in plant use by humans. Most pharmaceuticals are based on plant component structures, and secondary metabolites are widely used especially in Asia [<xref ref-type="bibr" rid="B2">2</xref>]. Phenolic compounds are famous group of secondary metabolites with wide pharmacological activities. Flavonoids are an important group of secondary metabolites and are a source of bioactive compounds in plants [<xref ref-type="bibr" rid="B3">3</xref>]. They are also a kind of natural product with antioxidant properties capable of scavenging free superoxide radicals, having anti-aging properties as well as reducing the risk of cancer. Park et al. [<xref ref-type="bibr" rid="B4">4</xref>] showed that some flavonoid components in green tea are effective in inhibiting cancer or induce mechanisms that may kill cancer cells and inhibit tumor invasion. It was found that flavonoids reduced blood-lipids and glucose, and enhanced human immunity [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. The effect of flavonoids on human health is the result of their ability to induce human protective enzyme systems [<xref ref-type="bibr" rid="B7">7</xref>]. Several studies have suggested that flavonoids such as catechin and rutin are able to control cancer cell growth in the human body [<xref ref-type="bibr" rid="B8">8</xref>-<xref ref-type="bibr" rid="B10">10</xref>].</p><p>Pandan (<italic>Pandanus amaryllifolius</italic> Roxb.) is a tropical plant of the family Pandanaceae in the screw pine genus. Pandan leaf, often known as screw pine, because they resemble the pineapple with the spiral arrangement of long, narrow and strap-shaped green leaves [<xref ref-type="bibr" rid="B11">11</xref>]. Even though the Pandanaceae family comprises approximately 600 species, there are only <italic>Pandanus amaryllifolius</italic> Roxb. <italic>and Pandanus odoratissimus</italic> Linn. that have fragrant leaves and flowers, respectively [<xref ref-type="bibr" rid="B12">12</xref>]. The genus Pandanus from the family Pandanaceae comprises approximately 600 species that are widely distributed in tropical and subtropical regions. The sweet and delightful flavour of pandan leaves, which is well-known as a source of natural flavouring, is widely used in various parts of South-East Asian countries including India, Thailand, Indonesia and Malaysia. For example, pandan leaves are commonly used when preparing rice dishes as a means of enhancing flavour. In addition, pandan leaves are also used in making other food, such as desserts, sweets, coconut jam and ice cream. Due to the high chlorophyll content, pandan leaves are also a popular green colourant for food [<xref ref-type="bibr" rid="B13">13</xref>]. In Malaysia, herbs and spices are generally eaten raw and fresh as vegetable (salad), especially among the Malay community. Most of these herbs and spice are believed to be associated with high antioxidant activities and have many benefits on human body. A systematic search for anticancer plants began in the middle of the 21^th century through the applications of appropriate biological screening assays. The chemopreventive properties of plants extracts are often investigated via screening against a panel of human cancer cell lines. In recent years, there has been increasing interest in organically grown food because people believe that they have less pesticide residues and are healthier [<xref ref-type="bibr" rid="B14">14</xref>]. However, most studies on this subject have reported conflicting results and it is unclear whether or not organically grown food contains more health-promoting phytochemicals as opposed to conventionally grown food. Information about flavonoid compounds of Malaysian pandan and their antioxidant and anticancer activity are still scarce and some information and such data would be useful to provide information on foods containing high levels of beneficial components. The present investigation was undertaken to screen phytochemical potential and their antioxidant activities in <italic>P. amaryllifolius</italic> collected from three different location of Malaysia. In addition <italic>in vitro</italic> anticancer properties of the extracts against breast cancer cell lines were also investigated.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Plant material and maintenance</title><p>Fresh leaves of <italic>P. amaryllifolius</italic> were collected locally from three different province of Malaysia namely: Johor (pontian, south), Selangor (Klang, Central) and Kelantan (Bachok, North). The samples were identified by Malaysian Agriculture Research and Development Institute (MARDI). Voucher specimens of <italic>P. amaryllifolius</italic> Kelantan (MTP008/1), Selangor (MTP008/2) and Johor (MTP008/3) were collected. Malaysian Agriculture Research and Development Institute (MARDI) verified and kept samples. The leaves were shade dried and were powdered using mechanical grinder. This powered material is used for further analysis.</p></sec><sec><title>Preparation of flavonoids extract</title><p>Leaf samples (0.25&#x000a0;g) were extracted with 20&#x000a0;mL of methanol on a shaker for 2&#x000a0;h at room temperature. The extract solution was treated with 6&#x000a0;M HCl (5&#x000a0;ml) and refluxed at 90&#x000b0;C for 2&#x000a0;h. The hydrolysed sample was cooled to room temperature and filtered through a 0.45&#x000a0;&#x003bc;m membrane [<xref ref-type="bibr" rid="B15">15</xref>].</p></sec><sec><title>Determination of total flavonoids</title><p>The TF were measured following a previously reported spectrophotometric method [<xref ref-type="bibr" rid="B16">16</xref>]. Briefly, extracts of each plant material (1&#x000a0;mL) were diluted with 4&#x000a0;mL water in a 10&#x000a0;mL volumetric flask. Initially, 5% NaNO₂ solution (0.3&#x000a0;mL) was added to each volumetric flask; after 5&#x000a0;min, 10% AlCl₃ (w/v) was added; and at 6&#x000a0;min, 1.0&#x000a0;M NaOH (2&#x000a0;mL) was added. Absorbance of the reaction mixture was read at 430&#x000a0;nm.</p></sec><sec><title>Separation and analysis of flavonoids by HPLC</title><p>Reversed-phase HPLC was used to assay flavonoid composition. The Agilent HPLC system used consisted of a Model 1100 pump equipped with a multi-solvent delivery system, an L-7400 ultraviolet (UV) detector, and fitted with an Agilent C18 (5&#x000a0;&#x003bc;m, 4.6&#x02009;&#x000d7;&#x02009;250&#x000a0;mm) column. The mobile phase consisted of: (A) 2% acetic acid (CH₃COOH) and (B) 0.5% acetic acid-acetonitrile (CH₃CN), (50:50&#x000a0;v/v). The mobile phase was filtered under vacuum through a 0.45 um membrane filter before use. Gradient elution was performed as follows: 0&#x000a0;min, 95:5; 10&#x000a0;min, 90:10; 40&#x000a0;min, 60:40, 55&#x000a0;min, 45:55; 60&#x000a0;min, 20:80; and 65&#x000a0;min, 0:100. The flow rate was maintained at 1&#x000a0;mL/min and UV absorbance was measured at 260&#x02013;360&#x000a0;nm. The operating temperature was maintained at room temperature [<xref ref-type="bibr" rid="B17">17</xref>]. Identification of the flavonoids was achieved by comparison of retention times with standards, UV spectra and UV absorbance ratios after co-injection of samples and standards. The standards [(+)-Catechin, (-)-Epicatechin, Naringin, Rutin and Kaempferol] were purchased from Sigma&#x02013;Aldrich (St. Louis, MO, USA).</p></sec><sec><title>Preparation of phenolic acids extract</title><p>Phenolics extracts were prepared by first carefully pipetting phosphoric acid (H₃PO₄, 1.2&#x000a0;mL) into about 950&#x000a0;mL water in a 1&#x000a0;L volumetric flask, mixing and bringing to volume with water. Then leaves (0.25&#x000a0;g) were extracted with 20&#x000a0;mL, of this phosphoric acid solution. Five mL of 6&#x000a0;M HC1 was added to each extract to give a 25&#x000a0;mL solution of 1.2&#x000a0;M HC1 in 50% MeOH. Extracts were refluxed at 90&#x000b0;C for 2&#x000a0;h and solution was filtered through a 0.45&#x000a0;&#x003bc;m filter [<xref ref-type="bibr" rid="B18">18</xref>].</p></sec><sec><title>Determination of total phenolic content</title><p>The total phenolic content was determined following the method of Kim et al. [<xref ref-type="bibr" rid="B19">19</xref>]. Briefly, 1&#x000a0;mL of extract was added to deionized water (10&#x000a0;mL) and Folin&#x02013;Ciocalteu phenol reagents (1.0&#x000a0;mL). After 5&#x000a0;min, 20% sodium carbonate (2.0&#x000a0;mL) was added to the mixture. The solution was kept in total darkness, and the absorbance was measured at 750&#x000a0;nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan).</p></sec><sec><title>Separation and analysis of phenolic acids by HPLC</title><p>An Agilent HPLC system (Tokyo, Japan) consisting of a Model 1100 pump equipped with a multisolvent delivery system and a L-7400 ultraviolet (UV) detector was used. The column was an Agilent C18 (5&#x000a0;&#x003bc;m, 4.6&#x02009;&#x000d7;&#x02009;250&#x000a0;mm). The mobile phase was composed of phosphoric acid (aqueous) and (B) acetonitrile and gradient elution was performed as follows: 0&#x000a0;min, 85:15; 12&#x000a0;min, 75:25; 20&#x000a0;min, 75:25; 22&#x000a0;min, 85:15 and 30&#x000a0;min, 85:15. The mobile phase was filtered under vacuum through a 0.45&#x000a0;lm membrane filter before use. The flow rate and injection volume were 1&#x000a0;mL/min and 20&#x000a0;&#x003bc;L. UV absorbance was measured at 220&#x02013;360&#x000a0;nm. The operating temperature was maintained at room temperature [<xref ref-type="bibr" rid="B18">18</xref>]. Identification of the phenolic acids were achieved by comparison with retention times of standards, UV spectra and calculation of UV absorbance ratios after co-injection of samples and standards. Commercial standards [Gallic acid, trans-Cinnamic acid and trans-Ferulic acid] were purchased from Sigma&#x02013;Aldrich.</p></sec><sec><title>Determination of antioxidant activity</title><sec><title>Ferric reducing antioxidant potential (FRAP) assay</title><p>The stock solutions consisted of 300&#x000a0;mM acetate buffer, 10&#x000a0;mM TPTZ (2,4,6-tripyridyl-S-triazine) solution in 40&#x000a0;mM HCl, and 20&#x000a0;mM FeCl₃ solution. Acetate buffer (25&#x000a0;mL) and TPTZ (2.5&#x000a0;mL) were mixed, and 2.5&#x000a0;mL FeCl₃ added. Leaf extract (150&#x000a0;&#x003bc;L) was added to 2850&#x000a0;&#x003bc;L of the FRAP solution and kept for 30&#x000a0;min in the dark place. The absorbance of solution was measured at 593&#x000a0;nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan) [<xref ref-type="bibr" rid="B20">20</xref>].</p></sec></sec><sec><title>1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay</title><p>1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma&#x02013;Aldrich. Butylated hydroxytoluene (BHT) and &#x003b1;-tocopherol were purchased from Merck. The radical scavenging ability was determined using the method described in Mensor et al. [<xref ref-type="bibr" rid="B21">21</xref>]. Briefly, an alcohol solution of DPPH (1&#x000a0;mL, 3&#x000a0;mg/mL) was added to 2.5&#x000a0;mL samples containing different concentrations of extracts. The samples were first kept in the dark at room temperature and their absorbance was read at 518&#x000a0;nm after 30&#x000a0;min. The antiradical activity was determined using the following formula:</p><p><disp-formula><mml:math id="M1" name="1472-6882-13-341-i1" overflow="scroll"><mml:mtable columnalign="left"><mml:mtr><mml:mtd><mml:mi mathvariant="italic">Percent</mml:mi><mml:mspace width="0.25em"/><mml:mfenced open="(" close=")"><mml:mi>%</mml:mi></mml:mfenced><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">inhibition</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">of</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">DPPH</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">activity</mml:mi></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mspace width="1em"/><mml:mo>=</mml:mo><mml:mfenced open="[" close="]"><mml:mrow><mml:mfenced open="(" close=")"><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>A</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:mfenced><mml:mo stretchy="true">/</mml:mo><mml:msub><mml:mi>A</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mrow></mml:mfenced><mml:mo>&#x000d7;</mml:mo><mml:mn>100</mml:mn><mml:mi>%</mml:mi></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula></p><p>Where A₀ is the absorbance value of the blank sample or control reaction, and A₁ is the absorbance value of the test sample. The optic density of the samples and controls were measured in comparison to ethanol. BHT (butylhydroxytoluene) and Vit C, were used as positive controls.</p></sec><sec><title>Determination of anticancer activity</title><sec><title>Cell culture and treatment</title><p>Human breast carcinoma (MCF-7) and normal (MCF-10A) cells were cultured in 100&#x000a0;&#x003bc;L of RPMI 1640 media (Roswell Park Memorial Institute) containing 10% fetal bovine serum (FBS). MCF-7 cells were incubated overnight at 37&#x000b0;C in 5% CO₂ for cell attachment.</p></sec><sec><title>MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay</title><p>The assay was conducted as follows: Cancer cells were seeded in 96-well plates at a density of 1&#x02009;&#x000d7;&#x02009;104 cells/well in 100&#x000a0;&#x003bc;L RPMI. At 24&#x000a0;h after seeding, the medium was removed and the cells were incubated for 3&#x000a0;days with RPMI in the absence or presence of various concentrations of extracts. Extract concentrations used ranged from 20, 40, 80, 160, 320 and 640&#x000a0;&#x003bc;g/mL. After incubation, 20&#x000a0;&#x003bc;L of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent was added into each well. The plate was incubated again for 4&#x000a0;h in a CO₂ incubator at 37&#x000b0;C. The resulting MTT&#x02013;products were determined by measuring the absorbance at 570&#x000a0;nm using ELISA reader [<xref ref-type="bibr" rid="B22">22</xref>]. Each point represents the mean of triplicate experiments. The cell viability was determined using the formula:</p><p><disp-formula><mml:math id="M2" name="1472-6882-13-341-i2" overflow="scroll"><mml:mtable columnalign="left"><mml:mtr><mml:mtd><mml:mi mathvariant="italic">Viability</mml:mi><mml:mspace width="0.25em"/><mml:mfenced open="(" close=")"><mml:mi>%</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced open="(" close=")"><mml:mrow><mml:mi mathvariant="italic">optical</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">density</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">of</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">sample</mml:mi><mml:mo stretchy="true">/</mml:mo><mml:mi mathvariant="italic">optical</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">density</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">of</mml:mi><mml:mspace width="0.25em"/><mml:mi mathvariant="italic">control</mml:mi></mml:mrow></mml:mfenced></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mspace width="7em"/><mml:mo>&#x000d7;</mml:mo><mml:mn>100</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula></p></sec></sec><sec><title>Statistical analysis</title><p>All analytical values shown represent the means of three replicates. Data were analysed using analysis of variance by Statistical Analysis System (SAS 9.0). Mean separation test between treatments was performed using Duncan multiple range test and a P-value&#x02009;&#x02264;&#x02009;0.05 was regarded as significant.</p></sec></sec><sec><title>Results and discussion</title><sec><title>The concentrations of TF and some flavonoid compounds</title><p>The results obtained from the preliminary analysis of flavonoid compounds are shown in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>. There was a significant difference between the three locations for TF production in pandan. Highest value of TF content in pandan was observed in Bachok (1.87&#x02009;&#x000b1;&#x02009;0.246&#x000a0;mg/g DW) location followed by klang (1.32&#x02009;&#x000b1;&#x02009;0.211&#x000a0;mg/g DW) and pontian (1.12&#x02009;&#x000b1;&#x02009;0.177&#x000a0;mg/g DW). In this research, 5 flavonoid compounds were detected and identified from pandan extract. There was a significant difference (P &#x02264; 0.05) between the three locations for TF content.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>The concentrations of TF and some flavonoid compounds detected in pandan extracts from three different locations</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left">&#x000a0;</th><th align="left"><bold>Bachok</bold></th><th align="left"><bold>Klang</bold></th><th align="left"><bold>Pontian</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">TF<hr/></td><td align="left" valign="bottom">1.87&#x02009;&#x000b1;&#x02009;0.246^a<hr/></td><td align="left" valign="bottom">1.32&#x02009;&#x000b1;&#x02009;0.211^b<hr/></td><td align="left" valign="bottom">1.12&#x02009;&#x000b1;&#x02009;0.177^c<hr/></td></tr><tr><td align="left" valign="bottom">Rutin<hr/></td><td align="left" valign="bottom">0.082&#x02009;&#x000b1;&#x02009;0.028^a<hr/></td><td align="left" valign="bottom">ND<hr/></td><td align="left" valign="bottom">ND<hr/></td></tr><tr><td align="left" valign="bottom">Epicatechin<hr/></td><td align="left" valign="bottom">0.035&#x02009;&#x000b1;&#x02009;0.045^a<hr/></td><td align="left" valign="bottom">0.022&#x02009;&#x000b1;&#x02009;0.039^a<hr/></td><td align="left" valign="bottom">0.008&#x02009;&#x000b1;&#x02009;0.042^a<hr/></td></tr><tr><td align="left" valign="bottom">Catechin<hr/></td><td align="left" valign="bottom">0.527&#x02009;&#x000b1;&#x02009;0.024^b<hr/></td><td align="left" valign="bottom">0.613&#x02009;&#x000b1;&#x02009;0.015^a<hr/></td><td align="left" valign="bottom">0.153&#x02009;&#x000b1;&#x02009;0.046^c<hr/></td></tr><tr><td align="left" valign="bottom">Kaempferol<hr/></td><td align="left" valign="bottom">0.158&#x02009;&#x000b1;&#x02009;0.033^b<hr/></td><td align="left" valign="bottom">0.278&#x02009;&#x000b1;&#x02009;0.029^a<hr/></td><td align="left" valign="bottom">ND<hr/></td></tr><tr><td align="left">Naringin</td><td align="left">0.325&#x02009;&#x000b1;&#x02009;0.025^a</td><td align="left">0.223&#x02009;&#x000b1;&#x02009;0.026^b</td><td align="left">ND</td></tr></tbody></table><table-wrap-foot><p>All analyses are the mean of triplicate measurements&#x02009;&#x000b1;&#x02009;standard deviation. Results expressed in mg/g DW. a,b,c represents Duncan multiple range test letters. Means not sharing a common letter were significantly different at P&#x02009;&#x02264;&#x02009;0.05. ND: not detected.</p></table-wrap-foot></table-wrap><p>As shown in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>, rutin detected just from one locations (Bachok) and high value of this flavonoid was recorded 0.082&#x02009;&#x000b1;&#x02009;0.028&#x000a0;mg/g DW. Bachok location also, showed high epicatechin content (0.035&#x02009;&#x000b1;&#x02009;0.045&#x000a0;mg/g DW) compared to Klang location but, there were no significant differences between locations for epicatechin content in pandan extracts. High concentration of catechin was observed in Klang location (0.613&#x02009;&#x000b1;&#x02009;0.015&#x000a0;mg/g DW).</p><p>Kaempferol is a rare flavonoid component in plants, but it was detected in the pandan extracts just from two locations (Bachok and Klang) with remarkable concentration. Klang showed highest concentration of kaempferol (0.278&#x02009;&#x000b1;&#x02009;0.029&#x000a0;mg/g DW) followed by Bachok (0.158&#x02009;&#x000b1;&#x02009;0.033&#x000a0;mg/g DW). These kaempferol contents were higher than those recorded in pegaga (0.0205&#x000a0;mg/g DW), sengkuang (0.037&#x000a0;mg/g DW), carrot (0.140&#x000a0;mg/g DW), green chilli (0.039&#x000a0;mg/g DW) and white radish (0.0383&#x000a0;mg/g DW) [<xref ref-type="bibr" rid="B23">23</xref>]. In other study, Tolonen et al. [<xref ref-type="bibr" rid="B24">24</xref>] identified kaempferol in white cabbages with concentration of 0.9 mg/kg FW, and it was the only flavonoid found. Meanwhile, Kim and Lee [<xref ref-type="bibr" rid="B25">25</xref>] detected about 0.1&#x02013;0.8&#x000a0;mg/g FW of kaempferol contents in green cabbages. In current study kaempferol was not detected in pandan extract from pontian location.</p><p>Naringin was also detected from pandan extract with substantial concentration. Between studied locations Bachok showed highest value 0.325&#x02009;&#x000b1;&#x02009;0.025&#x000a0;mg/g DW followed by Klang 0.223&#x02009;&#x000b1;&#x02009;0.026&#x000a0;mg/g DW. Naringin was not detected from pandan in Pontian location. Zhang et al. [<xref ref-type="bibr" rid="B26">26</xref>] identified rutin in pandan (location of Felorida, USA) with concentration of 0.356&#x000a0;mg/100&#x000a0;g DW. Generally, between identified flavonoid compounds the important compound based on concentration from high to low were: catechin&#x02009;&#x0003e;&#x02009;naringin&#x02009;&#x0003e;&#x02009;kaempferol&#x02009;&#x0003e;&#x02009;rutin&#x02009;&#x0003e;&#x02009;epicatechin. Results imply that catechin is abundant flavonoid compounds in pandan. Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref> shows the HPLC chromatogram of pandan extracts from Bachok location.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>HPLC chromatograms of flavonoid compounds extracted from pandan, Bachok location.</bold> The identified peaks are: catechin (1), epicatechin (2), naringin (3), rutin (4) and kaempferol (5).</p></caption><graphic xlink:href="1472-6882-13-341-1"/></fig></sec><sec><title>The concentrations of TP and some phenolic acids</title><p>Usually, phenolics that possess antioxidant activity are known to be mainly flavonoids and phenolic acids. Phenolic acids are a major class of phenolic compounds, widely occurring in the plant kingdom especially in herbs and vegetables. As shown in Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>, pandan extract from Bachok present highest content of TP (6.72&#x02009;&#x000b1;&#x02009;0.355&#x000a0;mg/g DW) followed by Klang (5.07&#x02009;&#x000b1; 0.406&#x000a0;mg/g DW) and Pontian (4.88&#x02009;&#x000b1;&#x02009;0.477&#x000a0;mg/g DW). A significant difference (P&#x02009;&#x02264;&#x02009;0.05) was observed between Bachok and Klang in TP content but, differences between Klang and Pontian was not significant. However, compared to some of potent herbs like as <italic>Melisa officinalis</italic> (13.2&#x000a0;mg/g DW), <italic>Taraxacum officinale</italic> (12.6&#x000a0;mg/g DW), <italic>Acorus calamus</italic> (12.45&#x000a0;mg/g DW), <italic>Echinacea purpurea</italic> (15.1&#x000a0;mg/g DW), <italic>Syzygium aromaticum</italic> (8.96&#x000a0;mg/g DW) and <italic>Salvia officinalis</italic> (8.25&#x000a0;mg/g DW) pandan recorded lowest contents of TP [<xref ref-type="bibr" rid="B27">27</xref>,<xref ref-type="bibr" rid="B28">28</xref>].</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>The concentrations of TP and some phenolic acids detected in pandan extracts from three different locations</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left">&#x000a0;</th><th align="left"><bold>Bachok</bold></th><th align="left"><bold>Klang</bold></th><th align="left"><bold>Pontian</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">TP<hr/></td><td align="left" valign="bottom">6.72&#x02009;&#x000b1;&#x02009;0.355^a<hr/></td><td align="left" valign="bottom">5.07&#x02009;&#x000b1;&#x02009;0.406^b<hr/></td><td align="left" valign="bottom">4.88&#x02009;&#x000b1;&#x02009;0.477^b<hr/></td></tr><tr><td align="left" valign="bottom">Gallic acid<hr/></td><td align="left" valign="bottom">0.423&#x02009;&#x000b1;&#x02009;0.052^a<hr/></td><td align="left" valign="bottom">0.325&#x02009;&#x000b1;&#x02009;0.041^a<hr/></td><td align="left" valign="bottom">0.214&#x02009;&#x000b1;&#x02009;0.019^b<hr/></td></tr><tr><td align="left" valign="bottom">Cinnamic acid<hr/></td><td align="left" valign="bottom">0.084&#x02009;&#x000b1;&#x02009;0.033^a<hr/></td><td align="left" valign="bottom">0.033&#x02009;&#x000b1;&#x02009;0.018^b<hr/></td><td align="left" valign="bottom">ND<hr/></td></tr><tr><td align="left">Ferulic acid</td><td align="left">0.281&#x02009;&#x000b1;&#x02009;0.037^a</td><td align="left">ND</td><td align="left">ND</td></tr></tbody></table><table-wrap-foot><p>All analyses are the mean of triplicate measurements&#x02009;&#x000b1;&#x02009;standard deviation. Results expressed in mg/g DW. Means not sharing a common letter were significantly different at P&#x02009;&#x02264;&#x02009;0.05.</p></table-wrap-foot></table-wrap><p>It is evident that the total phenolic content measured by the Folin&#x02013;Ciocalteu method does not give a full picture of the quality or quantity of the phenolic compounds in the plant extracts [<xref ref-type="bibr" rid="B29">29</xref>]. In current research three phenolic acids including gallic acid, cinnamic acid and ferulic acid were identified in pandan extracts from three locations. The extract of pandan from Bachok exhibited highest value (0.423&#x02009;&#x000b1;&#x02009;0.052&#x000a0;mg/g DW) of gallic acid compared to Klang (0.325&#x02009;&#x000b1;&#x02009;0.041&#x000a0;mg/g DW) and Pontian (0.214&#x02009;&#x000b1;&#x02009;0.019&#x000a0;mg/g DW) locations. In addition no significant difference was observed between Klang and Pontian locations for gallic acid production in pandan. As Table&#x000a0;<xref ref-type="table" rid="T2">2</xref> shows, among the studied phenolic compounds cinamic acid was detected from Bachok and Klang locations. Bachok location represent high value (0.084&#x02009;&#x000b1;&#x02009;0.033&#x000a0;mg/g DW) of cinnamic acid followed by Klang (0.033&#x02009;&#x000b1;&#x02009;0.018&#x000a0;mg/g DW). Cinnamic acid was not detected in pandan extract from Pontian location. Ferulic acid was detected just in pandan extract of Bachok location with value of 0.281&#x02009;&#x000b1;&#x02009;0.037&#x000a0;mg/g DW. Ferulic acid was shown to inhibit the photo peroxidation of linoleic acid which is potent fatty acid for decrease cancer risk, improve immune function, diabetes and heart disease prevention [<xref ref-type="bibr" rid="B23">23</xref>]. The most interesting finding was that ferulic acid just detected in pandan extracts from Bachok location. However, this result has not previously been described. Results imply that gallic acid is abundant pheolic acid between identified phenolics in pandan extracts.</p></sec><sec><title>Antioxidant activity</title><sec><title>Ferric reducing antioxidant potential (FRAP) assay</title><p>The FRAP assay depends upon the reduction of ferric tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. The FRAP assay has been used widely to estimate the antioxidant component/power in dietary polyphenols [<xref ref-type="bibr" rid="B30">30</xref>]. As Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref> shows, the reducing power for the pandan extracts from three different locations was in the range of 511.2 (Bachok) to 314.8&#x000a0;&#x003bc;m of Fe (II)/g (Pontian). The FRAP values for the pandan extract in three locations were significantly lower than those of BHT (672.4&#x000a0;&#x003bc;mol Fe (II)/g) and Vit C (1186.55&#x000a0;&#x003bc;mol Fe (II)/g). In this study, we used the FRAP assay because it is quick and simple to measure the antioxidant capacity of purpose compounds and not only plants, wines, and animal tissues [<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. In general, antioxidant activity of flavonoids belong to the substitution pattern and structure of hydroxyl groups. In flavonoids chemical structure 30,40-orthodihydroxy in ring B and 4-carbonyl group in ring C are the fundamental requirement for effective radical scavenging. The presence of 3- and 5-OH groups, giving a catechol-like structure in ring C, is also essential for the antioxidant activity of flavonoid compounds [<xref ref-type="bibr" rid="B28">28</xref>]. Furthermore, the presence of the C2&#x02013;C3 bond configured with a 4-keto arrangement is recognized to be responsible for electron delocalization from ring B and following that enhance the free radical scavenging activity. In the absence of the o-dihydroxy structure in ring B, a catechol structure in ring A can compensate for flavonoid antioxidant activity [<xref ref-type="bibr" rid="B33">33</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>FRAP activity of pandan extracts collected from three different locations compared to the positive controls butylated hydroxytoluene (BHT) and vitamin C.</p></caption><graphic xlink:href="1472-6882-13-341-2"/></fig></sec></sec><sec><title>1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay</title><p>The effect of antioxidants on DPPH scavenging is due to their hydrogen donating ability. Between studied locations Bachok exhibited highest value (64.27%) of DPPH activity followed by Klang (52.16%) and Pontian (50.10%), (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>). In addition, significant difference was observed between three location for DPPH activity. The results of the current study showed that DPPH radical scavenging abilities of the extracts of pandan from three locations were less than those of butylated hydroxytoluene (BHT) (83.7%) and Vit C (92.3%) at 35&#x000a0;mg/mL. The IC₅₀ (fifty percent free radical scavenging) value of pandan extract were 9.25, 11.6 and 12.5&#x000a0;mg/mL for Bachok, Klang and Pontian locations respectively. Sasikumar et al. [<xref ref-type="bibr" rid="B34">34</xref>] reported that the DPPH antioxidant activity in pandan extract were comparable with those obtained by Higher antioxidant potential was observed in both DPPH scavenging assay (EC&#x02009;=&#x02009;48.350&#x02009;&#x000b1;&#x02009;0.002&#x000a0;&#x003bc;g/mL) and reducing capacity (OD at 1000&#x000a0;&#x003bc;g/mL&#x02009;=&#x02009;0.787) by the methanolic root extract than by the aqueous extract. Marinova et al. [<xref ref-type="bibr" rid="B35">35</xref>] but higher than that of Odukoya et al. [<xref ref-type="bibr" rid="B36">36</xref>]. When a comparison betweeen Figures&#x000a0;<xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref> was made to appear the trend for ferric ions reducing activities of the pandan did not vary markedly from their DPPH free radical scavenging activities. Antioxidant compounds such as polyphenols may be more efficient reducing agents for ferric iron but some may not scavenge DPPH free radicals as efficiently due to steric hindrance [<xref ref-type="bibr" rid="B36">36</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>DPPH radical scavenging activity of pandan extracts collected from three different locations compared to the positive controls butylated hydroxytoluene (BHT) and vitamin C.</p></caption><graphic xlink:href="1472-6882-13-341-3"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Dose-dependent anticancer of pandan extracts from 3 different locations (Bachok, Klang and Pontian) towards MCF-7 cell line as determined by the MTT assay.</p></caption><graphic xlink:href="1472-6882-13-341-4"/></fig><p>Pulido et al. [<xref ref-type="bibr" rid="B37">37</xref>] reported that, in general, the ferric ion reducing ability of antioxidants correlates with the results from other methods used to estimate antioxidant capacity. Reducing DPPH radicals were also able to reduce ferric ions. Arnous et al. [<xref ref-type="bibr" rid="B38">38</xref>] reported a strong correlation between DPPH free radical scavenging ability and ferric ion reducing ability in wines.</p></sec><sec><title>Anticancer activity</title><p>The preliminary screening showed pandan extracts from 3 locations possessed anticancer promoting activity against MCF-7, with 78.3% 70.5% and 67.4 inhibition rate, respectively. Maximum MCF-7cell line inhibition was observed in pandan extract with values of 66.3% from Bachok location (Figure&#x000a0;<xref ref-type="fig" rid="F4">4</xref>). MCF-7 cell lines treated with tamoxifen (positive control) showed 92.5% inhibition at the same concentration. According to obtained results, at a concentration of 160&#x000a0;&#x003bc;g/ml, however, pandan extracts from Bachok locations exhibited IC₅₀ towards MCF-7 cells. The IC₅₀ values of pandan extract from Bachok, Klang and Pontian locations against MCF-7 cells were 210.4, 285.6.6 and 334.2&#x000a0;&#x003bc;g/mL, respectively. Our finding is consistent with Zan et al. [<xref ref-type="bibr" rid="B39">39</xref>] who is reported IC₅₀ value for <italic>P. amaryllifolius</italic> extract against breast cancer cell line obtain in &#x0003e;100&#x000a0;&#x003bc;g/mL. Flavonoids are among the best candidates for mediating the protective effect of diets rich in fruits and vegetables with respect to colorectal cancer [<xref ref-type="bibr" rid="B40">40</xref>]. Hence, flavonoid compounds could probably be responsible for the anticancer activity of curry leaf. Meanwhile, with the increase of extracts concentration, however, normal cell viability decreased in all extracts. In terms of toxicity to the normal cells (MCF-10A), <italic>P. amaryllifolius</italic> extracts from different locations were considered as not toxic as the IC₅₀ values were greater than 640&#x000a0;&#x003bc;g/ml (Figure&#x000a0;<xref ref-type="fig" rid="F5">5</xref>). In pandan extract from Bachok location at concentration of 210.4&#x000a0;&#x003bc;g/ml (IC₅₀) the normal cell viability was recorded about 78%. <italic>P. amaryllifolius</italic> extract was found to display selective antiproliferative activity against non hormone dependent breast cancer cells [<xref ref-type="bibr" rid="B39">39</xref>]. In other study, ethanol extract of <italic>P. amaryllifolius</italic> induced apoptosis on hormone independent breast cancer cell line MDA-MB-231 [<xref ref-type="bibr" rid="B41">41</xref>]. In the current study the highest values of flavonoid compounds were detected in pandan extract from Bachok location. Meanwhile, the highest anticancer activity against MCF-7 cell lines has been observed with extracts of pandan from Bachok location. This suggests that high anticancer activity in pandan extract may be attributed to the high concentrations of potent anticancer components such as rutin, epicatechin, kaempferol and gallic acid. However, more research needs to be undertaken before the association between these flavonoids and anticancer activity in curry leaf is more clearly understood.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>Effect of pandan extracts from three different locations (Bachok, Klang and Pontian) on normal cell (MCF-10A) viability.</p></caption><graphic xlink:href="1472-6882-13-341-5"/></fig></sec></sec><sec sec-type="conclusions"><title>Conclusion</title><p>In this study between three studied locations, the samples collected from the North (Bachok) which had a high content of phenolic acids (specially gallic acid) and flavonoids (specially catechin and kaempferol), had a very high antioxidant scavenging value in both FRAP and DPPH assay. Conversely, the samples collected from the Southern portion (Pontian) appeared to have the lowest content of TP, TF and antioxidant activity. Recently several plant derived natural compounds have been screened for their anticancer activity in order to identify putative compounds with novel structures or mechanism of action. In current study pandan extracts showed good potential of bioactive compounds such as catechin, gallic acid, kaempferol and naringin. It can be concluded that these bioactive compounds present in pandan extracts work synergistically in inhibiting proliferation of breast cancer cells and suggests that they may have potential for use as a natural additive in human diets. The wide ranges of the secondary metabolites content and antioxidant activities of pandan extracts could be due to many factors including locations, altitude, temperature, age of plant, climate and variation of plant variety. The ranges of phenolic acids and flavonoid content and antioxidant activity will be useful for standardization of pandan extracts for further pharmaceutical productions. These results also show the possibility of increasing the content of natural antioxidants by optimizing the growing conditions of pandan. More information on other bioactive component of pandan would help us to establish a greater degree of accuracy on this matter.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors&#x02019; contributions</title><p>Experimental work was done by AG under the supervision of HJ (post doctoral project). The first draft of the paper was written by AG and reviewed by HJ. Both authors read and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6882/13/341/prepub">http://www.biomedcentral.com/1472-6882/13/341/prepub</ext-link></p></sec>

Cystine nephrolithiasis

<p>Our understanding of the molecular basis of cystinuria has deepened as the result of the causative genes, <italic>SLC3A1</italic> and <italic>SLC7A9</italic>, being identified. The proteins coded for by these genes form a heterodimer responsible for reabsorption of filtered cystine in the proximal tubule. Failure of this transport system to be targeted to the apical membrane, as in the case of <italic>SLC3A1</italic> mutations, or failure of the transport system to function, as in the case of <italic>SLC7A9</italic> mutations, leads to abnormal urinary excretion of the relatively insoluble amino acid cystine. Stones and plugs of tubules result, with chronic kidney disease a frequent complication. Here we review the genetics, pathophysiology, pathology, clinical manifestations and clinical management. Increased fluid intake, restriction of sodium and animal protein ingestion, and urinary alkalinization are the standard therapies. Cystine binding thiol drugs tiopronin and D-penicillamine are reserved for patients for whom the conservative therapies are insufficient. New studies of cystine crystal inhibition are highlighted.</p>

<contrib contrib-type="author"><name><surname>Fattah</surname><given-names>Hasan</given-names></name><xref ref-type="aff" rid="aff1">¹</xref><xref ref-type="aff" rid="aff2">²</xref></contrib><contrib contrib-type="author"><name><surname>Hambaroush</surname><given-names>Yasmin</given-names></name><xref ref-type="aff" rid="aff1">¹</xref><xref ref-type="aff" rid="aff2">²</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Goldfarb</surname><given-names>David S.</given-names></name><xref ref-type="aff" rid="aff1">¹</xref><xref ref-type="aff" rid="aff2">²</xref></contrib><aff><target id="aff1" target-type="aff">¹</target>Nephrology Section, New York Harbor VA Healthcare System, New York, NY, <country>USA</country>; <target id="aff2" target-type="aff">²</target>Nephrology Division, NYU Langone Medical Center, New York, NY, <country>USA</country></aff>

Translational Andrology and Urology

<sec sec-type="intro"><title>Introduction</title><p>Cystinuria is a hereditary, heterogeneous disorder affecting the proximal tubule&#x02019;s cystine and dibasic amino acid transporter. It is one of the first inborn errors of metabolism recognized by Sir Archibald Garrod in 1908 (<xref rid="r1" ref-type="bibr">1</xref>). In contrast to the normal, near-complete reabsorption of cystine and the dibasic amino acids ornithine, arginine and lysine, (mnemonic: COAL), these amino acids are not reabsorbed and reach high concentrations in the urine of affected individuals. Only the abnormal excretion of cystine causes a clinical problem as it has low solubility in acidic and neutral urine and may lead to the formation of cystine crystals and stones in the urinary tract. Urinary stones may result clinically in hematuria, urinary obstruction and renal failure. Loss of ornithine, arginine and lysine does not cause any known phenotypic traits. Although cystinuria is one of the classic inborn errors of metabolism and has been described in several species, the disease is phenotypically heterogeneous, and its molecular basis is only imperfectly understood.</p></sec><sec><title>Physiology and pathophysiology of cystine transport</title><p>The luminal cystine transporter functions as an exchanger, transporting cystine and cationic amino acids (0, +) along their electrochemical gradient into the proximal tubular epithelial cells in exchange for intracellular neutral amino acids, in a Na⁺-independent manner (<xref rid="r2" ref-type="bibr">2</xref>). The cystine transporter, one of the heteromeric amino acid transporters (HATs), is a heterodimer of the heavy subunit known as rBAT, a type II membrane N-glycoprotein encoded by <italic>SLC3A1</italic>, and the eukaryotic light subunit b^0,+AT, a highly hydrophobic molecule encoded by <italic>SLC7A9</italic> (<xref rid="r3" ref-type="bibr">3</xref>). The two proteins are joined by a disulfide link. The co-expression of both subunits b^0,+AT and rBAT is essential for the functional expression of the whole system, named b^0,+, in the apical plasma membrane. The heavy subunit is needed to localize the transporter to the apical membrane, while the light subunit comprises the catalytic, transporting component (<xref rid="r4" ref-type="bibr">4</xref>).</p></sec><sec><title>Genetics of cystinuria</title><p>Two genes have been implicated in cystinuria: <italic>SLC3A1</italic> which contains 10 exons and encodes rBAT (<xref rid="r5" ref-type="bibr">5</xref>) and <italic>SLC7A9</italic> which contains 13 exons and encodes b^0,+AT (<xref rid="r6" ref-type="bibr">6</xref>). In large screening studies, more than 130 pathogenic variants in <italic>SLC3A1</italic> and nearly 100 mutations in <italic>SLC7A9</italic> have been reported. The observed variants cover the whole spectrum of mutations, ranging from nonsense, missense, splicing, and frame shift mutations to whole- and multi-exon imbalances. Mutations in <italic>SLC3A1</italic> are associated with an autosomal recessive mode of inheritance, whereas <italic>SLC7A9</italic> variants result in an incomplete dominant inheritance, meaning that a heterozygous patient is occasionally affected by stone disease (<xref rid="r7" ref-type="bibr">7</xref>). However, most patients have two mutated alleles. It is also possible that the stone-forming tendency of patients with cystinuria has a genetic component beyond mutation in both of the known genes and may include other yet-unidentified modifier genes.</p></sec><sec><title>Classification</title><p>In the past, cystinuria was divided into three subtypes according to the amount of cystine excreted by the obligate heterozygous parents of the affected children (phenotypic classification). The parents of type I patients excreted normal amounts of cystine, while the heterozygous parents of types II and III (non-type I) had abnormal excretion of cystine, indicating that the disease is transmitted in a dominant mode with incomplete penetrance (<xref rid="r8" ref-type="bibr">8</xref>).</p><p>In the International Cystinuria Consortium (ICC), which included patients from Europe and Israel, it was recognized that there was a poor genotype-phenotype correlation. Although type I was largely caused by mutations in <italic>SLC3A1</italic>, a small proportion of these patients had two mutated <italic>SLC7A9</italic> alleles (<xref rid="r9" ref-type="bibr">9</xref>). In contrast almost all cases of non-type I cystinuria were caused by mutations in <italic>SLC7A9</italic>, with the exception of a few cases with a complex, in-frame duplication of <italic>SLC3A1</italic>. Cases previously termed types II and III based on clinical testing were genetically indistinguishable. The result was a new classification, currently in use, based on genetic data. Type A is due to mutations in both alleles <italic>SLC3A1</italic> (genotype AA) and type B disease is due to mutations in both alleles <italic>SLC7A9</italic> (genotype BB) (<xref rid="r10" ref-type="bibr">10</xref>). Rare cases of digenic inheritance (type AB) have also been described, but individuals with this pattern do not produce stones. Other digenic stone formers were shown to have ABB or AAB patterns. Considering that in many studies the detection rates for mutations in <italic>SLC3A1</italic> or <italic>SLC7A9</italic> do not reach 100%, and due to the complex nature of renal amino acid transport, the role of further genes and modifying factors in the etiology of cystinuria has been postulated.</p><p>In clinical practice, genotyping of affected patients is not currently necessary as it scarcely influences the prognosis and therapy of the disease. While about 94% of people homozygous for <italic>SLC3A1</italic> or <italic>SLC7A9</italic> mutations will develop kidney stones in their lives, the age of onset of kidney stone formation is difficult to predict and shows a broad intrafamilial variability (<xref rid="r9" ref-type="bibr">9</xref>). The urinary excretion pattern in heterozygote <italic>SLC7A9</italic> mutation carriers is variable and does not allow prediction of the clinical course; most will not have stones. The majority of <italic>SLC3A1</italic> heterozygotes do not exhibit an abnormal amino aciduria phenotype (<xref rid="r11" ref-type="bibr">11</xref>).</p></sec><sec><title>Prevalence, clincal features and presentation</title><p>The prevalence of cystinuria has been estimated to vary from 1:2,500 in a Libyan-Jewish population to 1:100,000 in Swedes (<xref rid="r12" ref-type="bibr">12</xref>). Screening tests for cystinuria in Japan identified 6 patients among 110,000 students. None of these showed stone formation after 7 years of follow-up (<xref rid="r13" ref-type="bibr">13</xref>). The relative rarity of this condition accounts for the paucity of data involving large series.</p><p>The median age of onset of stones was 12 years in a cohort of over 200 patients (<xref rid="r14" ref-type="bibr">14</xref>). For unclear reasons, males appear to be more severely affected than females. Substantial variability existed in the age of onset of stones, as 40% of patients had their first stone between 11 and 20 years, while 14% of females, and 28% of males formed calculi before the age of 3 (<xref rid="r15" ref-type="bibr">15</xref>). In a database of 224 patients from 150 families there were no clinical differences in urinary amino acid excretion patterns between cystinuria type A and B (<xref rid="r10" ref-type="bibr">10</xref>). Stone formation did not correlate with amino acid urinary excretion, suggesting the importance of other modifiers of stone formation such as fluid intake, diet and modifier genes.</p><p>Like non-cystine stone formers, cystinuria presents with symptoms related to stone formation, such as renal colic and hematuria. However, people with cystinuria were almost 5 times as likely to have undergone a nephrectomy, sometimes presenting with a non-functioning kidney containing a large staghorn calculus (<xref rid="r16" ref-type="bibr">16</xref>). Cystinuria is associated with reduced glomerular filtration rates (GFR). In the ICC data, GFR was reduced in 17% of those patients. Factors which might contribute to reduce GFR include the need for multiple urological procedures such as extracorporeal shock-wave lithotripsy, intranephronal crystal formation and repeated episodes of urinary tract obstruction.</p></sec><sec><title>Diagnosis</title><p>Cystinuria is diagnosed among patients with nephrolithiasis and one or more of the following findings:</p><list list-type="bullet" id="L1"><list-item><p>Stone analysis showing cystine (<xref ref-type="fig" rid="f1"><italic>Figure 1</italic></xref> depicts a large, typical, yellow cystine stone passed spontaneously);<fig id="f1" fig-type="figure" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Spontaneously passed cystine stone. Scale is in inches.</p></caption><graphic xlink:href="tau-03-03-228-f1"/></fig> </p></list-item><list-item><p>Positive family history of cystinuria;</p></list-item><list-item><p>Positive laboratory screening of urine with sodium nitroprusside;</p></list-item><list-item><p>Identification of pathognomonic hexagonal cystine crystals on urinalysis (seen on initial urinalysis in about 25% of patients);</p></list-item><list-item><p>Cystinuria should also be suspected in patients with large branched (staghorn) calculi, filling the collecting system and requiring surgical management.</p></list-item></list></sec><sec><title>Laboratory studies</title><p>Quantitative testing of urinary cystine excretion should follow the diagnosis of cystinuria. Sodium nitroprusside detects more than 75 mg/L, while normal cystine excretion is 30 mg/day (0.13 mmol/day). Patients with cystinuria generally excrete more than 400 mg/day (1.7 mmol/day). However, the accuracy of cystine testing by most labs is imperfect. Among the problems is that these tests depend on cystine solubility, and can under certain circumstances lead to misleading results. Cystine is relatively insoluble at urine pH below 6.5, remaining in non-measurable, crystalline form. The result is artifactually low values if urine samples are not alkalinized after voiding before measurement of cystine (<xref rid="r17" ref-type="bibr">17</xref>). Another problem is that cystine assays do not distinguish cystine from soluble thiol drug-cysteine complexes. In patients taking tiopronin or d-penicillamine, thiol-cysteine bonds can be broken by sample preparation, releasing cysteine which recombines with itself to form the insoluble dimer cystine. The result is inaccurate measurement in patients taking thiol drugs and inability to judge drug efficacy.</p><p>These problems led to the development of a solid-phase assay which is reliable in the presence of thiol drugs (<xref rid="r18" ref-type="bibr">18</xref>). Cystine crystals are added to a patient&#x02019;s urine and after incubation for 48 h, the remaining crystals are collected. In supersaturated urine, cystine precipitates onto the added crystals, so the solid phase grows and is greater than that which was added; the urine is said to have a &#x02018;negative cystine capacity&#x02019;. If urine is undersaturated, it can dissolve the added cystine crystals; such urine has a &#x02018;positive cystine capacity&#x02019; (<xref rid="r19" ref-type="bibr">19</xref>). Whether the assay predicts stone-forming activity has not yet been established but is a topic of study by the Rare Kidney Stone Consortium (<xref rid="r20" ref-type="bibr">20</xref>).</p></sec><sec><title>Pathology</title><p>Biopsies of renal papillae have been performed on well characterized cystine stone formers (<xref rid="r21" ref-type="bibr">21</xref>). Histopathological findings varied from normal in some participants, to plugging, dilatation and injury of ducts of Bellini. Inner medullary collecting ducts were also affected. The large plugs of crystals in ducts of Bellini were always composed of cystine, whereas in the inner medulla and thin loop of Henle, deposits were invariably composed of calcium phosphate in the form of hydroxyapatite. Whether treatment with potassium citrate for urinary alkalinization was responsible for these hydroxyapatite precipitates is not clear. An alternative explanation is that these crystals are the result of the relatively alkaline urine pH often observed in patients with cystinuria.</p><p>Renal stone disease is often considered to be a mechanical problem with nephronal obstruction by cystine crystals the primary driver of renal injury. This microscopic obstruction could cause upstream cell injury and interstitial reaction, glomerular loss, and cortical fibrosis. However, crystal nephropathies have recently been recognized to cause significant intra-renal inflammation that can cause acute kidney injury (<xref rid="r22" ref-type="bibr">22</xref>). For example, inflammation and tissue remodeling have been seen as the result of calcium oxalate crystals. Activation of the NLRP3 inflammasome leads to secretion of the pro-inflammatory cytokine IL-1&#x003b2; (<xref rid="r23" ref-type="bibr">23</xref>). Whether cystine is capable of stimulating these inflammatory pathways has not yet been determined.</p></sec><sec><title>Management</title><p>Treatment is focused on reducing the absolute amount of cystine in the urine and increasing the solubility of cystine. Initial conservative measures should be attempted in all patients focusing on decreasing cystine concentration and supersaturation by increasing fluid intake and pH; and decreasing the cystine excretion and production by limiting sodium and protein intake. If these measures fail, cystine-binding thiol drugs are considered (<xref rid="r24" ref-type="bibr">24</xref>).</p></sec><sec><title>Increasing oral fluid intake</title><p>Increasing oral fluid intake to increase urine volume is an essential measure for all types of nephrolithiasis, including cystinuria. The goal is to reduce urine cystine supersaturation to less than 1.0 at a urine pH above 7.0. Patients with cystinuria often need to drink 3-4 L per day to achieve a urine cystine concentration of less than 243 mg/L (1 mmol/L) and thus decrease the concentration of urinary cystine below the level of saturation (<xref rid="r19" ref-type="bibr">19</xref>). Fluid intake throughout the day, at night, and augmented with higher ambient temperatures or exercise, should all be stressed. Given the barriers to achieve such a high fluid intake, especially the subsequent need to void frequently, prescription of an antidiuretic hormone (ADH) antagonist was proposed (<xref rid="r25" ref-type="bibr">25</xref>). In these two reported cases, tolvaptan 15 mg once daily was used for 5 days. By provoking thirst, urine dilution was achieved with no significant side effects. However the drug is extremely costly and currently has a black box warning associated with it due to hepatotoxicity seen in treatment of polycystic kidney disease (<xref rid="r26" ref-type="bibr">26</xref>). We do not consider prescription of vasopressin antagonists a reasonable method of increasing urine volume.</p></sec><sec><title>Urinary alkalinization</title><p>Cystine solubility increases with increasing pH, best achieved with oral administration of potassium citrate (<xref rid="r27" ref-type="bibr">27</xref>). A starting dose of 20 meq twice daily might or might not be sufficient with increases based on periodic pH testing. Hyperkalemia is unusual unless GFR is reduced. Taking the supplements after meals reduces the risk of dyspepsia. Potassium bicarbonate might have fewer adverse gastric effects for some patients. Sodium citrate or sodium bicarbonate may also be appropriate but are second line choices as increased urinary sodium excretion leads to increased cystine excretion.</p><p>Although acetazolamide has been recommended to increase urinary pH, it was not shown to be superior to citrate and the associated metabolic acidosis with presumed risk to bone mineral density make it less than an ideal method to raise urine pH (<xref rid="r28" ref-type="bibr">28</xref>).</p></sec><sec><title>Dietary manipulation</title><p>Although the tubular reabsorption of cystine in the proximal tubule occurs via a sodium-independent transporter, cystine excretion can be reduced by decreasing dietary sodium (<xref rid="r19" ref-type="bibr">19</xref>,<xref rid="r29" ref-type="bibr">29</xref>). The physiology of this effect is not known. We therefore recommend restricting salt intake to about 2 g per day. Reduction of animal protein intake to 1 g/kg body weight may be useful as well for two reasons. First, reduction of intake of cystine and its precursor methionine is associated with reduction in cystine excretion (<xref rid="r30" ref-type="bibr">30</xref>). In addition, as animal protein is an important source of protons in the Western diet, reducing protein intake leads to an increase in urine pH and should lead to a reduction in the amount of citrate required to achieve urinary alkalinization to pH greater than 7 (<xref rid="r31" ref-type="bibr">31</xref>). Despite the evidence that protein and salt restriction lead to less cystine excretion, there are no long-term studies demonstrating that these prescriptions reduce formation of cystine stones.</p></sec><sec><title>Cystine-binding thiol drugs</title><p>In patients who are refractory to increased fluid intake, urinary alkalinization and dietary restriction of protein and salt, CBTDs are recommended. The two in use currently are D-penicillamine and alpha-mercaptopropionylglycine, or tiopronin. These drugs work by reducing the disulfide bond of cystine, producing mixed disulfides with cysteine that are more soluble than cystine. The solubility of the cysteine-penicillamine complex, for example, is 50-fold higher than of cystine (<xref rid="r32" ref-type="bibr">32</xref>).</p><p>The incidence of adverse effects such as allergy is slightly lower with tiopronin, making it our usual first-line agent. D-penicillamine is reserved for people with adverse events related to tiopronin and for those who have taken it for a long time without problems. Adverse events can be limited if one starts with low doses. Monitoring of liver enzymes, complete blood count, and urinary protein excretion should be performed at 4-6-month intervals. The angiotensin converting enzyme inhibitor captopril also contains a thiol group and can increase solubility of cystine <italic>in vitro</italic>. However urinary excretion of captopril may not be adequate to cause meaningful changes in cystine solubility (<xref rid="r19" ref-type="bibr">19</xref>). A recent <italic>in vitro</italic> study found that increasing urine pH was associated with increased thiol drug efficacy in a clinically relevant time frame (<xref rid="r33" ref-type="bibr">33</xref>). Using CBTDs without alkali would not be recommended based on this non-clinical study.</p></sec><sec><title>Prospects for new drugs</title><p>Atomic force microscopy (AFM) is a technique that allows study of crystal growth. Computer modeling led to selection of molecules that are candidates for inhibition of cystine crystallization, with AFM used to confirm (<xref rid="r34" ref-type="bibr">34</xref>). Cystine crystal growth was significantly inhibited at low concentrations by L-cystine dimethylester (CDME), a structural analog of cystine that probably provides steric inhibition of cystine crystal growth. We extended this work by demonstrating that CDME can be given to knock-out mice with cystinuria and effectively limits cystine stone formation (<xref rid="r35" ref-type="bibr">35</xref>). Toxicity was not apparent. Despite the encouraging findings, toxicity remains of concern as CDME has been used to produce an animal model of cystinosis, a disorder in which intracellular lysosomal cystine accumulation occurs (<xref rid="r36" ref-type="bibr">36</xref>).</p><p>Another promising strategy for the treatment of cystinuria might be chaperone therapy, which is useful for some diseases caused by mutations that lead to misfolded proteins (<xref rid="r37" ref-type="bibr">37</xref>). A pharmacological chaperone may stabilize an already folded protein against proteolytic degradation or thermal denaturation. This process accelerates the escape of mutated proteins from the endoplasmic reticulum associated protein degradation pathway, increasing the level of residual protein activity (<xref rid="r38" ref-type="bibr">38</xref>,<xref rid="r39" ref-type="bibr">39</xref>). As several mutations affecting the rBAT protein cause protein misfolding, chaperone therapy could be an attractive treatment strategy for patients with cystinuria in the future.</p></sec>

Reactive oxygen species, inflammation and calcium oxalate nephrolithiasis

<p>Calcium oxalate (CaOx) kidney stones are formed attached to Randall&#x02019;s plaques (RPs) or Randall&#x02019;s plugs. Mechanisms involved in the formation and growth are poorly understood. It is our hypothesis that stone formation is a form of pathological biomineralization or ectopic calcification. Pathological calcification and plaque formation in the body is triggered by reactive oxygen species (ROS) and the development of oxidative stress (OS). This review explores clinical and experimental data in support of ROS involvement in the formation of CaOx kidney stones. Under normal conditions the production of ROS is tightly controlled, increasing when and where needed. Results of clinical and experimental studies show that renal epithelial exposure to high oxalate and crystals of CaOx/calcium phosphate (CaP) generates excess ROS, causing injury and inflammation. Major markers of OS and inflammation are detectable in urine of stone patients as well as rats with experimentally induced CaOx nephrolithiasis. Antioxidant treatments reduce crystal and oxalate induced injury in tissue culture and animal models. Significantly lower serum levels of antioxidants, alpha-carotene, beta-carotene and beta-cryptoxanthine have been found in individuals with a history of kidney stones. A diet rich in antioxidants has been shown to reduce stone episodes. ROS regulate crystal formation, growth and retention through the timely production of crystallization modulators. In the presence of abnormal calcium, citrate, oxalate, and/or phosphate, however, there is an overproduction of ROS and a decrease in the antioxidant capacity resulting in OS, renal injury and inflammation. Cellular degradation products in the urine promote crystallization in the tubular lumen at a faster rate thus blocking the tubule and plugging the tubular openings at the papillary tips forming Randall&#x02019;s plugs. Renal epithelial cells lining the loops of Henle/collecting ducts may become osteogenic, producing membrane vesicles at the basal side. In addition endothelial cells lining the blood vessels may also become osteogenic producing membrane vesicles. Calcification of the vesicles gives rise to RPs. The growth of the RP&#x02019;s is sustained by mineralization of collagen laid down as result of inflammation and fibrosis.</p>

<contrib contrib-type="author" corresp="yes"><name><surname>Khan</surname><given-names>Saeed R.</given-names></name></contrib><aff id="aff1">Department of Pathology and Department of Urology, College of Medicine, University of Florida, Gainesville, Florida, <country>USA</country></aff>

Translational Andrology and Urology

<p>Kidney stones are comprised of mineral and organic components. Approximately 80% of the kidney stones contain calcium oxalate (CaOx) as the major mineral phase mixed mostly with calcium phosphate (CaP) and sometime uric acid (<xref rid="r1" ref-type="bibr">1</xref>). Stone formation involves crystal nucleation, growth, aggregation and their retention in the kidneys (<xref rid="r2" ref-type="bibr">2</xref>). These processes are modulated by a variety of urinary macromolecules which become incorporated in the growing crystals and stones and eventually constitute stones&#x02019; organic component or matrix. A better understanding of the pathogenesis of kidney stone formation has been developed through examination of clinical data and the use of animal models and tissue cultures. Based on clinical and experimental data, it is becoming obvious that stone formation is not a simple physicochemical disorder. Renal epithelial cells as well as others respond to changing urinary environment; dysregulated mineral metabolism and in the case of CaOx nephrolithiasis, abnormal calcium, citrate, oxalate, phosphate, and CaOx/CaP crystals, by increased production of a variety of crystallization modulating macromolecules, epithelial to mesenchymal transition (EMT), epithelial to osteoblast transformation (EOT), and remodeling of extracellular matrix (ECM). It appears that reactive oxygen species (ROS) are intimately involved as signaling molecules as well as agents of injury and inflammation during stone formation (<xref rid="r3" ref-type="bibr">3</xref>-<xref rid="r6" ref-type="bibr">6</xref>).</p><sec><title>Reactive oxygen species (ROS)</title><p>ROS comprising free radicals, atoms or molecules with unpaired electrons, and their metabolites, are highly reactive and play a critical role as signaling molecules. But they can also produce chemical modifications of, and damage to proteins, lipids, carbohydrates and nucleotides (<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>). Major cellular ROS include superoxide anion (O₂^-&#x02022;), nitric oxide radical (NO^&#x02022;), hydroxyl radical (OH^&#x02022;), and hydrogen peroxide (H₂O₂), which are generated by several pathways (<xref ref-type="fig" rid="f1"><italic>Figure 1</italic></xref>). O₂^-&#x02022; anions are produced by NADPH oxidases, xanthine oxidase, lipooxigenase, cyclooxygenase, hemeoxygenase and as a byproduct of mitochondrial respiratory chain. Lipid radicals can also produce O₂^-&#x02022;. NO^&#x02022; radicals are produced by the endothelial nitric oxide synthase (eNOS) mediated oxidation of L-arginine. In addition, eNOS can also produce O₂^-&#x02022; rather than NO. The reaction between superoxide and nitric oxide can produce the highly reactive peroxynitrite ONOO^-.</p><fig id="f1" fig-type="figure" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Sources and reactions involved in the production of superoxide (O₂^-&#x02022;), nitric oxide (NO^&#x02022;), hydrogen peroxide (H₂O₂), peroxynitrie (ONOO^{&#x000b7;&#x000af;}), and hydroxyl radicals (OH^&#x02022;). GSH, glutathione; GPx, glutathione peroxidase; GSGG, oxidised glutathione; NOS, nitric oxide synthase; SOD, superoxide dismutase.</p></caption><graphic xlink:href="tau-03-03-256-f1"/></fig><p>Cells are equipped with a number of scavenging systems to control ROS availability (<xref ref-type="fig" rid="f1"><italic>Figure 1</italic></xref>). These include superoxide dismutase (SOD) to eliminate O₂^-&#x02022;, and glutathione (GSH) peroxidase (GPx) and catalase to detoxify H₂O₂ (<xref ref-type="fig" rid="f1"><italic>Figure 1</italic></xref>). Superoxide has a short half-life and spontaneously converts to H₂O₂ which is long-lasting and far more reactive than superoxide ions_. The reaction is noticeably enhanced by SOD. Moreover, in a more complex transition metal catalyzed reaction called metal catalyzed Haber-Weiss reaction, H₂O₂ yields an even more reactive hydroxyl radical, which is however, short lived and works at short range. Initially, superoxide anions donate single electrons to ferric ions resulting in molecular oxygen and ferrous ions. The Fenton Reaction between ferrous ions and H₂O_2, leads to the formation of OH^&#x02022;. H₂O₂ is subsequently metabolized to water via catalase or by glutathione peroxidase in the presence of reduced glutathione.</p><p>Under normal conditions the superoxide anions (O₂^-&#x02022;), NO radicals (NO^&#x02022;) and their metabolites are generated by tightly controlled enzymes and serve as mediators in a variety of regulatory processes and signaling pathways including proliferation, activation or inactivation of regulatory biomolecules, and regulation of transcriptional activities. ROS regulate many calcium signals as well as such genes as <italic>c-fos, c-myc,</italic> and <italic>c-jun</italic> and transcription factor activation protein-1 (AP-1) and nuclear factor &#x003ba;B (NF-&#x003ba;B).</p><p>ROS and reactive nitrogen species (RNS) normally occur at steady state levels, generated when needed and then cleared by activities of various antioxidants and scavengers. But uncontrolled generation of the reactive oxygen or nitrogen species and/or a reduction in the endogenous antioxidant capacity creates oxidative stress (OS). Most cells respond to OS by boosting the levels of intracellular antioxidants such as glutathione. The oxidants can react with all the basic constituents of cells: lipids, carbohydrates, proteins and nucleic acids severely affecting their structure and function. Pathological changes may result from the damaging effects of ROS and from ROS-mediated changes in gene expression and signal transduction.</p></sec><sec><title>Sources of ROS in CaOx nephrolithiasis</title><p>ROS are produced through the involvement of both mitochondria (<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r9" ref-type="bibr">9</xref>-<xref rid="r12" ref-type="bibr">12</xref>) and NADPH oxidase (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>) (<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>). NADPH oxidase is a major source of ROS in the kidneys (<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>), particularly in the presence of Angiotensin II (<xref rid="r17" ref-type="bibr">17</xref>). NADPH oxidase consists of six subunits, the two transmembrane units, p22^phox and gp91^phox; and four cytosolic units, p47^phox, p67^phox, p40^phox and the small GTPase rac1 or rac2 (<xref rid="r18" ref-type="bibr">18</xref>). The two transmembrane units, gp91^phox and p22^phox and a flavin make cytochrome b₅₅₈. The cytosolic units translocate to the membrane and assemble with the cytochrome to activate the enzyme.</p><fig id="f2" fig-type="figure" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Proposed scheme for NADPH oxidase and mitochonrial involvement in the generation of reactive oxygen species (ROS) and their role in CaOx nephrolithiasis. Hyperoxaluria/CaOx/CaP crystal deposition causes renin upregulation and generation of angiotensin II which activates NADPH oxidase and production of ROS. Mitochondrial permeability transition pores are opened. The activation of phospholipase A2 (cPLA2) and neutral sphingomyelinase (N-Smase) affect mitochondria through lipid products archidonic acid, lysophosphatidylcholine (Lyso-PC) and ceramide. The production of cytochrome C (Cyt-C) goes up. There is a reduction in mitochondrial membrane potential (&#x00394;&#x003a8;) and reduced glutathione (GSH). These actions lead to activation of caspases and apoptosis. ROS activate transcription factors such as nuclear factor &#x003ba;B (NF&#x003ba;B), activated protein-1 (AP-1) and growth factors including TGF&#x003b2; through P38 mitogen-activated protein kinase (-MAPK)/JNK. Runt-related transcription factor-2 (RUNX-2) and Osterix which are associated with osteoblast differentiation are also upregulated. Secondary mediators such as isoprostanes and prostaglandins are generated. Macromolecules involved in chemoattraction of monocyte-macrophages, crystallization modulation, bone morphogenesis, and fibrosis are produced. O₂^&#x02022;-, superoxide; OH^&#x02022;, hydroxyl radical; H₂O₂, hydrogen peroxide; OPN, osteopontin; BK, bikunin; MCP-1, monocyte chemoattractant protein-1; MGP, matrix gla protein; BMP, bone morphogenetic protein; BSP, bone sialoprotein [Modified from Khan SR (<xref rid="r4" ref-type="bibr">4</xref>)].</p></caption><graphic xlink:href="tau-03-03-256-f2"/></fig><p>ROS in response to oxalate and CaOx crystals are in part produced with the involvement of NADPH oxidase through the activation of the rennin angiotensin system (RAS). Reduction of angiotensin production, by inhibiting the angiotensin converting enzyme as well as blocking the angiotensin receptor, increased renin expression, reduced osteopontin (OPN) expression, crystal deposition and ameliorated the associated inflammatory response (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>) (<xref rid="r19" ref-type="bibr">19</xref>-<xref rid="r21" ref-type="bibr">21</xref>). NADPH oxidase inhibition by apocynin treatment reduced the production of ROS, urinary excretion of kidney injury molecule (KIM) and renal deposition of CaOx crystals in hyperoxaluric rats (<xref rid="r22" ref-type="bibr">22</xref>). Atrovastatin, which has been shown to reduce the expression of gp91^phox and p22^phox subunits of NADPH oxidase (<xref rid="r23" ref-type="bibr">23</xref>), also inhibited crystal deposition in rats with experimentally induced hyperoxaluria (<xref rid="r24" ref-type="bibr">24</xref>).</p><p>Mitochondria are generally the most common source of superoxide and H₂O₂ in most cells and tissues. Hyperoxaluria and CaOx crystal deposition in rat kidneys causes mitochondrial damage. Treatment with taurine which has been shown to prevent oxidative injury of the mitochondria, reversed mitochondrial changes in the hyperoxaluric rat kidneys and decreased crystal deposition (<xref rid="r25" ref-type="bibr">25</xref>). Selective probes, substrates and inhibitors show mitochondria to be significant site of CaOx crystal induced superoxide production and glutathione depletion in both LLC-PK1 and MDCK cells (<xref rid="r9" ref-type="bibr">9</xref>). Exposure of LLC-PK1 cells to oxalate significantly increased cellular ceramides (<xref rid="r26" ref-type="bibr">26</xref>), however, pretreatment with glutathione precursor N-acetylcysteine (NAC) blocked this increase. Isolated mitochondria responded to oxalate exposure by the accumulation of ROS, lipid peroxides and oxidized thiol proteins (<xref rid="r11" ref-type="bibr">11</xref>). Citrate is also involved in maintaining endogenous antioxidant defenses. Administration of exogenous citrate to LLC-PK1 and MDCK cells bolstered these defenses and diminished the cellular injury inflicted by exposure to increased Ox and CaOx crystals (<xref rid="r27" ref-type="bibr">27</xref>). The presence of citrate in the culture medium was associated with a significant increase in GSH peroxidase and a drop in the production of H₂O₂ and 8-isoprostane (8-IP), which is an end product of lipid breakdown. There was a significant improvement in cell viability as demonstrated by decreased LDH release and increased trypan blue exclusion.</p><p>Mitochondrial damage is suggested to be induced by the opening of mitochondrial permeability transition pore (mPTP). mPTP opening depends upon the activation of cyclophilin D in the mitochondrial matrix by ROS produced by NADPH oxidase and is inhibited by cyclosporine A (CSA) (<xref rid="r28" ref-type="bibr">28</xref>). CSA prevented the depolarization of mitochondrial membrane, decrease in SOD expression, increase in 4-hydroxy-2-nonenal (4HNE) and release of cytochrome-c into the cytosol in NR52E renal epithelial cells exposed to CaOx monohydrate crystals <italic>in vitro.</italic> CSA treatment of hyperoxaluric rats resulted in reduced mitochondrial damage, OS and CaOx crystal deposition in the kidneys.</p></sec><sec><title>Association of inflammation and injury with human stone formation</title><p>Most idiopathic stones are formed attached to Randall&#x02019;s plaques (RPs), the sub-epithelial deposits of CaP on renal papillary surfaces (<xref rid="r29" ref-type="bibr">29</xref>). RPs are postulated to start as deposits of poorly crystalline biological apatite in the basement membrane of the loops of Henle (<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref>) or collecting ducts (<xref rid="r32" ref-type="bibr">32</xref>) or vasa recta (<xref rid="r33" ref-type="bibr">33</xref>,<xref rid="r34" ref-type="bibr">34</xref>). The deposits, consisting of aggregated CaP spherules, grow through the interstitium towards the renal papillary epithelium, where they eventually ulcerate to the surface (<xref rid="r35" ref-type="bibr">35</xref>). Interestingly, all RPs are not connected to stones and kidneys of non-stone formers also contain interstitial plaques (<xref rid="r36" ref-type="bibr">36</xref>).</p><p>Stones such as cystine, brushite, CaOx in primary hyperoxaluria and after bariatric surgery, some idiopathic stones and CaP in primary hyperparathyroidism are found attached to Randall&#x02019;s plugs (the tubular crystal deposits in the ducts of Bellini (<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r37" ref-type="bibr">37</xref>,<xref rid="r38" ref-type="bibr">38</xref>), which were most likely formed as a result of higher supersaturation with respect to the precipitating salt (<xref rid="r39" ref-type="bibr">39</xref>). Crystal deposition is associated with renal cell injury, cell loss, inflammation and fibrosis (<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r40" ref-type="bibr">40</xref>-<xref rid="r45" ref-type="bibr">45</xref>). The inflammation is generally localized to areas around crystal deposits in the renal papillae. In brushite stone formers, however, inflammation and fibrosis reach the cortex showing wide spread renal tubular atrophy and glomerular pathology (<xref rid="r42" ref-type="bibr">42</xref>).</p><p>It has been suggested that RPs are formed without causing renal injury and inflammation (<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r46" ref-type="bibr">46</xref>). But a close examination of published illustrations (<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r32" ref-type="bibr">32</xref>,<xref rid="r36" ref-type="bibr">36</xref>,<xref rid="r47" ref-type="bibr">47</xref>) clearly demonstrate the presence of necrotic tubules with thickened and layered basal lamina, along with perfectly normal ones in association with the CaP spherules embedded in a matrix of collagen and other fibers. Similarly we have found injured tubules associated with the interstitial deposits of apatitic mineral (<xref rid="r35" ref-type="bibr">35</xref>,<xref rid="r38" ref-type="bibr">38</xref>). The molecules generally involved in inflammatory pathways, such as OPN (<xref rid="r48" ref-type="bibr">48</xref>,<xref rid="r49" ref-type="bibr">49</xref>), heavy chain of inter-&#x003b1;- inhibitor (<xref rid="r50" ref-type="bibr">50</xref>,<xref rid="r51" ref-type="bibr">51</xref>), collagen (<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r36" ref-type="bibr">36</xref>,<xref rid="r47" ref-type="bibr">47</xref>), and zinc (<xref rid="r52" ref-type="bibr">52</xref>) have been seen in the interstitial plaques strongly suggesting that inflammation may have been an early and local participant (<xref rid="r4" ref-type="bibr">4</xref>), which was resolved by the time stone was discovered. Biopsies are taken at the time of stone removal, many months after stone formation. Moreover, only a very small amount of tissue from limited number of patients has so far been investigated. &#x0201c;It is important to emphasize that urolithiasis is merely a final manifestation of diverse and systemic etiological and pathogenic events&#x0201d; (<xref rid="r53" ref-type="bibr">53</xref>). Inflammation is a complex biological response to various irritants. Osteopotin and inter-&#x003b1;-inhibitor are protective mediators, which are most likely produced to inhibit crystallization and protect the kidneys. In normal human kidneys, OPN is localized primarily to the distal nephron and is strongly expressed in the thick ascending limbs of the loops of Henle and papillary surface epithelium. OPN expression is increased during inflammation and interstitial fibrosis (<xref rid="r54" ref-type="bibr">54</xref>).</p><p>Renal CaOx crystal deposits have been seen in a variety of disorders with increased production and excretion of oxalate. In biopsies from a patient with primary hyperoxaluria, crystals were seen within tubular epithelial cells as well as in the interstitium of the transplanted kidney (<xref rid="r55" ref-type="bibr">55</xref>) and were associated with vascular and interstitial inflammation, cell proliferation and multinucleated giant cells. Similar observations have been made in other cases of increased urinary excretion of Ox secondary to enteric hyperoxaluria, Crohn&#x02019;s disease, and after intestinal bypass (<xref rid="r56" ref-type="bibr">56</xref>,<xref rid="r57" ref-type="bibr">57</xref>).</p><p>Higher than normal levels of renal enzymes, gamma-glutamyl transpeptidase (GGTP), angiotensin 1 converting enzyme (ACE), &#x003b2;-galactosidase (GAL), and N-acetyl-&#x003b2;-glucoseaminidase (NAG) were found in the urine of idiopathic CaOx stone formers (<xref rid="r58" ref-type="bibr">58</xref>), indicative of renal proximal tubular injury. The urine had significantly increased NAG, &#x003b2;-GAL, &#x003b1;-glutathione S-transferase (&#x003b1;-GST), malondialdehyde (MDA) and thiobarbituric acid-reactive substances (TBARS) (<xref rid="r59" ref-type="bibr">59</xref>), suggesting that stone-associated injury was most likely caused by the production of ROS. Urinary 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative damage of DNA, was increased in stone patients and was positively correlated with tubular damage as assessed by urinary excretion of NAG (<xref rid="r60" ref-type="bibr">60</xref>). Recurrent idiopathic calcium stone formers with and without stones, exhibited antioxidant deficiency. Investigators concluded that lithiasis started with oxidatively damaged cells (<xref rid="r61" ref-type="bibr">61</xref>). Anti-inflammatory proteins calgranulin, &#x003b1;-defensin, and myeloperoxidase (<xref rid="r62" ref-type="bibr">62</xref>), were increased in urine of stone patients and were also found in the inner core of the CaOx stones.</p><p>Members of I&#x003b1;I family of proteins, which are important participants in wound healing, were significantly increased in the urine of male stone formers (<xref rid="r63" ref-type="bibr">63</xref>), and found in the stone matrix as well as the matrix of CaOx and CaP crystals induced in the urine (<xref rid="r64" ref-type="bibr">64</xref>,<xref rid="r65" ref-type="bibr">65</xref>). Hyaluronan, which plays an important role in renal injury and repair, was a major constituent of the organic matrix of stones (<xref rid="r66" ref-type="bibr">66</xref>). Prothrombin fragment-1, a member of thrombin family of proteins which are extensively involved in tissue repair, was also excreted in urine, present in stone matrix and preferentially bound to CaOx crystals (<xref rid="r67" ref-type="bibr">67</xref>). Kidneys of stone formers expressed mRNA for MCP-1 as well as IL-6 (<xref rid="r68" ref-type="bibr">68</xref>).</p></sec><sec><title>Nephrolithiasis and chronic kidney diseases (CKDs)</title><p>Kidney stone formation has been linked with a number of chronic diseases (<xref rid="r69" ref-type="bibr">69</xref>), such as obesity (OB) (<xref rid="r70" ref-type="bibr">70</xref>), diabetes mellitus (DM) (<xref rid="r71" ref-type="bibr">71</xref>), hypertension (HTN) (<xref rid="r70" ref-type="bibr">70</xref>), metabolic syndrome (MS) (<xref rid="r72" ref-type="bibr">72</xref>), and CKD (<xref ref-type="fig" rid="f3"><italic>Figure 3</italic></xref>) (<xref rid="r73" ref-type="bibr">73</xref>). Nephrolithiasis is a risk factor for the development of hypertension (<xref rid="r74" ref-type="bibr">74</xref>), while similarly hypertensive patients are at greater risk to develop nephrolithiasis (<xref rid="r75" ref-type="bibr">75</xref>). There is an association between stone disease and DM (<xref rid="r71" ref-type="bibr">71</xref>,<xref rid="r76" ref-type="bibr">76</xref>-<xref rid="r81" ref-type="bibr">81</xref>), as diabetics persistently produce acidic pH and have a greater risk to form uric acid stones (<xref rid="r82" ref-type="bibr">82</xref>). Kidney stone formers are also at greater risk for coronary artery disease (<xref rid="r83" ref-type="bibr">83</xref>), myocardial infarction (<xref rid="r84" ref-type="bibr">84</xref>) and CKD (<xref rid="r83" ref-type="bibr">83</xref>,<xref rid="r85" ref-type="bibr">85</xref>). Not surprisingly, stone formation is common in adults with metabolic syndrome and the frequency of stone formation is directly correlated with weight and BMI (<xref rid="r86" ref-type="bibr">86</xref>-<xref rid="r88" ref-type="bibr">88</xref>).</p><fig id="f3" fig-type="figure" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Diagram showing associations between nephrolithiasis and other diseases with renal involvement.</p></caption><graphic xlink:href="tau-03-03-256-f3"/></fig><p>Both clinical and experimental investigations indicate that OS and inflammation play a significant role in the development of cardiovascular diseases (<xref rid="r89" ref-type="bibr">89</xref>). OS is a common feature of HTN, DM, atherosclerosis, and myocardial infarct (<xref ref-type="fig" rid="f4"><italic>Figure 4</italic></xref>) (<xref rid="r69" ref-type="bibr">69</xref>). An increase in the production of ROS/RNS, and/or decrease in the extra and intracellular antioxidants has been demonstrated in both clinical and experimental HTNs (<xref rid="r90" ref-type="bibr">90</xref>) and leads to OS which may not only initiate HTN but also be developed by the hypertensive state (<xref rid="r91" ref-type="bibr">91</xref>). Experimental studies have shown the involvement of NADPH oxidase in the development of hypertension (<xref rid="r91" ref-type="bibr">91</xref>-<xref rid="r93" ref-type="bibr">93</xref>). OB associated OS eventually leads to systemic inflammation and endothelial cell dysfunction (<xref rid="r94" ref-type="bibr">94</xref>,<xref rid="r95" ref-type="bibr">95</xref>). Proper endothelial performance requires NO which acts on pericytes, and is depleted during OS because of its inactivation by O₂^-&#x02022; (<xref rid="r96" ref-type="bibr">96</xref>,<xref rid="r97" ref-type="bibr">97</xref>). Oxidation of the NO also results in the formation of highly active ONOO^- and enhancement of OS. NADPH oxidase is a major source of ROS in the kidneys and is activated by Ang II, mostly through the AT1 receptor. Both NO and O₂^-&#x02022; are produced by the renal epithelial cells, in addition, NO is also produced by endothelial cells. There is a tubulovascular cross talk, whereby NO produced by the epithelial cells of the medullary thick ascending limb affect the interstitial pericytes and endothelial cells.</p><fig id="f4" fig-type="figure" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Role of ROS in nephrolithiasis and co-morbidities. ROS play a role in the progression of many disorders including nephrolithiasis. ROS produced during one disorder may instigate the other under conditions suitable for the specific disease. ROS, reactive oxygen species.</p></caption><graphic xlink:href="tau-03-03-256-f4"/></fig><p>NADPH oxidase is the major source of ROS in the kidneys and cardiovascular system (<xref rid="r93" ref-type="bibr">93</xref>). The kidneys of spontaneously hypertensive rats (SHR) showed increased production of O₂^-&#x02022; and upregulation of p47^phox subunit (<xref rid="r98" ref-type="bibr">98</xref>). Administration of SOD mimetic tempol produced a reduction of blood pressure and renal vascular resistance (<xref rid="r99" ref-type="bibr">99</xref>). Significantly higher p22^phox mRNA levels and NADPH oxidase driven O₂^{&#x02013;&#x02022;} production were found in the aorta of SHR which were ameliorated by treatment with irbesartan, an angiotensin II receptor antagonist (<xref rid="r100" ref-type="bibr">100</xref>). The importance of p47^phox is also shown by moderate hypertensive response to angiotensin II in mice lacking the p47^phox (<xref rid="r101" ref-type="bibr">101</xref>,<xref rid="r102" ref-type="bibr">102</xref>). Inhibition by membrane permeable gp91ds-tat of p47^phox assembly with gp91^phox in Dahl salt sensitive (DS) rats fed a 4% salt diet, normalized ROS production and endothelium dependent relaxation as well as expression of LOX-1 and MCP-1 (<xref rid="r103" ref-type="bibr">103</xref>). Administration of apocynin, an antioxidant and an inhibitor of the p47^phox assembly with gp91^phox, to DS rats on high salt diet produced significant reductions in the mRNA expression of gp91^phox, p47^phox, p22^phox, and p67^phox subunits in addition to significantly reducing insterstitial superoxide and mean arterial pressure (<xref rid="r104" ref-type="bibr">104</xref>). Apocynin also reduced NADPH oxidase activity, renal cortical O₂^-&#x02022;, monocyte/macrophage infiltration and glomerular and interstitial damage (<xref rid="r105" ref-type="bibr">105</xref>). Experimental studies involving other animal models of hypertension have similarly shown the involvement of NADPH oxidase in the development of hypertension (<xref rid="r91" ref-type="bibr">91</xref>-<xref rid="r93" ref-type="bibr">93</xref>).</p><p>NaDPH oxidase also plays an important role in diabetic nephropathy (<xref rid="r106" ref-type="bibr">106</xref>-<xref rid="r108" ref-type="bibr">108</xref>), particularly in the presence of high glucose. Levels of Nox 4 as well as p22^phox mRNA were increased in kidneys of rats with STZ-induced diabetes along with an increase in immunostaining of 8-OHdG (<xref rid="r109" ref-type="bibr">109</xref>). Insulin treatment reduced them to control levels. STZ induced diabetes also increased excretion of H₂O₂, lipid peroxidation (LPO) products, and nitric oxide products (Nox) (<xref rid="r110" ref-type="bibr">110</xref>). Kidneys showed increased expression of gp91^phox and p47^phox and endothelial eNOS, increased mesangial matrix, fibronectin and type I collagen. The treatment with apocynin, which inhibits assembly of the cytosolic p47^phox with the membranous gp91^phox, inhibited the increases in membrane fraction of p47^phox, and excretion of H₂O₂, LPO and Nox.</p><p>Dietary Approaches to Stop Hypertension (DASH) diet, which reduces the risks for stroke and cardiovascular diseases, also reduced the risk for stone formation by up to 45% (<xref rid="r111" ref-type="bibr">111</xref>). The relationship between DASH type diet and the incident symptomatic kidney stones was examined in a prospective analysis of data from Health Professionals Follow up Study (n=45,821), Nurses&#x02019; Health Study-1 (n=94,108) and 2 (n=101,837) and found that men and women with higher DASH scores were significantly less likely to develop kidney stones than those with lower DASH scores. Low sodium DASH diet reduces OS and improves vascular functions, lowers blood pressure (<xref rid="r112" ref-type="bibr">112</xref>,<xref rid="r113" ref-type="bibr">113</xref>). Analysis of data from National Health and Nutrition Examination Survey (NHANESIII) showed that serum levels of antioxidants alpha-carotene, beta-carotene and beta-cryptoxanthin were significantly lower in stone patients. Lower levels of these antioxidants were associated with decreasing incidents of stone disease (<xref rid="r114" ref-type="bibr">114</xref>).</p><p>The association between nephrolithiasis, CKD, DM, OB, HTN, and MS is most likely a result of common pathophysiological mechanisms (<xref rid="r70" ref-type="bibr">70</xref>). ROS and OS are common feature of CKD, nephrolithiasis, DM, OB, HTN, and MS. It is conceivable that ROS produced by one disease may lead to another under appropriate circumstances (<xref ref-type="fig" rid="f4"><italic>Figure 4</italic></xref>) (<xref rid="r69" ref-type="bibr">69</xref>,<xref rid="r115" ref-type="bibr">115</xref>,<xref rid="r116" ref-type="bibr">116</xref>). For example mild hypercalciuria, hyperoxaluria, hypocitraturia which under normal conditions may just be a curiosity or nuisance, can promote crystallization and stone formation when cells are injured by ROS produced through another disorder.</p></sec><sec><title>Epithelial exposure to oxalate or calcific crystals, inflammation and injury</title><p>Tissue culture studies in which renal epithelial cells are exposed to Ox and/or CaOx or CaP crystals have provided new insights into mechanisms involved in stone formation. Cell response is time and concentration dependent and cell specific. Exposure to high concentrations of Ox as well as CaOx and CaP crystals for longer duration is injurious to renal epithelial cells (<xref rid="r117" ref-type="bibr">117</xref>-<xref rid="r121" ref-type="bibr">121</xref>). Crystals bind rapidly to the surface of epithelial cells and are internalized (<xref rid="r122" ref-type="bibr">122</xref>-<xref rid="r127" ref-type="bibr">127</xref>). Cells of proximal tubular origin are more susceptible to injury then cells originating from distal sections of the renal tubules. Lower Ox levels induce expression of immediate early genes, stimulate DNA synthesis and promote cellular proliferation, while higher Ox levels induce cell damage and death.</p><p>The response of renal epithelial cells to COM crystals is characterized by increased expression of specific genes that encode transcriptional activators, regulators of the ECM, and growth factors (<xref rid="r118" ref-type="bibr">118</xref>,<xref rid="r128" ref-type="bibr">128</xref>), and production of pro and anti-inflammatory molecule, such as OPN, monocyte chemoattractant-1 (MCP-1), prostaglandin E2 (PGE2), bikunin and components of inter-&#x003b1;-inhibitor (I&#x003b1;I), &#x003b1;-1 microglobulin, CD-44, calgranulin, heparin sulfate, osteonectin, fibronectin and matrix-gla-protein (MGP) (<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r129" ref-type="bibr">129</xref>-<xref rid="r134" ref-type="bibr">134</xref>). Even though many of these molecules are integral to inflammation and fibrosis, they are also modulators of calcification (<xref rid="r135" ref-type="bibr">135</xref>,<xref rid="r136" ref-type="bibr">136</xref>). Gene expression of vimentin, a mesenchymal marker, is also increased (<xref rid="r137" ref-type="bibr">137</xref>).</p><p>Tissue culture studies have also provided the evidence for the involvement of free radicals in toxicity, production of various crystallization modulators, and inflammatory and anti-inflammatory macromolecules (<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r138" ref-type="bibr">138</xref>-<xref rid="r141" ref-type="bibr">141</xref>). Renal cells exposed to CaOx crystals secrete superoxide (<xref rid="r142" ref-type="bibr">142</xref>) and cellular injury could be ameliorated by antioxidants and free radical scavenger (<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r138" ref-type="bibr">138</xref>-<xref rid="r140" ref-type="bibr">140</xref>,<xref rid="r143" ref-type="bibr">143</xref>). Free radical scavengers, catalase and SOD provided protection from oxalate induced injury to LLC-PK1 and MDCK cells (<xref rid="r138" ref-type="bibr">138</xref>). Catalase treatment also reduced MCP-1 mRNA as well as protein in the oxalate, CaOx, CaP and uric acid treated NRK52E renal epithelial cells (<xref rid="r132" ref-type="bibr">132</xref>,<xref rid="r133" ref-type="bibr">133</xref>).</p></sec><sec><title>Inflammation and injury in animal models of CaOx nephrolithiasis</title><p>A number of rat and mice models (<xref rid="r144" ref-type="bibr">144</xref>,<xref rid="r145" ref-type="bibr">145</xref>), have been developed to investigate the pathogenesis of kidney stones. None of the models completely replicate the process of idiopathic stone formation and develop stones on papillary surface attached to the RPs. Instead the crystals are intraluminal, reminiscent of Randall&#x02019;s plugs. CaOx nephrolithiasis is produced by inducing hyperoxaluria through the administration of oxalate or its precursors such as glyoxylate, ethylene glycol (EG) and hydroxyl-L-proline (HLP). Both hyperoxaluria and CaOx crystal deposition trigger morphological and pathophysiological changes in the kidneys and alter urinary composition (<xref rid="r22" ref-type="bibr">22</xref>,<xref rid="r146" ref-type="bibr">146</xref>). Renal expression of OPN (<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r147" ref-type="bibr">147</xref>), Tamm-Horsfall Protein (THP) (<xref rid="r148" ref-type="bibr">148</xref>-<xref rid="r150" ref-type="bibr">150</xref>), bikunin (<xref rid="r130" ref-type="bibr">130</xref>,<xref rid="r131" ref-type="bibr">131</xref>), I&#x003b1;I (<xref rid="r151" ref-type="bibr">151</xref>), &#x003b1;-1microglobulin (<xref rid="r152" ref-type="bibr">152</xref>), prothrombin fragment-1 (PTF-1) (<xref rid="r153" ref-type="bibr">153</xref>), calgranulin, heparin sulfate (HS) (<xref rid="r154" ref-type="bibr">154</xref>), matrix gla protein (MGP) (<xref rid="r155" ref-type="bibr">155</xref>,<xref rid="r156" ref-type="bibr">156</xref>), are generally increased (<xref ref-type="table" rid="t1"><italic>Table 1</italic></xref>) and often found at locations not normally seen. For example THP is specifically produced by epithelial cells lining the thick ascending limbs of the loops of Henle. However, in the rat model of CaOx nephrolithiasis THP is seen associated with crystals throughout the kidneys including the cortex (<xref rid="r150" ref-type="bibr">150</xref>,<xref rid="r157" ref-type="bibr">157</xref>). In addition the expression of NF&#x003ba;B, KIM, proliferating cell nuclear antigen (PCNA), and CD 44, e-cadherin, is also increased indicating both injury and proliferation (<xref rid="r22" ref-type="bibr">22</xref>,<xref rid="r158" ref-type="bibr">158</xref>). Urinary excretion of many of these molecules is increased as well (<xref rid="r22" ref-type="bibr">22</xref>,<xref rid="r131" ref-type="bibr">131</xref>,<xref rid="r146" ref-type="bibr">146</xref>,<xref rid="r147" ref-type="bibr">147</xref>,<xref rid="r151" ref-type="bibr">151</xref>,<xref rid="r153" ref-type="bibr">153</xref>,<xref rid="r159" ref-type="bibr">159</xref>). There is migration of monocyte and macrophages to the sites of crystal deposition. We examined kidneys at different times after induction of acute hyperoxaluria in male Sprague-Dawley rats, and found that CaOx crystals appeared first in the tubular lumen, then moved to inter- and intracellular locations, eventually relocating into the interstitium, where they became surrounded by macrophages. After a few weeks, the interstitial crystals disappeared, indicating the existence of a mechanism to remove the CaOx crystals (<xref rid="r160" ref-type="bibr">160</xref>). Multinucleated giant cells were also identified in the interstitium (<xref rid="r161" ref-type="bibr">161</xref>).</p><table-wrap id="t1" orientation="portrait" position="float"><label>Table 1</label><caption><title>Urinary macromolecules and their role in crystallization and inflammation</title></caption><table frame="hsides" rules="groups"><col width="24.13%" span="1"/><col width="41.52%" span="1"/><col width="34.35%" span="1"/><thead><tr><th valign="top" align="left" scope="col" rowspan="1" colspan="1">Name</th><th valign="top" align="left" scope="col" rowspan="1" colspan="1">Role in nephrolithiasis</th><th valign="top" align="left" scope="col" rowspan="1" colspan="1">Role in inflammation and repair</th></tr></thead><tbody><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Tamm-Horsfall protein (THP)</td><td valign="top" align="left" rowspan="1" colspan="1">Modulator of CaOx crystal nucleation, growth and aggregation as well as adherence to epithelium</td><td valign="top" align="left" rowspan="1" colspan="1">Renoprotective, elicits immune response</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Osteopontin (OPN)</td><td valign="top" align="left" rowspan="1" colspan="1">Modulator of CaOx crystallization, aggregation and crystal attachment</td><td valign="top" align="left" rowspan="1" colspan="1">Calcium binding, renoprotective, anti-inflammatory, chemoattractant for monocytes</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Prothrombin fragment-1 (PTF-1)</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of crystal growth and aggregation</td><td valign="top" align="left" rowspan="1" colspan="1">Calcium binding, coagulation</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Bikunin and inter-&#x003b1;-inhibitor family (I&#x003b1;I)</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of CaOx crystallization and attachment</td><td valign="top" align="left" rowspan="1" colspan="1">Metastasis, tissue repair and remodeling</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">&#x003b1;-1-microglobulin (&#x003b1;1m)</td><td valign="top" align="left" rowspan="1" colspan="1">Modulator of crystallization</td><td valign="top" align="left" rowspan="1" colspan="1">Immunosuppressive, protective against oxidative stress</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Hyaluronic acid (HA)</td><td valign="top" align="left" rowspan="1" colspan="1">A major constituent of stone matrix, modulator of crystallization and adherence to renal epithelium</td><td valign="top" align="left" rowspan="1" colspan="1">Major constituent of extracellular matrix</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">CD-44</td><td valign="top" align="left" rowspan="1" colspan="1">Promoter of crystal attachment</td><td valign="top" align="left" rowspan="1" colspan="1">Tissue repair and remodeling</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Calgranulin (Calprotectin)</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of crystal growth and aggregation</td><td valign="top" align="left" rowspan="1" colspan="1">Calcium binding, tissue remodeling and inflammation</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Heparan sulfate (HS)</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of crystal aggregation and attachment</td><td valign="top" align="left" rowspan="1" colspan="1">Tissue remodeling</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Osteonectin</td><td valign="top" align="left" rowspan="1" colspan="1"/><td valign="top" align="left" rowspan="1" colspan="1">Calcium binding, tissue remodeling</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Fibronectin</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of crystal aggregation, attachment and endocytosis</td><td valign="top" align="left" rowspan="1" colspan="1">Morphogenesis, wound healing and metastasis</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Matrix gla protein (MGP)</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of crystal deposition</td><td valign="top" align="left" rowspan="1" colspan="1">Inhibitor of biomineralization</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Fetuin</td><td valign="top" align="left" rowspan="1" colspan="1">Increased urinary excretion by stone patients</td><td valign="top" align="left" rowspan="1" colspan="1">Anti-inflammatory</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Albumin</td><td valign="top" align="left" rowspan="1" colspan="1">Modulator of crystal nucleation</td><td valign="top" align="left" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Interleukin-6</td><td valign="top" align="left" rowspan="1" colspan="1">Increased urinary excretion by stone patients</td><td valign="top" align="left" rowspan="1" colspan="1">Mediator of inflammation</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">Monocyte chemoattractant protein-1 (MCP-1)</td><td valign="top" align="left" rowspan="1" colspan="1">No known role in crystallization</td><td valign="top" align="left" rowspan="1" colspan="1">Attracts monocytes, memory T cells, dendritic cells to site of inflammation</td></tr></tbody></table><table-wrap-foot><p>CaOx, calcium oxalate.</p></table-wrap-foot></table-wrap><p>Lipid peroxides increased in both the renal tissue and urine in rats with hyperoxaluria and CaOx nephrolithiasis (<xref rid="r162" ref-type="bibr">162</xref>). Additionally, treatment with antioxidant vitamin E improved the tissue levels of antioxidant enzymes, reduced injury and totally eliminated CaOx crystal deposition in the kidneys (<xref rid="r163" ref-type="bibr">163</xref>). Deposition of CaOx crystals in the kidneys was associated with reduction of total renal cellular glutathione and an increase in lipid peroxides (<xref rid="r19" ref-type="bibr">19</xref>). Rats who received ACE inhibitor losartan, known to reduce OS, showed a significant increase in glutathione concentration and a decrease in the thiobarbituric acid reactive substances in the kidneys. The activities of catalase and MnSOD increased in kidneys while &#x003b1;- and &#x003bc;-glutathione-S-transferase (GST) levels increased in the urine of hyperoxaluric rats (<xref rid="r164" ref-type="bibr">164</xref>). Microarray analysis of the kidneys of hyperoxaluric rats also revealed the development of OS during hyperoxaluria and CaOx crystals deposition. Expression of genes for SOD, GPx, GST, aldehyde dehydrogenase, mitochondrial uncoupling protein and ceruloplasmin was increased in hyperoxaluric rats (<xref rid="r165" ref-type="bibr">165</xref>). Administration of apocyanin, an antioxidant and inhibitor of NADPH oxidase, to rats with hydroxypropline induced hyperoxaluria nearly completely reversed the effects of hyperoxaluria (<xref rid="r146" ref-type="bibr">146</xref>), in addition, the deposition of CaOx crystals in the kidneys was also significantly reduced (<xref ref-type="fig" rid="f5"><italic>Figure 5</italic></xref>), and the urinary excretion of OPN, KIM, MCP-1 was significantly reduced without any effect on hyperoxaluria (<xref ref-type="fig" rid="f6"><italic>Figure 6</italic></xref>).</p><fig id="f5" fig-type="figure" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Hematoxylin and eosin stained paraffin section of a kidney from a rat made hyperoxaluric by feeding hydroxyl-L-proline for 28 days (<xref rid="r146" ref-type="bibr">146</xref>). Sections were examined by light microscope equipped with polarizing optics (&#x000d7;4). (A) Hyperoxaluric rat. Renal tubules are full with birefringent CaOx crystals; (B) hyperoxaluric rat receiving apocynin. Only a few birefringent CaOx crystals are present. CaOx, calcium oxalate.</p></caption><graphic xlink:href="tau-03-03-256-f5"/></fig><fig id="f6" fig-type="figure" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Urinary excretion of oxalate, osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), and kidney injury molecule (KIM-1) by rats with hyperoxaluria induced by feeding hydroxyl-L-proline (HLP) (<xref rid="r146" ref-type="bibr">146</xref>) with or without apocyanin (HLP/Apo). There were significant reductions in the production of OPN, KIM-1, and MCP-1 by treatment with apocynin, an inhibitor of NADPH oxidase as well as an antioxidant.</p></caption><graphic xlink:href="tau-03-03-256-f6"/></fig><p>CaOx crystal deposition caused inflammation and attracted many inflammatory cells including leukocytes, monocytes, and macrophages (<xref rid="r146" ref-type="bibr">146</xref>,<xref rid="r161" ref-type="bibr">161</xref>,<xref rid="r166" ref-type="bibr">166</xref>,<xref rid="r167" ref-type="bibr">167</xref>), and multinucleated giant cells were identified around the crystals. The mechanism by which inflammatory cells enter the renal interstitium is not known, but chemotactic factors and adhesion molecules are involved. Leukocytes (neutrophils, monocytes, and lymphocytes) infiltrate the kidneys during a variety of inflammatory diseases. They mediate renal injury and subsequent sclerosis induced by such pathologies. Chemotactic factors are produced by renal cells and are found in the kidney and urine during inflammation. Results show that approximately 70-80% of monocyte chemotactic activity produced by cultured human mesangial cells (<xref rid="r168" ref-type="bibr">168</xref>), renal cortical epithelial cells (<xref rid="r169" ref-type="bibr">169</xref>), proximal tubular epithelial cells (<xref rid="r170" ref-type="bibr">170</xref>), and bovine glomerular endothelial cells (<xref rid="r171" ref-type="bibr">171</xref>), is accounted for by MCP-1. As discussed earlier, exposure to oxalate and both the CaP and CaOx crystals is associated with the production of MCP-1 by rat kidney cells culture (<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r132" ref-type="bibr">132</xref>,<xref rid="r133" ref-type="bibr">133</xref>).</p></sec><sec><title>Epithelial to osteoblast transformation (EOT)</title><p>Vascular calcification, which plays a major role in the development of CKD, was considered to occur by a passive, unregulated physicochemical mechanism as an irreversible degenerative process. Now however, it is considered to be an actively regulated process in which vascular smooth cells (VSMC) acquire osteogenic phenotype (<xref rid="r172" ref-type="bibr">172</xref>-<xref rid="r174" ref-type="bibr">174</xref>). Exposure of VSMC to elevated levels of calcium and phosphate triggers osteogenic transformation of VSMC (<xref rid="r175" ref-type="bibr">175</xref>-<xref rid="r178" ref-type="bibr">178</xref>), which involves an increased expression of osteoblast specific genes and a decrease in smooth muscle cell markers (<xref rid="r179" ref-type="bibr">179</xref>,<xref rid="r180" ref-type="bibr">180</xref>). Bone morphogenetic proteins, BMP 2 and BMP 4, and Wnt signaling pathways are activated through up-regulation of transcription factor, Runt-related transcription factor 2 (RUNX2)/msh homeobox 2 (MSX-2). Cells produce matrix proteins. Crystallization starts in membrane bound matrix vesicles produced by the viable transformed vascular smooth muscle cells or apoptotic bodies produced on their death (<xref rid="r178" ref-type="bibr">178</xref>,<xref rid="r181" ref-type="bibr">181</xref>,<xref rid="r182" ref-type="bibr">182</xref>). The vesicles are similar in composition to the matrix vesicles derived from chondrocytes and provide sites for the nucleation of CaP crystals (<xref rid="r176" ref-type="bibr">176</xref>). Once mineralized, the crystals poke through the limiting membrane of the vesicles and help mineralize the nearby ECM which sustains calcification. In addition to abnormal mineral metabolism, OS, inflammation and aberrant crystallization inhibition play significant role in vascular calcification. ROS are likely involved in the VSMC transformation to osteogenic phenotype by regulating RUNX-2 transcription factor (<xref rid="r183" ref-type="bibr">183</xref>,<xref rid="r184" ref-type="bibr">184</xref>). Advanced glycation end-products commonly seen in blood and arteries of diabetic patients and older individuals can promote vascular calcification mediated by NADPH oxidase induced ROS (<xref rid="r185" ref-type="bibr">185</xref>). Cytokines such as interleukin (IL)-1&#x003b2;, IL-6, IL-8, tumor necrosis factor (TNF)-&#x003b1;, transforming growth factor (TGF)-&#x003b2; produced by macrophages induce transformation of VSMCs (<xref rid="r186" ref-type="bibr">186</xref>). Inflammatory cells also produce proteolytic enzymes such as metalloproteinases (MMP)-2 and -9 which degrade matrix and promote calcification (<xref rid="r187" ref-type="bibr">187</xref>-<xref rid="r190" ref-type="bibr">190</xref>).</p><p>Calcification of vascular smooth cell is inhibited by MGP, pyrophosphate, OPN and Fetuin-A (<xref rid="r179" ref-type="bibr">179</xref>). MGP is a vitamin K-dependent protein functioning primarily as an inhibitor of vascular calcification (<xref rid="r191" ref-type="bibr">191</xref>). MGP also regulates BMP-2 activity (<xref rid="r192" ref-type="bibr">192</xref>). Mutations in the MGP gene lead to keutel syndrome, a disorder associated with extensive soft tissue and vascular calcification (<xref rid="r193" ref-type="bibr">193</xref>). MGP knockout mice die within two months as a result of arterial calcification and blood vessel rupture (<xref rid="r194" ref-type="bibr">194</xref>) while restoration of MGP in these mice prevents arterial calcification (<xref rid="r195" ref-type="bibr">195</xref>). Polymorphism of MGP may play a role in vascular calcification (<xref rid="r196" ref-type="bibr">196</xref>) and has shown an association with myocardial infarction (<xref rid="r197" ref-type="bibr">197</xref>).</p><p>Fetuin A, a member of cystatin family of protease inhibitors, is a serum protein, produced by the liver and specifically enriched in mineralized tissues (<xref rid="r198" ref-type="bibr">198</xref>-<xref rid="r200" ref-type="bibr">200</xref>). Irrespective of its origin and posttranslational modifications fetuin-A prevents precipitation of hydroxyapatite <italic>in vitro</italic> (<xref rid="r201" ref-type="bibr">201</xref>). <italic>In vivo</italic>, serum fetuin A levels are lower in patients with CKD (<xref rid="r202" ref-type="bibr">202</xref>), and ectopic calcification is seen in fetuin A -/- mice (<xref rid="r200" ref-type="bibr">200</xref>).</p><p>Cardiovascular complications are significantly increased in patients with CKD (<xref rid="r203" ref-type="bibr">203</xref>,<xref rid="r204" ref-type="bibr">204</xref>), and coronary artery calcification is considered an independent predictor of future cardiac event (<xref rid="r205" ref-type="bibr">205</xref>). Carotid atherosclerotic lesions of CKD patients frequently become calcified (<xref rid="r206" ref-type="bibr">206</xref>,<xref rid="r207" ref-type="bibr">207</xref>). The calcified plaques are more unstable and contain significantly lower amounts of collagen. Serum levels of MMPs are significantly increased (<xref rid="r206" ref-type="bibr">206</xref>-<xref rid="r209" ref-type="bibr">209</xref>). Enhanced calcification and reduced collagen, perhaps through the activities of MMPs, lead to plaque instability and rupture (<xref rid="r206" ref-type="bibr">206</xref>).</p><p>It is our hypothesis that stone formation is yet another case of pathological biomineralization. Renal epithelial cells under OS may become osteogenic (<xref rid="r210" ref-type="bibr">210</xref>) as happens to vascular smooth muscle cells during vascular calcification (<xref rid="r177" ref-type="bibr">177</xref>). The production of OPN (<xref rid="r147" ref-type="bibr">147</xref>,<xref rid="r149" ref-type="bibr">149</xref>), MGP (<xref rid="r155" ref-type="bibr">155</xref>,<xref rid="r211" ref-type="bibr">211</xref>), collagen, fribronectin, osteonectin and fetuin (unpublished results) by renal epithelial cells of rats with experimentally induced CaOx nephrolithiasis are indicative of such a transition. The presence of OPN, osteocalcin, fibronectin, and collagen (<xref rid="r212" ref-type="bibr">212</xref>) in stone matrices also suggests their increased production and excretion into the urine. Renal crystals in a CaOx stone patient were also associated with bone sialoprotein (BSP) (<xref rid="r213" ref-type="bibr">213</xref>). EMT (<xref rid="r214" ref-type="bibr">214</xref>) as well as endothelial to mesenchymal transition (<xref rid="r215" ref-type="bibr">215</xref>,<xref rid="r216" ref-type="bibr">216</xref>) are regularly seen in the diseased kidneys. Mesenchymal stromal cells have the ability to differentiate into osteoblast. Interestingly, perivascular cells or pericyte were heavily stained for MGP in kidneys of hyperoxaluric rats (<xref rid="r211" ref-type="bibr">211</xref>).</p><p>Stone patients excrete lower amounts of fetuin-A (<xref rid="r217" ref-type="bibr">217</xref>), and more BMP-2 (<xref rid="r218" ref-type="bibr">218</xref>). Single nucleotide polymorphism of MGP gene is associated with CaOx kidney stones disease in the Japanese (<xref rid="r219" ref-type="bibr">219</xref>) and Chinese populations (<xref rid="r220" ref-type="bibr">220</xref>). Brush border membrane vesicles of renal epithelium promote nucleation of CaOx and CaP crystals <italic>in vivo</italic> as well as <italic>in vitro</italic> (<xref rid="r221" ref-type="bibr">221</xref>-<xref rid="r223" ref-type="bibr">223</xref>).</p></sec><sec><title>Discussion and concluding remarks</title><p>Supersaturation is the driving force behind crystallization and in most idiopathic CaOx stone formers hypercalciuria, hyperoxaluria and hypocitraturia alone or in combination are the main abnormalities. As a result, most stone therapies attempt to lower urinary supersaturation with respect to the crystallizing salt, yet 30% to 50% stone patients still continue to form stones (<xref rid="r224" ref-type="bibr">224</xref>). The risk of stone recurrence increases with each new episode (<xref rid="r225" ref-type="bibr">225</xref>), and nearly all stone formers are expected to form another provided they live long enough after the first episode (<xref rid="r226" ref-type="bibr">226</xref>). Even initial interventions do not stop stone recurrence in about fifty percent of the patients (<xref rid="r227" ref-type="bibr">227</xref>,<xref rid="r228" ref-type="bibr">228</xref>). Apparently stone formation does not occur by a passive unregulated physicochemical mechanism, but by a regulated process, similar to pathological biomineralization at other sites in the body including kidneys during vascular calcification (<xref rid="r172" ref-type="bibr">172</xref>, <xref rid="r229" ref-type="bibr">229</xref>,<xref rid="r230" ref-type="bibr">230</xref>).</p><p>Renal epithelial cells are exposed to high oxalate and/or CaOx and/or CaP crystals during stone formation. Experimental studies suggest that renal cellular exposure to high oxalate and/or CaOx or CaP crystals results in increased gene expression and production of molecules involved in tissue remodeling, inflammation and biomineralization (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>). Hyperoxaluria and CaOx crystal deposition induce rennin upregulation and generation of angiotensin II (<xref rid="r21" ref-type="bibr">21</xref>). Non phagocytic NADPH oxidase is activated (<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r22" ref-type="bibr">22</xref>,<xref rid="r231" ref-type="bibr">231</xref>,<xref rid="r232" ref-type="bibr">232</xref>) leading to the production of ROS (<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r231" ref-type="bibr">231</xref>,<xref rid="r233" ref-type="bibr">233</xref>) which is mediated by protein kinase C (PKC) (<xref rid="r232" ref-type="bibr">232</xref>). The activation involves phosphorylation of p47phox and translocation of Rac1 (<xref rid="r234" ref-type="bibr">234</xref>) and p47 phox to the membrane. P-38 MAPK/JNK transduction pathway is turned on (<xref rid="r235" ref-type="bibr">235</xref>,<xref rid="r236" ref-type="bibr">236</xref>), in addition to a variety of transcriptional and growth factors, including NF&#x003ba;B, AP-1, TGF&#x003b2;, become involved (<xref rid="r19" ref-type="bibr">19</xref>,<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r237" ref-type="bibr">237</xref>). There is the generation of secondary mediators such as isoprostanes, cytoplasmic phospholipase A2 and prostaglandins (<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r238" ref-type="bibr">238</xref>), and an increased production of chemoattractants such as MCP-1 (132-134) and crystallization modulators OPN (<xref rid="r134" ref-type="bibr">134</xref>), bikunin (<xref rid="r130" ref-type="bibr">130</xref>,<xref rid="r131" ref-type="bibr">131</xref>), &#x003b1;1-microglobulin (<xref rid="r152" ref-type="bibr">152</xref>), I&#x003b1;I (<xref rid="r151" ref-type="bibr">151</xref>) and prothrombin fragment-1 (<xref rid="r153" ref-type="bibr">153</xref>). Macrophages infiltrate the renal interstitium around the crystal deposits and phagocytic NADPH oxidase is also activated producing additional ROS resulting in inflammation, ECM production and fibrosis. Clinical data also provide the evidence of ROS generation and byproducts of their activities have been detected in both the kidneys and urine of the patients who form CaOx kidney stones.</p><p>The macromolecules, produced on exposure to oxalate and or various types of crystals through ROS dependent pathways, have dual functions (<xref ref-type="table" rid="t1"><italic>Table 1</italic></xref>). They regulate crystallization and are also involved in inflammatory processes. For example, HS, an inhibitor of crystal aggregation and attachment, regulates ECM production (<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r239" ref-type="bibr">239</xref>). Bikunin, a constituent of ITI, an inhibitor of CaOx crystal formation and attachment, is a proteinase inhibitor, and stabilizes the ECM (<xref rid="r240" ref-type="bibr">240</xref>-<xref rid="r242" ref-type="bibr">242</xref>). THP a modulator of CaOx crystal nucleation, growth and aggregation, is renoprotective and present in the renal interstitium during many tubulointerstitial diseases (<xref rid="r243" ref-type="bibr">243</xref>). OPN, an inhibitor of crystal nucleation, growth and aggregation, is also a chemoattractant involved in inflammation and fibrosis (<xref rid="r244" ref-type="bibr">244</xref>,<xref rid="r245" ref-type="bibr">245</xref>). Prothrombin is the precursor of thrombin and fragments 1 and 2 and plays a major role in the recruitment and activation of infiltrating immune cells. Fragment-1 is inhibitor of CaOx crystal growth and aggregation. Inflammation is primarily a protective response, therefore in the presence of impending mineralization, the body responds by producing macromolecules to inhibit crystallization and once crystal are formed to attract the inflammatory cells for their elimination (<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r161" ref-type="bibr">161</xref>,<xref rid="r246" ref-type="bibr">246</xref>). Crystals are phagocytosed and eliminated (<xref rid="r246" ref-type="bibr">246</xref>) or fenced in by a &#x0201c;wall&#x0201d; of macromolecules adsorbed to crystal surfaces rendering them harmless (<xref rid="r247" ref-type="bibr">247</xref>). This is likely the case with the interstitial plaques which are not attached to kidney stones and are common in the kidneys (<xref rid="r32" ref-type="bibr">32</xref>,<xref rid="r36" ref-type="bibr">36</xref>,<xref rid="r47" ref-type="bibr">47</xref>,<xref rid="r248" ref-type="bibr">248</xref>).</p><p>In summary, disturbance in the physiochemical milieu leads to the production of ROS and development of OS. ROS start a signaling cascade culminating in the production of macromolecules to inhibit crystal nucleation, growth and aggregation. In case of transitory disorder, either no crystal will form or crystals formed will stay small, well dispersed and passed out as crystalluria particles. In the face of persistent disorder, for example, hyperoxaluria, hypercalciuria and hypocitraturia, there is a loss of the balance between oxidative and antioxidative forces. ROS induced damage to the cells leads to cell death and the formation of membrane bound vesicles which support crystal nucleation (<xref rid="r221" ref-type="bibr">221</xref>,<xref rid="r249" ref-type="bibr">249</xref>). As a result crystallization inhibitors which are produced may be defective or damaged by exposure to the free radicals and thus not able to provide adequate protection resulting in crystal growth and aggregation. Cell death also leads to the formation of new cells to repopulate the epithelium. The surfaces of the new cells as well as the exposed basement membrane are conducive to crystal attachment and retention (<xref rid="r250" ref-type="bibr">250</xref>). Crystals retained in the terminal collecting ducts produce Randall&#x02019; plugs (<xref rid="r38" ref-type="bibr">38</xref>,<xref rid="r251" ref-type="bibr">251</xref>) which will act as stone nidus when exposed to the pelvic urine. A recent study has shown that plugging is quite common in stones patients (<xref rid="r37" ref-type="bibr">37</xref>).</p><p>As far as interstitial RPs are concerned, CaP crystals may originate in the tubular lumen, endocytose on the luminal side and exocytose on the basolateral side (<xref rid="r213" ref-type="bibr">213</xref>,<xref rid="r251" ref-type="bibr">251</xref>,<xref rid="r252" ref-type="bibr">252</xref>), to initiate the formation of the plaque. Alternatively renal epithelial cells under OS may become osteogenic (<xref rid="r210" ref-type="bibr">210</xref>). In addition to epithelium to osteoblast transformation, vascular endothelial cells may also become osteogenic (<xref rid="r216" ref-type="bibr">216</xref>). The transformed epithelial or endothelial cells will produce a membrane bound vesicle on the basolateral side of the epithelium. Renal epithelial cells have been shown to produce <italic>in vitro</italic>, CaP crystal deposits in the basement membrane under a variety of growing conditions (<xref rid="r213" ref-type="bibr">213</xref>,<xref rid="r253" ref-type="bibr">253</xref>,<xref rid="r254" ref-type="bibr">254</xref>).</p><p>Calcification of the membrane vesicles and their aggregation produces calcified RP&#x02019;s in the basement membrane of the renal tubules. ROS induced inflammation results in the formation of collagen which is deposited during fibrosis (<xref rid="r255" ref-type="bibr">255</xref>), and is an excellent nucleator of CaP (<xref rid="r182" ref-type="bibr">182</xref>,<xref rid="r256" ref-type="bibr">256</xref>) playing a critical role in biomineralization processes in the body (<xref rid="r182" ref-type="bibr">182</xref>,<xref rid="r257" ref-type="bibr">257</xref>). Calcification which starts with membrane bound vesicles is propagated through the mineralization of collagen (<xref rid="r35" ref-type="bibr">35</xref>). Mineralization of collagen leads to the growth of the plaque, which eventually reaches the papillary epithelium, ulcerates to the surface and develops into a stone nidus. Once exposed to the pelvic urine, the plaque is overgrown by CaOx crystals, and promotes the formation of an idiopathic CaOx kidney stone attached to the sub-epithelial RP (<xref rid="r258" ref-type="bibr">258</xref>).</p></sec>

Socio-economic predictors of performance in the Undergraduate Medicine and Health Sciences Admission Test (UMAT)

<sec><title>Background</title><p>Entry from secondary school to Australian and New Zealand undergraduate medical schools has since the late 1990&#x02019;s increasingly relied on the Undergraduate Medicine and Health Sciences Admission Test (UMAT) as one of the selection factors. The UMAT consists of 3 sections &#x02013; logical reasoning and problem solving (UMAT-1), understanding people (UMAT-2) and non-verbal reasoning (UMAT-3). One of the goals of using this test has been to enhance equity in the selection of students with the anticipation of an increase in the socioeconomic diversity in student cohorts. However there has been limited assessment as to whether UMAT performance itself might be influenced by socioeconomic background.</p></sec><sec><title>Methods</title><p>Between 2000 and 2012, 158,909 UMAT assessments were completed. From these, 118,085 cases have been identified where an Australian candidate was sitting for the first time during that period. Predictors of the total UMAT score, UMAT-1, UMAT-2 and UMAT-3 scores were entered into regression models and included gender, age, school type, language used at home, deciles for the Index of Relative Socioeconomic Advantage and Disadvantage score, the Accessibility/Remoteness Index of Australia (ARIA), self-identification as being of Aboriginal or Torres Strait Islander origin (ATSI) and current Australian state or territory of abode.</p></sec><sec><title>Results</title><p>A lower UMAT score was predicted by living in an area of relatively higher social disadvantage and lower social advantage. Other socioeconomic indicators were consistent with this observation with lower scores in those who self-identified as being of ATSI origin and higher scores evident in those from fee-paying independent school backgrounds compared to government schools. Lower scores were seen with increasing age, female gender and speaking any language other than English at home. Divergent effects of rurality were observed, with increased scores for UMAT-1 and UMAT-2, but decreasing UMAT-3 scores with increasing ARIA score. Significant state-based differences largely reflected substantial socio-demographic differences across Australian states and territories.</p></sec><sec><title>Conclusions</title><p>Better performance by Australian candidates in the UMAT is linked to an increase in socio-economic advantage and reduced disadvantage.This observation provides a firm foundation for selection processes at medical schools in Australia that have incorporated affirmative action pathways to quarantine places for students from areas of socio-economic disadvantage.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Puddey</surname><given-names>Ian B</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Ian.Puddey@uwa.edu.au</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Mercer</surname><given-names>Annette</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>Annette.Mercer@uwa.edu.au</email></contrib>

BMC Medical Education

<sec><title>Background</title><p>The Undergraduate Medicine and Health Sciences Admission Test (UMAT) has been developed to assist with the selection of students into medicine, dentistry and health science degree programs at an undergraduate level in Australian and New Zealand universities. It comprises 3 subtests which are developed each year by the Australian Council for Educational Research (ACER) on behalf of a group of Australian universities which form the UMAT Consortium [<xref ref-type="bibr" rid="B1">1</xref>]. The test is promoted as enhancing a focus on selection based on general attributes and non-academic personal skills gained through prior experience and learning and is designed to complement academic results used in selection processes. In section 1 (UMAT-1) candidates are required to exercise logical reasoning and problem solving skills using both inductive and deductive reasoning with an emphasis on logical argument in working to a solution. The construct and the nature of the items in this section have been consistent over the years. In section 2 (UMAT-2) the emphasis has always been on assessing empathy and emotional intelligence with candidates required to show an understanding of the thoughts, feelings, behaviour and intentions portrayed within each question. Between 2003 and 2004 the section was changed in name from Interaction Skills to Understanding People together with a change in the format of some items. Section 3 (UMAT-3) evaluates a candidate&#x02019;s non-verbal reasoning skills. It also changed at the same time as Section 2, when the use of &#x02018;embedded figures&#x02019; was removed with items subsequently consisting solely of patterns or sequences of shapes. This was consistent with recent literature on the construct and the desire to obtain a measure of cognitive ability which was relatively independent of language ability and specific cultural knowledge.</p><p>Changes in selection strategies for admission to medical schools that have incorporated aptitude tests have at least in part been in the belief that they might serve to redress the under representation of students from a lower socio-economic background in medical schools [<xref ref-type="bibr" rid="B2">2</xref>-<xref ref-type="bibr" rid="B4">4</xref>]. This imbalance has been longstanding and is consistently reported globally [<xref ref-type="bibr" rid="B5">5</xref>-<xref ref-type="bibr" rid="B7">7</xref>]. It is attributed to a lower number of applicants from those from a more disadvantaged socio-economic background. For example, in the UK those applying to study medicine are more likely to be of higher socio-economic status and from fee paying independent secondary schools and in particular independent schools that exhibit higher levels of academic achievement [<xref ref-type="bibr" rid="B8">8</xref>]. As a further example, only 8% of applicants to the 1999 University of Newcastle medical course originated from postcodes linked to those in the lowest socio-economic quartile [<xref ref-type="bibr" rid="B9">9</xref>].</p><p>In a review of potential approaches to widening access for a broader spread of students across the socio-economic spectrum, Powis et al. [<xref ref-type="bibr" rid="B10">10</xref>] recommend the application of tests that measure a range of cognitive skills and non-cognitive personal qualities, with the implication that these tests are diversity neutral. However, with respect to the UMAT we have previously reported that a socio-economic index linked to the secondary school of origin of students entering our medical school predicted higher UMAT scores in those who attended schools with higher socio-economic advantage [<xref ref-type="bibr" rid="B5">5</xref>]. We also reported that the introduction of both a structured interview and the score from UMAT had not served to increase socio-economic diversity in secondary school leaver entrants to our medical course [<xref ref-type="bibr" rid="B5">5</xref>].</p><p>Others who have introduced attribute-based admission criteria as an alternative to grades-based selection in an attempt to increase medical student socio-economic diversity have also failed to see any significant change [<xref ref-type="bibr" rid="B11">11</xref>]. These observations question the assumption that simply utilising measures of aptitude as an additional selection tool can help overcome the potential for socio-economic background to influence selection processes that are based entirely on secondary school results or grade point average during tertiary studies. Furthermore, they raise the question as to whether there may be potential socio-economic influences on performance in aptitude tests that may have been previously overlooked. In this respect, recent reports have indicated that socio-economic factors may be determinants of performance in both the Medical College Admission Test (MCAT) [<xref ref-type="bibr" rid="B12">12</xref>], widely used for medical student selection in North America, and the UK Clinical Aptitude test (UKCAT), used in medical student selection in the UK since 2006 [<xref ref-type="bibr" rid="B13">13</xref>].</p><p>An association of a number of demographic variables with overall performance in the UMAT and each of its sections has been regularly observed in annual reports on performance prepared for ACER on behalf of the UMAT Consortium [<xref ref-type="bibr" rid="B1">1</xref>] and have also been reported by others [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Generally, males perform better than females in total UMAT score, UMAT-1 and UMAT-3 but less well than females in UMAT-2. Older students underperform relative to their younger counterparts and those from non-English speaking backgrounds perform less well than those from an English speaking background. Those from rural backgrounds perform less well in UMAT-1 and UMAT-3 but not in UMAT-2. Mulivariate linear regression suggests these associations are relatively weak, accounting for only 4.1% of the variance for total UMAT score, 5.4% of the variance in UMAT-1, 11.1% for UMAT-2 and 3.7% for UMAT-3 [<xref ref-type="bibr" rid="B1">1</xref>]. However, these annual reports have not considered the potential contribution of socio-economic background in predicting UMAT performance.</p><p>We have therefore identified all Australian candidates who sat the UMAT on a first occasion between 2000 and 2012 and linked their postcodes to the Socio-Economic Indexes for Areas generated from the 2006 census data [<xref ref-type="bibr" rid="B16">16</xref>]. We have then investigated the associations of relative socio-economic advantage and disadvantage scores with total UMAT score or performance in each of its 3 sections and now report these findings in relation to other already established demographic predictors of UMAT performance. As a further window on any potential socio-economic influences we also report UMAT performance in relation to background secondary school, rural background or self-identification as an Aboriginal or Torres Strait Islander (ATSI).</p></sec><sec sec-type="methods"><title>Methods</title><p>The UMAT results for 158,909 candidates who sat the UMAT between 2000 and 2012 were obtained from ACER. From these, 118,085 cases were identified where an Australian candidate was sitting for the first time during that period. Demographic data collected on enrolment for the UMAT included date of birth, gender, postal address, language spoken at home, type of high school attended and self-identification as being of ATSI origin.</p><p>Language spoken at home was classified according to the Australian Standard Classification of Languages (ASCL), 2011 [<xref ref-type="bibr" rid="B17">17</xref>]. For multivariate analysis this was collapsed into 4 groups &#x02013; English, European languages, Asian languages and all Other languages. Type of school was classified into one of 5 groups &#x02013; government (publicly funded), independent (fee paying), Catholic, Technical and Further Education institutions (TAFE &#x02013; public provider of predominantly vocational tertiary education courses) and Other.</p><p>Socio-economic status was imputed from each candidate&#x02019;s correspondence postcode at the time of first sitting the UMAT by linking it to the Socio-Economic Indexes for Areas (SEIFA) generated from the 2006 census data [<xref ref-type="bibr" rid="B16">16</xref>]. We used the deciles generated from the Index of Relative Socioeconomic Advantage and Disadvantage Score (IRSAD Score) as the index of choice for this study. It is derived by principal components analysis of 21 separate variables such as low or high income, internet connection, unemployment, occupation and education. It does not include age or self-identification as of ATSI origin. The score is standardised against a mean of 1000 with a standard deviation of 100 with two thirds of SEIFA scores falling between 900 and 1100.</p><p>Remoteness of an area and relative access to infrastructure are also not included in the information used to construct SEIFA codes [<xref ref-type="bibr" rid="B16">16</xref>]. We have therefore also linked each candidate&#x02019;s postcode to the Accessibility/Remoteness Index of Australia (ARIA) [<xref ref-type="bibr" rid="B18">18</xref>]. ARIA calculates remoteness as accessibility to some 201 service centres based on road distances. Remoteness values for 11,340 populated localities are derived from the road distance to service centres in four categories. Remoteness values for each populated locality are then interpolated to a 1&#x000a0;km grid that covers the whole of Australia and averages calculated for larger areas. ARIA values are grouped into one of five categories within the 0 &#x02013; 12 continuous variable: Highly Accessible (ARIA score 0&#x02013;1.84) - relatively unrestricted accessibility to a wide range of goods and services and opportunities for social interaction, Accessible (ARIA score &#x0003e;1.84 - 3.51) - some restrictions to accessibility of some goods, services and opportunities for social interaction, Moderately Accessible (ARIA score &#x0003e;3.51 -5.80) - significantly restricted accessibility of goods, services and opportunities for social interaction. Remote (ARIA score &#x0003e;5.80 - 9.08) - very restricted accessibility of goods, services and opportunities for social interaction, and Very Remote (ARIA score &#x0003e;9.08 - 12) - very little accessibility of goods, services and opportunities for social interaction.</p><p>Even though the total UMAT score alone is usually used in the ranking process at medical schools in Australia, each of the three component scores, UMAT-1 (Logical reasoning and problem solving), UMAT-2 (Understanding people) and UMAT-3 (Non-verbal reasoning) have different and independent constructs [<xref ref-type="bibr" rid="B19">19</xref>] and have therefore been independently evaluated in this study together with the total score. They are presented as percentile values to provide a more meaningful understanding of the relative magnitude of the associations of each score with predictor socio-demographic variables.</p><p>The project has been approved by the Human Research Ethics Committee at the University of Western Australia (file reference RA/4/1/2178).</p><sec><title>Statistics</title><p>Univariate comparisons of each demographic characteristic or each selection criteria utilised either independent sample T-tests, one-way analysis of variance (with post-hoc comparisons by Bonferroni correction), or cross-tabulation with generation of the chi-squared statistic, as appropriate. Multivariate analyses utilised linear regression to assess the independent relationships of total UMAT, UMAT-1, UMAT-2 and UMAT-3 with age, gender, type of secondary school, language spoken at home, country of origin, IRSAD decile, ARIA accessibility index and self-identification as ATSI. Further adjustment of each linear regression model by inclusion of 11 dummy variables for each year the UMAT was sat resulted in minimal change in both the B regression coefficients for each of the socio-economic predictor variables and the total variance explained by each model and have therefore not been included in the final analyses. All analyses were carried out utilising IBM SPSS Statistics Version 20.0.</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>Age</title><p>Most Australians sitting the UMAT were school leavers with 84% of the sample 17 or 18&#x000a0;years of age. Performance in the UMAT decreased linearly with age (Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref>) with those 30&#x000a0;yr and older predicted in multivariate linear regression (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>) to have a score 22.7 percentiles (95% CI 20.7,24.6) lower than those&#x02009;&#x02264;&#x02009;16&#x000a0;yr in age (P&#x02009;&#x0003c;&#x02009;0.001). This trend was present for UMAT-1 (27.2, 95% CI 25.3, 29.1, P&#x02009;&#x0003c;&#x02009;0.001) (Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>), not present for UMAT-2 (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>) and present for UMAT-3 (29.4, 95% CI 27.4, 31.4, P&#x02009;&#x0003c;&#x02009;0.001) (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>). For UMAT-2, scores were significantly lower at all ages compared to those aged&#x02009;&#x02264;&#x02009;16&#x000a0;yr (P&#x02009;&#x0003c;&#x02009;0.001) except in those 30&#x000a0;yr and older where scores were not significantly different (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by age for all Australian subjects first sitting the UMAT from 2000 to 2012 (N&#x02009;=&#x02009;118,029).</bold> (*** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-1"/></fig><table-wrap position="float" id="T1"><label>Table 1</label><caption><p><bold>Multivariate linear regression for total UMAT score (N&#x02009;=&#x02009;109,880, r</bold>^{<bold>2</bold>}&#x02009;<bold>=&#x02009;0.120)</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Predictor variable (reference group in brackets)</italic> </bold></th><th align="center" valign="bottom"><bold>B Coefficient</bold></th><th align="center" valign="bottom"><bold>95% CI for B</bold></th><th align="center" valign="bottom"><bold>Beta</bold></th><th align="center" valign="bottom"><bold>P-Value</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><bold> <italic>Age</italic> </bold><italic>(&#x02264; 16&#x000a0;yr&#x000a0;yr of age)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>17&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;8.7<hr/></td><td align="center" valign="bottom">&#x02212;10.2, -7.2<hr/></td><td align="center" valign="bottom">&#x02212;0.147<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>18&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.0<hr/></td><td align="center" valign="bottom">&#x02212;13.5, -10.5<hr/></td><td align="center" valign="bottom">&#x02212;0.207<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>19&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;19.4<hr/></td><td align="center" valign="bottom">&#x02212;21.0, -17.7<hr/></td><td align="center" valign="bottom">&#x02212;0.133<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>20 - 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;19.3<hr/></td><td align="center" valign="bottom">&#x02212;20.9, -17.7<hr/></td><td align="center" valign="bottom">&#x02212;0.183<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>&#x0003e; 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;22.7<hr/></td><td align="center" valign="bottom">&#x02212;24.6, -20.7<hr/></td><td align="center" valign="bottom">&#x02212;0.100<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Gender</italic> </bold><italic>(Females)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Males</italic><hr/></td><td align="center" valign="bottom">3.6<hr/></td><td align="center" valign="bottom">3.3, 3.9<hr/></td><td align="center" valign="bottom">0.062<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Language spoken at home</italic> </bold><italic>(English)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Asian languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;10.4<hr/></td><td align="center" valign="bottom">&#x02212;10.8, -10.0<hr/></td><td align="center" valign="bottom">&#x02212;0.159<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>European languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.7<hr/></td><td align="center" valign="bottom">&#x02212;13.8, -11.6<hr/></td><td align="center" valign="bottom">&#x02212;0.063<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Other languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;18.4<hr/></td><td align="center" valign="bottom">&#x02212;19.6, -17.2<hr/></td><td align="center" valign="bottom">&#x02212;0.083<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>School Type</italic> </bold><italic>(Government)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Catholic</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.0<hr/></td><td align="center" valign="bottom">&#x02212;5.5, -4.6<hr/></td><td align="center" valign="bottom">&#x02212;0.068<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Independent</italic><hr/></td><td align="center" valign="bottom">3.9<hr/></td><td align="center" valign="bottom">3.5, 4.3<hr/></td><td align="center" valign="bottom">0.064<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Other</italic><hr/></td><td align="center" valign="bottom">&#x02212;10.7<hr/></td><td align="center" valign="bottom">&#x02212;11.9, -9.4<hr/></td><td align="center" valign="bottom">&#x02212;0.049<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>TAFE</italic><hr/></td><td align="center" valign="bottom">&#x02212;13.3<hr/></td><td align="center" valign="bottom">&#x02212;15.7, -10.8<hr/></td><td align="center" valign="bottom">&#x02212;0.030<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>State or territory</italic> </bold><italic>(NSW)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>ACT</italic><hr/></td><td align="center" valign="bottom">3.1<hr/></td><td align="center" valign="bottom">1.7, 4.5<hr/></td><td align="center" valign="bottom">0.013<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>NT</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.1<hr/></td><td align="center" valign="bottom">&#x02212;14.4, -9.8<hr/></td><td align="center" valign="bottom">&#x02212;0.031<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>QLD</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.9<hr/></td><td align="center" valign="bottom">&#x02212;6.5, -5.3<hr/></td><td align="center" valign="bottom">&#x02212;0.065<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>SA</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.0<hr/></td><td align="center" valign="bottom">&#x02212;9.6, -8.3<hr/></td><td align="center" valign="bottom">&#x02212;0.089<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>TAS</italic><hr/></td><td align="center" valign="bottom">1.3<hr/></td><td align="center" valign="bottom">0.2, 2.5<hr/></td><td align="center" valign="bottom">0.007<hr/></td><td align="center" valign="bottom">0.022<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>VIC</italic><hr/></td><td align="center" valign="bottom">&#x02212;6.6<hr/></td><td align="center" valign="bottom">&#x02212;7.0, -6.2<hr/></td><td align="center" valign="bottom">&#x02212;0.110<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>WA</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.9<hr/></td><td align="center" valign="bottom">&#x02212;6.5, -5.3<hr/></td><td align="center" valign="bottom">&#x02212;0.062<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>IRSAD Decile</italic> </bold><italic>(Deciles 1 and 2)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 3 and 4</italic><hr/></td><td align="center" valign="bottom">3.9<hr/></td><td align="center" valign="bottom">3.2, 4.9<hr/></td><td align="center" valign="bottom">0.035<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 5 and 6</italic><hr/></td><td align="center" valign="bottom">4.5<hr/></td><td align="center" valign="bottom">3.9, 5.4<hr/></td><td align="center" valign="bottom">0.053<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 7 and 8</italic><hr/></td><td align="center" valign="bottom">7.1<hr/></td><td align="center" valign="bottom">6.6, 8.0<hr/></td><td align="center" valign="bottom">0.102<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 9 and 10</italic><hr/></td><td align="center" valign="bottom">13.0<hr/></td><td align="center" valign="bottom">12.4, 13.8<hr/></td><td align="center" valign="bottom">0.224<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>ARIA Accessibility code</italic> </bold><italic>(Highly accessible)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Accessible</italic><hr/></td><td align="center" valign="bottom">3.0<hr/></td><td align="center" valign="bottom">2.2, 3.8<hr/></td><td align="center" valign="bottom">0.022<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Moderately accessible</italic><hr/></td><td align="center" valign="bottom">1.9<hr/></td><td align="center" valign="bottom">0.4, 3.4<hr/></td><td align="center" valign="bottom">0.007<hr/></td><td align="center" valign="bottom">0.015<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Remote</italic><hr/></td><td align="center" valign="bottom">1.9<hr/></td><td align="center" valign="bottom">&#x02212;1.4, 3.9<hr/></td><td align="center" valign="bottom">0.004<hr/></td><td align="center" valign="bottom">0.161<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Very remote</italic><hr/></td><td align="center" valign="bottom">&#x02212;1.5<hr/></td><td align="center" valign="bottom">&#x02212;6.8, 4.7<hr/></td><td align="center" valign="bottom">&#x02212;0.001<hr/></td><td align="center" valign="bottom">0.614<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Aboriginal &#x00026; Torres Strait Islander</italic> </bold><italic>(non-ATSI)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left">&#x02003;&#x02009;<italic>ATSI</italic></td><td align="center">&#x02212;9.6</td><td align="center">&#x02212;11.9, -7.3</td><td align="center">&#x02212;0.023</td><td align="center">&#x0003c;0.001</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T2"><label>Table 2</label><caption><p><bold>Multivariate linear regression for UMAT-1 score (Logical reasoning and problem solving) (N&#x02009;=&#x02009;109,880, r</bold>^{<bold>2</bold>}&#x02009;<bold>=&#x02009;0.135)</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Predictor variable (reference group in brackets)</italic> </bold></th><th align="center" valign="bottom"><bold>B Coefficient</bold></th><th align="center" valign="bottom"><bold>95% CI for B</bold></th><th align="center" valign="bottom"><bold>Beta</bold></th><th align="center" valign="bottom"><bold>P-Value</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><bold> <italic>Age</italic> </bold><italic>(&#x02264; 16&#x000a0;yr&#x000a0;yr of age)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;17&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.2<hr/></td><td align="center" valign="bottom">&#x02212;10.7, -7.7<hr/></td><td align="center" valign="bottom">&#x02212;0.156<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;18&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.5<hr/></td><td align="center" valign="bottom">&#x02212;14.0, -11.1<hr/></td><td align="center" valign="bottom">&#x02212;0.216<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;19&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;18.9<hr/></td><td align="center" valign="bottom">&#x02212;20.5, -17.2<hr/></td><td align="center" valign="bottom">&#x02212;0.130<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;20 - 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;20.3<hr/></td><td align="center" valign="bottom">&#x02212;21.9, -18.7<hr/></td><td align="center" valign="bottom">&#x02212;0.192<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;&#x0003e; 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;27.2<hr/></td><td align="center" valign="bottom">&#x02212;29.1, -25.3<hr/></td><td align="center" valign="bottom">&#x02212;0.120<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Gender</italic> </bold><italic>(Females)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Males</italic><hr/></td><td align="center" valign="bottom">7.0<hr/></td><td align="center" valign="bottom">6.7, 7.3<hr/></td><td align="center" valign="bottom">0.119<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Language spoken at home</italic> </bold><italic>(English)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Asian&#x000a0;languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.5<hr/></td><td align="center" valign="bottom">&#x02212;12.9, -12.1<hr/></td><td align="center" valign="bottom">&#x02212;0.192<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;European languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.9<hr/></td><td align="center" valign="bottom">&#x02212;14.0, -11.8<hr/></td><td align="center" valign="bottom">&#x02212;0.064<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Other languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;21.2<hr/></td><td align="center" valign="bottom">&#x02212;22.4, -19.9<hr/></td><td align="center" valign="bottom">&#x02212;0.096<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>School</italic> </bold><italic>(Government)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Catholic</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.5<hr/></td><td align="center" valign="bottom">&#x02212;4.9, -4.0<hr/></td><td align="center" valign="bottom">&#x02212;0.061<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Independent</italic><hr/></td><td align="center" valign="bottom">3.6<hr/></td><td align="center" valign="bottom">3.2, 4.0<hr/></td><td align="center" valign="bottom">0.060<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Other</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.4<hr/></td><td align="center" valign="bottom">&#x02212;10.6, -8.1<hr/></td><td align="center" valign="bottom">&#x02212;0.043<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;TAFE</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.7<hr/></td><td align="center" valign="bottom">&#x02212;15.2, -10.3<hr/></td><td align="center" valign="bottom">&#x02212;0.029<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>State or territory</italic> </bold><italic>(NSW)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;ACT</italic><hr/></td><td align="center" valign="bottom">2.6<hr/></td><td align="center" valign="bottom">1.2, 3.4<hr/></td><td align="center" valign="bottom">0.011<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;NT</italic><hr/></td><td align="center" valign="bottom">&#x02212;12.7<hr/></td><td align="center" valign="bottom">&#x02212;15.0, -10.5<hr/></td><td align="center" valign="bottom">&#x02212;0.033<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;QLD</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.4<hr/></td><td align="center" valign="bottom">&#x02212;6.0, -4.8<hr/></td><td align="center" valign="bottom">&#x02212;0.059<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;SA</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.8<hr/></td><td align="center" valign="bottom">&#x02212;10.4, -9.2<hr/></td><td align="center" valign="bottom">&#x02212;0.098<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;TAS</italic><hr/></td><td align="center" valign="bottom">1.3<hr/></td><td align="center" valign="bottom">0.1, 2.4<hr/></td><td align="center" valign="bottom">0.006<hr/></td><td align="center" valign="bottom">0.028<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;VIC</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.0<hr/></td><td align="center" valign="bottom">&#x02212;5.4, -4.6<hr/></td><td align="center" valign="bottom">&#x02212;0.082<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;WA</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.2<hr/></td><td align="center" valign="bottom">&#x02212;4.8, -3.6<hr/></td><td align="center" valign="bottom">&#x02212;0.044<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>IRSAD Decile</italic> </bold><italic>(Deciles 1 and 2)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Deciles 3 and 4</italic><hr/></td><td align="center" valign="bottom">3.9<hr/></td><td align="center" valign="bottom">3.1, 4.8<hr/></td><td align="center" valign="bottom">0.035<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Deciles 5 and 6</italic><hr/></td><td align="center" valign="bottom">4.3<hr/></td><td align="center" valign="bottom">3.5, 5.1<hr/></td><td align="center" valign="bottom">0.051<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Deciles 7 and 8</italic><hr/></td><td align="center" valign="bottom">6.6<hr/></td><td align="center" valign="bottom">5.9, 7.3<hr/></td><td align="center" valign="bottom">0.094<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Deciles 9 and 10</italic><hr/></td><td align="center" valign="bottom">11.5<hr/></td><td align="center" valign="bottom">10.8, 12.2<hr/></td><td align="center" valign="bottom">0.199<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>ARIA Accessibility code</italic> </bold><italic>(Highly accessible)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Accessible</italic><hr/></td><td align="center" valign="bottom">4.2<hr/></td><td align="center" valign="bottom">3.4, 5.0<hr/></td><td align="center" valign="bottom">0.031<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Moderately accessible</italic><hr/></td><td align="center" valign="bottom">3.3<hr/></td><td align="center" valign="bottom">1.8, 4.8<hr/></td><td align="center" valign="bottom">0.013<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Remote</italic><hr/></td><td align="center" valign="bottom">2.7<hr/></td><td align="center" valign="bottom">0.1, 5.3<hr/></td><td align="center" valign="bottom">0.006<hr/></td><td align="center" valign="bottom">0.040<hr/></td></tr><tr><td align="left" valign="bottom"><italic>&#x02003;&#x000a0;Very remote</italic><hr/></td><td align="center" valign="bottom">0.4<hr/></td><td align="center" valign="bottom">&#x02212;5.2, 6.0<hr/></td><td align="center" valign="bottom">0.000<hr/></td><td align="center" valign="bottom">0.888<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Aboriginal &#x00026; Torres Strait Islander</italic> </bold><italic>(non-ATSI)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left"><italic>&#x02003;&#x000a0;ATSI</italic></td><td align="center">&#x02212;8.9</td><td align="center">&#x02212;11.2, -6.7</td><td align="center">&#x02212;0.022</td><td align="center">&#x0003c;0.001</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p><bold>Multivariate linear regression for UMAT-2 score (Understanding people) (N&#x02009;=&#x02009;109,880, r</bold>^{<bold>2</bold>}&#x02009;<bold>=&#x02009;0.113)</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Predictor variable (reference group in brackets)</italic> </bold></th><th align="center" valign="bottom"><bold>B Coefficient</bold></th><th align="center" valign="bottom"><bold>95% CI for B</bold></th><th align="center" valign="bottom"><bold>Beta</bold></th><th align="center" valign="bottom"><bold>P-Value</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><bold> <italic>Age</italic> </bold><italic>(&#x02264; 16&#x000a0;yr&#x000a0;yr of age)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>17&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;3.8<hr/></td><td align="center" valign="bottom">&#x02212;5.3, -2.3<hr/></td><td align="center" valign="bottom">&#x02212;0.064<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>18&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.1<hr/></td><td align="center" valign="bottom">&#x02212;6.6, -3.6<hr/></td><td align="center" valign="bottom">&#x02212;0.087<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>19&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;11.1<hr/></td><td align="center" valign="bottom">&#x02212;12.7, -9.4<hr/></td><td align="center" valign="bottom">&#x02212;0.076<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>20 - 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.7<hr/></td><td align="center" valign="bottom">&#x02212;6.3, -3.1<hr/></td><td align="center" valign="bottom">&#x02212;0.044<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>&#x0003e; 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">1.5<hr/></td><td align="center" valign="bottom">&#x02212;0.5, 3.4<hr/></td><td align="center" valign="bottom">0.006<hr/></td><td align="center" valign="bottom">0.143<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Gender</italic> </bold><italic>(Females)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Males</italic><hr/></td><td align="center" valign="bottom">&#x02212;7.3<hr/></td><td align="center" valign="bottom">&#x02212;7.6, -7.1<hr/></td><td align="center" valign="bottom">&#x02212;0.125<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Language spoken at home</italic> </bold><italic>(English)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Asian languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;15.6<hr/></td><td align="center" valign="bottom">&#x02212;15.9, -15.2<hr/></td><td align="center" valign="bottom">&#x02212;0.238<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>European languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.7<hr/></td><td align="center" valign="bottom">&#x02212;10.8, -8.5<hr/></td><td align="center" valign="bottom">&#x02212;0.048<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Other languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;14.9<hr/></td><td align="center" valign="bottom">&#x02212;16.1, -13.7<hr/></td><td align="center" valign="bottom">&#x02212;0.067<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>School</italic> </bold><italic>(Government)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Catholic</italic><hr/></td><td align="center" valign="bottom">&#x02212;1.3<hr/></td><td align="center" valign="bottom">&#x02212;1.8, -0.9<hr/></td><td align="center" valign="bottom">&#x02212;0.018<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Independent</italic><hr/></td><td align="center" valign="bottom">3.4<hr/></td><td align="center" valign="bottom">3.0, 3.8<hr/></td><td align="center" valign="bottom">0.056<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Other</italic><hr/></td><td align="center" valign="bottom">&#x02212;10.1<hr/></td><td align="center" valign="bottom">&#x02212;11.3, -8.8<hr/></td><td align="center" valign="bottom">&#x02212;0.046<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>TAFE</italic><hr/></td><td align="center" valign="bottom">&#x02212;10.7<hr/></td><td align="center" valign="bottom">&#x02212;13.2, -8.2<hr/></td><td align="center" valign="bottom">&#x02212;0.025<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>State or territory</italic> </bold><italic>(NSW)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>ACT</italic><hr/></td><td align="center" valign="bottom">1.9<hr/></td><td align="center" valign="bottom">0.5, 3.2<hr/></td><td align="center" valign="bottom">0.008<hr/></td><td align="center" valign="bottom">0.008<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>NT</italic><hr/></td><td align="center" valign="bottom">&#x02212;8.6<hr/></td><td align="center" valign="bottom">&#x02212;10.9, -6.2<hr/></td><td align="center" valign="bottom">&#x02212;0.022<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>QLD</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.6<hr/></td><td align="center" valign="bottom">&#x02212;5.2, -4.0<hr/></td><td align="center" valign="bottom">&#x02212;0.051<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>ISA</italic><hr/></td><td align="center" valign="bottom">&#x02212;6.3<hr/></td><td align="center" valign="bottom">&#x02212;6.9, -5.7<hr/></td><td align="center" valign="bottom">&#x02212;0.063<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>TAS</italic><hr/></td><td align="center" valign="bottom">1.4<hr/></td><td align="center" valign="bottom">0.3, 2.6<hr/></td><td align="center" valign="bottom">0.007<hr/></td><td align="center" valign="bottom">0.014<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>IVIC</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.7<hr/></td><td align="center" valign="bottom">&#x02212;6.1, -5.3<hr/></td><td align="center" valign="bottom">&#x02212;0.094<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>WA</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.5<hr/></td><td align="center" valign="bottom">&#x02212;6.1, -4.9<hr/></td><td align="center" valign="bottom">&#x02212;0.057<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>IRSAD Decile</italic> </bold><italic>(Deciles 1 and 2)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Deciles 3 and 4</italic><hr/></td><td align="center" valign="bottom">2.9<hr/></td><td align="center" valign="bottom">2.1, 3.8<hr/></td><td align="center" valign="bottom">0.026<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Deciles 5 and 6</italic><hr/></td><td align="center" valign="bottom">3.6<hr/></td><td align="center" valign="bottom">2.8, 4.4<hr/></td><td align="center" valign="bottom">0.042<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Deciles 7 and 8</italic><hr/></td><td align="center" valign="bottom">5.4<hr/></td><td align="center" valign="bottom">4.7, 6.1<hr/></td><td align="center" valign="bottom">0.077<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Deciles 9 and 10</italic><hr/></td><td align="center" valign="bottom">8.1<hr/></td><td align="center" valign="bottom">7.4, 8.8<hr/></td><td align="center" valign="bottom">0.140<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>ARIA Accessibility code</italic> </bold><italic>(Highly accessible)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Accessible</italic><hr/></td><td align="center" valign="bottom">3.7<hr/></td><td align="center" valign="bottom">2.8, 4.5<hr/></td><td align="center" valign="bottom">0.027<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Moderately accessible</italic><hr/></td><td align="center" valign="bottom">2.6<hr/></td><td align="center" valign="bottom">1.0, 4.1<hr/></td><td align="center" valign="bottom">0.010<hr/></td><td align="center" valign="bottom">0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Remote</italic><hr/></td><td align="center" valign="bottom">4.4<hr/></td><td align="center" valign="bottom">1.8, 7.1<hr/></td><td align="center" valign="bottom">0.010<hr/></td><td align="center" valign="bottom">0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Very Remote</italic><hr/></td><td align="center" valign="bottom">1.4<hr/></td><td align="center" valign="bottom">&#x02212;4.3, 7.0<hr/></td><td align="center" valign="bottom">0.001<hr/></td><td align="center" valign="bottom">0.635<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Aboriginal &#x00026; Torres Strait Islander</italic> </bold><italic>(non-ATSI)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left">&#x02003;&#x000a0;<italic>ATSI</italic></td><td align="center">&#x02212;6.2</td><td align="center">&#x02212;8.5, -3.9</td><td align="center">&#x02212;0.015</td><td align="center">&#x0003c;0.001</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T4"><label>Table 4</label><caption><p><bold>Multivariate linear regression for UMAT-3 score (Non-verbal reasoning) (N&#x02009;=&#x02009;109,880, r</bold>^{<bold>2</bold>}&#x02009;<bold>=&#x02009;0.098)</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Predictor variable (reference group in brackets)</italic> </bold></th><th align="center" valign="bottom"><bold>B Coefficient</bold></th><th align="center" valign="bottom"><bold>95% CI for B</bold></th><th align="center" valign="bottom"><bold>Beta</bold></th><th align="center" valign="bottom"><bold>P-Value</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><bold> <italic>Age</italic> </bold><italic>(&#x02264; 16&#x000a0;yr&#x000a0;yr of age)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>17&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;7.2<hr/></td><td align="center" valign="bottom">&#x02212;8.8, -5.7<hr/></td><td align="center" valign="bottom">&#x02212;0.122<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>18&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;10.4<hr/></td><td align="center" valign="bottom">&#x02212;11.9, -8.8<hr/></td><td align="center" valign="bottom">&#x02212;0.178<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>19&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;15.6<hr/></td><td align="center" valign="bottom">&#x02212;17.3, -13.9<hr/></td><td align="center" valign="bottom">&#x02212;0.107<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>20 - 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;21.0<hr/></td><td align="center" valign="bottom">&#x02212;22.6, -19.4<hr/></td><td align="center" valign="bottom">&#x02212;0.198<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>&#x0003e; 30&#x000a0;yr</italic><hr/></td><td align="center" valign="bottom">&#x02212;29.4<hr/></td><td align="center" valign="bottom">&#x02212;31.4, -27.4<hr/></td><td align="center" valign="bottom">&#x02212;0.130<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Gender</italic> </bold><italic>(Females)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Males</italic><hr/></td><td align="center" valign="bottom">8.1<hr/></td><td align="center" valign="bottom">7.8, 8.4<hr/></td><td align="center" valign="bottom">0.138<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Language spoken at home</italic> </bold><italic>(English)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Asian languages</italic><hr/></td><td align="center" valign="bottom">2.4<hr/></td><td align="center" valign="bottom">2.0, 2.8<hr/></td><td align="center" valign="bottom">0.037<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>European languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;7.2<hr/></td><td align="center" valign="bottom">&#x02212;8.3, -6.0<hr/></td><td align="center" valign="bottom">&#x02212;0.036<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Other languages</italic><hr/></td><td align="center" valign="bottom">&#x02212;8.9<hr/></td><td align="center" valign="bottom">&#x02212;10.1, -7.6<hr/></td><td align="center" valign="bottom">&#x02212;0.040<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>School</italic> </bold><italic>(Government)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Catholic</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.4<hr/></td><td align="center" valign="bottom">&#x02212;5.8, -4.9<hr/></td><td align="center" valign="bottom">&#x02212;0.073<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Independent</italic><hr/></td><td align="center" valign="bottom">2.4<hr/></td><td align="center" valign="bottom">2.0, 2.8<hr/></td><td align="center" valign="bottom">0.039<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Other</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.9<hr/></td><td align="center" valign="bottom">&#x02212;7.2, -4.7<hr/></td><td align="center" valign="bottom">&#x02212;0.027<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>TAFE</italic><hr/></td><td align="center" valign="bottom">&#x02212;9.1<hr/></td><td align="center" valign="bottom">&#x02212;11.6, -6.6<hr/></td><td align="center" valign="bottom">&#x02212;0.021<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>State or territory</italic> </bold><italic>(NSW)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>ACT</italic><hr/></td><td align="center" valign="bottom">2.0<hr/></td><td align="center" valign="bottom">0.6, 3.3<hr/></td><td align="center" valign="bottom">0.008<hr/></td><td align="center" valign="bottom">0.005<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>NT</italic><hr/></td><td align="center" valign="bottom">&#x02212;7.5<hr/></td><td align="center" valign="bottom">&#x02212;10.1, -5.4<hr/></td><td align="center" valign="bottom">&#x02212;0.020<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>QLD</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.0<hr/></td><td align="center" valign="bottom">&#x02212;4.6, -3.4<hr/></td><td align="center" valign="bottom">&#x02212;0.044<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>SA</italic><hr/></td><td align="center" valign="bottom">&#x02212;5.0<hr/></td><td align="center" valign="bottom">&#x02212;5.6, -4.4<hr/></td><td align="center" valign="bottom">&#x02212;0.050<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>TAS</italic><hr/></td><td align="center" valign="bottom">&#x02212;0.04<hr/></td><td align="center" valign="bottom">&#x02212;1.1, 1.2<hr/></td><td align="center" valign="bottom">0.000<hr/></td><td align="center" valign="bottom">0.945<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>VIC</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.8<hr/></td><td align="center" valign="bottom">&#x02212;5.2, -4.4<hr/></td><td align="center" valign="bottom">&#x02212;0.080<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>WA</italic><hr/></td><td align="center" valign="bottom">&#x02212;3.9<hr/></td><td align="center" valign="bottom">&#x02212;4.6, -3.3<hr/></td><td align="center" valign="bottom">&#x02212;0.041<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>IRSAD Decile</italic> </bold><italic>(Deciles 1 and 2)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 3 and 4</italic><hr/></td><td align="center" valign="bottom">2.7<hr/></td><td align="center" valign="bottom">1.9, 3.7<hr/></td><td align="center" valign="bottom">0.025<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 5 and 6</italic><hr/></td><td align="center" valign="bottom">3.0<hr/></td><td align="center" valign="bottom">2.4, 3.9<hr/></td><td align="center" valign="bottom">0.037<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 7 and 8</italic><hr/></td><td align="center" valign="bottom">5.5<hr/></td><td align="center" valign="bottom">4.8, 6.3<hr/></td><td align="center" valign="bottom">0.079<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;<italic>Deciles 9 and 10</italic><hr/></td><td align="center" valign="bottom">11.6<hr/></td><td align="center" valign="bottom">11.0, 12.4<hr/></td><td align="center" valign="bottom">0.202<hr/></td><td align="center" valign="bottom">&#x0003c;0.001<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>ARIA Accessibility code</italic> </bold><italic>(Highly accessible)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Accessible</italic><hr/></td><td align="center" valign="bottom">&#x02212;0.08<hr/></td><td align="center" valign="bottom">&#x02212;0.9, 0.8<hr/></td><td align="center" valign="bottom">0.000<hr/></td><td align="center" valign="bottom">0.852<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Moderately accessible</italic><hr/></td><td align="center" valign="bottom">&#x02212;1.0<hr/></td><td align="center" valign="bottom">&#x02212;2.6, 0.5<hr/></td><td align="center" valign="bottom">&#x02212;0.004<hr/></td><td align="center" valign="bottom">0.182<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Remote</italic><hr/></td><td align="center" valign="bottom">&#x02212;2.5<hr/></td><td align="center" valign="bottom">&#x02212;5.5, 0.2<hr/></td><td align="center" valign="bottom">&#x02212;0.006<hr/></td><td align="center" valign="bottom">0.068<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x000a0;<italic>Very remote</italic><hr/></td><td align="center" valign="bottom">&#x02212;4.1<hr/></td><td align="center" valign="bottom">&#x02212;9.8, 1.6<hr/></td><td align="center" valign="bottom">&#x02212;0.003<hr/></td><td align="center" valign="bottom">0.163<hr/></td></tr><tr><td align="left" valign="bottom"><bold> <italic>Aboriginal &#x00026; Torres Strait Islander</italic> </bold><italic>(non-ATSI)</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left">&#x02003;&#x000a0;<italic>ATSI</italic></td><td align="center">&#x02212;7.7</td><td align="center">&#x02212;10.1, -5.4</td><td align="center">&#x02212;0.019</td><td align="center">&#x0003c;0.001</td></tr></tbody></table></table-wrap></sec><sec><title>Gender</title><p>Females comprised 58% of the sample. Males performed better for total UMAT score by 3.6 percentiles (95% CI 3.3, 3.9, P&#x02009;&#x0003c;&#x02009;0.001), UMAT-1 by 7.0 percentiles (95% CI 6.7, 7.3, P&#x02009;&#x0003c;&#x02009;0.001) and UMAT-3 by 8.1 percentiles (95% CI 7.8, 8.4, P&#x02009;&#x0003c;&#x02009;0.001), but worse than females in UMAT-2 with scores lower by 7.3 percentiles (95% CI 7.1, 7.6, P&#x02009;&#x0003c;&#x02009;0.001) (Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>) (Tables&#x000a0;<xref ref-type="table" rid="T1">1</xref>, <xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T4">4</xref>).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by gender for all Australian subjects first sitting the UMAT from 2000 to 2012 (N&#x02009;=&#x02009;118,078).</bold> (*** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-2"/></fig></sec><sec><title>Language at home</title><p>When compared to those who only spoke English at home (69.4% of the sample), scores for total UMAT were lower by 10.4 percentiles (95% CI 10.0, 10.8, P&#x02009;&#x0003c;&#x02009;0.001) for those speaking Asian languages (26.7% of the sample), lower by 12.7 percentiles (95% CI 11.6, 13.8, P&#x02009;&#x0003c;&#x02009;0.001) for those speaking European languages (2.1% of the sample) and lower by 18.4 percentiles (95% CI 17.2, 19.6, P&#x02009;&#x0003c;&#x02009;0.001) for those speaking any other language (1.8% of the sample) (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>) (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>). Similar differentials were evident for UMAT-1 and UMAT-2 for anyone speaking a language other than English at home (Tables <xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref>) (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>). However for UMAT-3, for those speaking Asian languages the score was higher by 2.4 percentiles (95% CI 2.0, 2.8, P&#x02009;&#x0003c;&#x02009;0.001) but remained lower for those speaking European languages by 7.2 percentiles (95% CI 6.0, 8.3, P&#x02009;&#x0003c;&#x02009;0.001) and by 8.9 percentiles (95% CI 7.6, 10.1, P&#x02009;&#x0003c;&#x02009;0.001) for those speaking any other language (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>) (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>).</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by language spoken at home for all Australian subjects first sitting the UMAT from 2000 to 2012 (total number of applicants N&#x02009;=&#x02009;110,136).</bold> (*** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-3"/></fig></sec><sec><title>Secondary school type</title><p>The largest proportion of students were from predominantly a government secondary school background (43.6%) followed by independent secondary schools (35.1%), catholic secondary schools (19.1%) and other secondary school or TAFE (2.2%). Students attending government schools scored lower for total UMAT by 3.9 percentiles (95% CI 3.5, 4.3) compared to those from independent schools (P&#x02009;&#x0003c;&#x02009;0.001). Those attending government schools scored higher than those attending Catholic schools (by 5.0 percentiles, 95% CI 4.6, 5.5) (P&#x02009;&#x0003c;&#x02009;0.001), TAFE colleges (13.3 percentiles, 95% CI 10.8, 15.7) (P&#x02009;&#x0003c;&#x02009;0.001) and all Other institutions (10.7 percentiles, 95% CI 9.4, 11.9) (P&#x02009;&#x0003c;&#x02009;0.001) (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>, Figure&#x000a0;<xref ref-type="fig" rid="F4">4</xref>). Nearly identical results were seen for each section of the UMAT (Tables&#x000a0;<xref ref-type="table" rid="T2">2</xref>,<xref ref-type="table" rid="T3"> 3</xref>, <xref ref-type="table" rid="T4">4</xref>).</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by type of secondary school for all Australian subjects first sitting the UMAT from 2000 to 2012 (total number of applicants N&#x02009;=&#x02009;110,011).</bold> (*** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-4"/></fig></sec><sec><title>Accessibility/remoteness index of Australia</title><p>Students from areas defined by ARIA score as highly accessible comprised 93.4% of the sample. Those from accessible areas comprised 4.9%, moderately accessible 1.2% and remote or very remote only 0.5%. The associations with ARIA score were inconsistent across the 3 sections of UMAT. Compared to highly accessible areas, UMAT-1 scores were higher in those from areas that were defined as accessible (4.2 percentiles, 95% CI 3.4, 5.0) (P&#x02009;&#x0003c;&#x02009;0.001) or moderately accessible (3.3 percentiles, 95% CI 1.8, 4.8) (P&#x02009;&#x0003c;&#x02009;0.001) (Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>, Figure&#x000a0;<xref ref-type="fig" rid="F5">5</xref>). UMAT-2 scores were higher in those from areas defined as accessible (3.7 percentiles, 95% CI 2.8, 4.5) (P&#x02009;&#x0003c;&#x02009;0.001), moderately accessible (2.6 percentiles, 95% CI 1.0, 4.1) (P&#x02009;=&#x02009;0.001) or remote (4.4 percentiles, 95% CI 1.8, 7.1) (P&#x02009;=&#x02009;0.001) (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>, Figure&#x000a0;<xref ref-type="fig" rid="F5">5</xref>). In contrast, compared to highly accessible areas, UMAT-3 scores were progressively lower in the univariate analysis (Figure&#x000a0;<xref ref-type="fig" rid="F5">5</xref>) with this trend no longer statistical significance in the multivariate analysis (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>). These divergent associations overall resulted in the multivariate analysis predicting total UMAT scores which were higher in those from areas that were defined as accessible (3.0 percentiles, 95% CI 2.2, 3.8) (P&#x02009;&#x0003c;&#x02009;0.001) or moderately accessible (1.9 percentiles, 95% CI 0.4, 3.4) (P&#x02009;=&#x02009;0.015) compared to those from highly accessible areas (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>).</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by accessibility/remoteness index of Australia for all Australian subjects first sitting the UMAT from 2000 to 2012 (N&#x02009;=&#x02009;118,085).</bold> (* P&#x02009;&#x0003c;&#x02009;0.05, ** P&#x02009;&#x0003c;&#x02009;0.01, *** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-5"/></fig></sec><sec><title>Aboriginal or Torres Strait Islander</title><p>Only a relatively small number of those self-identifying as Aboriginal or Torres Strait Islander (ATSI) (N&#x02009;=&#x02009;556, 0.5%) sat the UMAT during the time period. Their scores on total UMAT were lower by 9.6 percentiles (95% CI 7.3, 11.9, P&#x02009;&#x0003c;&#x02009;0.001), UMAT-1 by 8.9 percentiles (95% CI 6.7, 11.2, P&#x02009;&#x0003c;&#x02009;0.001) and UMAT-2 by 6.2 percentiles (95% CI 3.9, 8.5, P&#x02009;&#x0003c;&#x02009;0.001) and UMAT-3 by 7.7 percentiles (95% CI 5.4, 10.1, P&#x02009;&#x0003c;&#x02009;0.001) (Tables&#x000a0;<xref ref-type="table" rid="T1">1</xref>, <xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>).</p></sec><sec><title>Socio-economic advantage and disadvantage</title><p>Australians sitting the UMAT largely came from the top 2 deciles for IRSAD score (50.8%) with only 6.9% from the bottom 2 deciles. For every UMAT section, scores diminished progressively with increasing socio-economic disadvantage and decreasing socio-economic advantage. This translated into IRSAD decile being the strongest predictor of total UMAT score in the final multivariate model. It resulted in total UMAT scores in those in the highest 2 deciles that were 13.0 percentiles higher (95% CI, 12.4, 13.8) (P&#x02009;&#x0003c;&#x02009;0.001) than those achieved by candidates from the lowest 2 deciles (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>, Figure&#x000a0;<xref ref-type="fig" rid="F6">6</xref>). In subsequent analyses the magnitude and profile of this relationship remained similar for each language group (data not shown).</p><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold>Percentile score for total UMAT, UMAT-1, UMAT-2 and UMAT-3 by deciles for index of relative socio-economic advantage and disadvantage score for all Australian subjects first sitting the UMAT from 2000 to 2012 (N&#x02009;=&#x02009;117,347).</bold> (** P&#x02009;&#x0003c;&#x02009;0.01, *** P&#x02009;&#x0003c;&#x02009;0.001 &#x02013; one way ANOVA post-hoc comparisons with Bonferroni correction).</p></caption><graphic xlink:href="1472-6920-13-155-6"/></fig><p>Many of the predictor variables were also related to the IRSAD decile of origin of each candidate. Approximately 49% females were in the top 2 deciles compared to 53% of males (&#x003c7;²&#x02009;=&#x02009;173, P&#x02009;&#x0003c;&#x02009;0.001). With increasing age there was a progressively diminishing proportion in the top 2 deciles (from 53% of 17&#x000a0;year-olds down to 46% of those &#x0003e;30&#x000a0;yr, &#x003c7;²&#x02009;=&#x02009;126, P&#x02009;&#x0003c;&#x02009;0.001). For those who attended independent secondary schools, 63% were from the highest 2 IRSAD deciles compared to 46% from government schools and 39% from Catholic schools (&#x003c7;²&#x02009;=&#x02009;4170, P&#x02009;&#x0003c;&#x02009;0.001). Approximately 54% of those in the most highly accessible areas were in the top 2 deciles compared to only 3 to 10% of those in any area with higher ARIA scores (&#x003c7;²&#x02009;=&#x02009;7653, P&#x02009;&#x0003c;&#x02009;0.001). Approximately 53% of those where English was the language spoken at home were in the top 2 deciles compared to 46% of those speaking Asian languages, 42% of those speaking European languages and 37% of those speaking any other language (&#x003c7;²&#x02009;=&#x02009;703, P&#x02009;&#x0003c;&#x02009;0.001). Of those identifying as ATSI, 32% were within the highest 2 IRSAD deciles compared to 51% of non-ATSI (&#x003c7;²&#x02009;=&#x02009;82.7, P&#x02009;&#x0003c;&#x02009;0.001).</p></sec><sec><title>State or territory of origin</title><p>Nearly two thirds of the cohort (64.7%) were from Australia&#x02019;s 2 most populous states, New South Wales and Victoria. With New South Wales as the comparator state, higher mean scores were seen in candidates from the Australian capital Territory (ACT) and Tasmania (TAS) while lower mean scores were seen in Victoria (VIC), Queensland (QLD), South Australia (SA), Western Australia (WA) and the Northern Territory (NT) (Tables&#x000a0;<xref ref-type="table" rid="T1">1</xref>, <xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref>).</p><p>Many of these state-based differences were confounded by substantial socio-demographic differences across the states. For example TAS had the largest proportion of subjects speaking English at home (93% of all TAS candidates) while those with an Asian language background were heavily concentrated in NSW and VIC (38% and 35% of all NSW and VIC candidates respectfully) (&#x003c7;²&#x02009;=&#x02009;2338, P&#x02009;&#x0003c;&#x02009;0.001). When broken down by state, 99.8% of those from the ACT were within the top 2 IRSAD deciles compared to 63% of those from WA, 54% of those from QLD, 51% of those from VIC, 50% of those from NSW, 35% of those from SA, 28% of those from TAS and 17% of those from the NT (&#x003c7;²&#x02009;=&#x02009;4402, P&#x02009;&#x0003c;&#x02009;0.001). The relative profile of government vs independent vs catholic school education also varied significantly across states as did the relative proportion of candidates from a rural background. Finally the age first sitting the UMAT was different across states with more 17 yo candidates from WA and QLD and more 18 yo candidates from VIC, TAS and the ACT.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>We have observed a consistent relationship between a number of socioeconomic indices and performance over more than a decade by Australian candidates in the UMAT. The UMAT total score and performance in each of its subsections was linked to an index of relative socio-economic advantage and disadvantage generated from the postcode of the correspondence address at the time of sitting the UMAT. Being from the top 2 socio-economic deciles as determined by a broad spectrum of indices generated from Australian census data was positively associated with UMAT performance. In addition, it was positively associated with prior secondary education in a fee-paying independent school and negatively associated with self-identification as being of ATSI origin. These results raise the prospect of a diversity limiting effect of selection processes at universities that utilise the UMAT.</p><p>A recent Canadian study of applicants to 6 medical schools also utilised a measure of socio-economic status linked by postcode to community size and income levels [<xref ref-type="bibr" rid="B12">12</xref>]. They identified an association between lower performance in the MCAT in those from smaller communities but saw no relationship with income levels. Academic performance as measured by GPA was linked to income levels but not to community size while interview scores were unrelated to either of these socio-economic measures. Similar to our finding, they also reported lower MCAT scores in applicants of self-declared aboriginal origin. Although this was interpreted as another indication that performance in aptitude tests might be influenced by socio-economic status, the authors also allowed that a contributing factor may have been the widespread availability of facilitative admissions processes for indigenous Canadians which could have created a larger pool of candidates applying with generally lower admission test scores [<xref ref-type="bibr" rid="B20">20</xref>].</p><p>A study of all UK candidates who sat the inaugural UKCAT test in 2006 [<xref ref-type="bibr" rid="B13">13</xref>] is probably more relevant in comparing our observations of a potential socio-economic influence on UMAT performance. It similarly is administered predominantly to secondary school leaver applicants to medical and dental schools and comprises 4 sections with some similarities to the UMAT &#x02013; verbal reasoning, quantitative reasoning, abstract reasoning and decision analysis [<xref ref-type="bibr" rid="B13">13</xref>]. They used 3 potential indicators of socioeconomic status &#x02013; ethnicity, parental occupation and education at independent/grammar schools &#x02013; and found that male sex, white ethnicity, having parents from a professional/managerial background and independent or grammar schooling were each independent indicators of more favourable UKCAT performance. This test like the UMAT had been introduced in the expectation of increasing diversity and fairness in selection but the authors have concluded on the basis of this data that a significant socio-economic influence on test results may still remain.</p><p>The more important outcome of course, is whether such an association between test performance and socio-economic background ultimately translates into an actual impact on medical school selection. In this regard a follow-up study [<xref ref-type="bibr" rid="B4">4</xref>] has assessed the impact of use of the UKCAT in the 2009 cohort on subsequent selection into medical school. If it was utilised as a weighted factor in selection or as a tie-breaker for borderline applicants, those candidates from the lowest socioeconomic background were approximately 30-50% less likely to be given a conditional or unconditional offer of a place at medical school. However, if it was used as a threshold score to decide whether or not a candidate was to receive an interview, socio-economic background was not a significant predictor of whether an offer would be made. This translated into medical schools that used the UKCAT as a threshold being 3.6 times more likely to offer a place to someone from a low socioeconomic background than schools that used it as a weighted factor or as a borderline tie-breaker. The corollary however, was that where offers were made to students conditional on a certain level of academic achievement, conversion from a conditional to an unconditional offer for those of low socio-economic status was nearly 60% less likely in applicants to schools that used the UKCAT as a threshold parameter for selection. While this study was able to compare these different approaches across 22 medical schools that utilised the UKCAT in selection, it was not able to compare the relative socio-demographic makeup of selected students in schools not using the UKCAT. It also did not report on the actual change in the socio-demographic make-up of participating medical schools before and after incorporation of the UKCAT into selection processes [<xref ref-type="bibr" rid="B4">4</xref>].</p><p>The UMAT has been correlated, albeit weakly, with academic performance [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Given that academic performance is a consistently utilised selection factor for medical schools globally, it is likely that UMAT attracts a cohort of students who are academically performing at the higher end of the spectrum. Better academic performance by school leavers has been repeatedly linked to increased socio-economic advantage with academic achievement highest at entry into medical school in those from less materially disadvantaged households [<xref ref-type="bibr" rid="B20">20</xref>]. The association of UMAT scores with IRSAD decile therefore may at least in part be dictated by a stronger academic performance in those who choose to sit the UMAT. In this cohort we did not have any data on candidate&#x02019;s prior academic performance, the UMAT for the most part being sat in the final year of secondary school before tertiary entrance academic results are known. However, we have been able to investigate this hypothesis in a cohort of students who have entered our medical school from secondary school over a 12&#x000a0;year period using a combination of UMAT, academic performance and a score from a structured interview [<xref ref-type="bibr" rid="B5">5</xref>]. At least in part the hypothesis was supported in this much smaller and highly selected cohort by the finding of a substantial attenuation of the relationship between UMAT score and IRSAD decile when prior academic performance was taken into account, with parameter estimates for the magnitude of the association reducing by 30 to 50 percent (Puddey IB &#x02013; personal communication).</p><p>In the UK, ethnicity has been recognized as a potential confounding factor when attempting to unpick the relationship between socio-economic background and access into medical schools [<xref ref-type="bibr" rid="B21">21</xref>]. We were unable to assess ethnicity but by using language spoken at home as a surrogate, we found that approximately 27% of those who sat the UMAT from 2000 to 2012 came from Asian language backgrounds. We have previously reported an over-representation of Asian students applying to and being admitted into medical schools in Australia relative to background prevalence in the population [<xref ref-type="bibr" rid="B5">5</xref>]. A similar phenomenon has been observed in both the UK [<xref ref-type="bibr" rid="B22">22</xref>] and in New Zealand [<xref ref-type="bibr" rid="B23">23</xref>]. In our cohort, fewer Asian language students were in the top 2 IRSAD deciles compared to English language students and their overall UMAT performance was generally weaker. To an extent therefore, ethnicity may have been an additional factor in the pathway linking socio-economic background to performance in the UMAT. However, when we studied each major language group separately, the magnitude and pattern of the relationship between IRSAD decile and UMAT performance was almost identical.</p><p>It is recognised that a high percentage of candidates for aptitude tests for medical school entry undergo some prior commercial coaching in an attempt to optimise performance. In an Australian context this has been reported to be as high as 56% [<xref ref-type="bibr" rid="B14">14</xref>] while for the MCAT a review identified 4 studies in the area where the prevalence of coaching varied between 22%, 25%, 38% and 72% of candidates respectively [<xref ref-type="bibr" rid="B24">24</xref>]. Given this high prevalence and that UMAT coaching courses are relatively expensive it is possible that prior coaching could have confounded our results. More students from higher socio-economic backgrounds may have had access to coaching which enhanced their subsequent UMAT performance. Also UMAT coaching and practice may be more systematic and organised within fee-paying independent schools [<xref ref-type="bibr" rid="B14">14</xref>]. Whether this has influenced our results remains controversial, however, with recent reports indicating either no effect of preparation courses on UMAT performance or only a weak effect in improving Section 3 - non-verbal reasoning in selected students [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B25">25</xref>].</p><p>The findings of significant differences in performance by Australian state or territory of abode were a somewhat surprising finding but look likely to be linked to considerable differences in socio-demographic profiles across the states. The top performing states were those with the highest proportion of students in the top 2 IRSAD deciles. Age of students at entry to and exit from primary and secondary schooling varies across states and would have contributed to a changing age profile across states for age first sitting the UMAT. Relative proportions of students from Asian language versus English language backgrounds were markedly dissimilar across states and there were differences in the proportions of those from rural backgrounds. Finally, the mix of those receiving government vs independent vs Catholic school education also differed significantly by state.</p><p>Our estimates of the relative contribution that socio-demographic factors make to the overall variance in UMAT scores were substantially higher than those previously identified in the 2012 ACER report on the UMAT [<xref ref-type="bibr" rid="B1">1</xref>] with estimates from our linear regression models of 12% for total UMAT score, 13.5% for UMAT-1, 11.3% for UMAT2 and 9.8% for UMAT-3. This represents a nearly 3-fold higher estimate for the total UMAT score and relates to both the broader range of variables included in our analysis as well as the increase in power afforded by including all those who sat for the test over a 13&#x000a0;year period.</p><sec><title>Study limitations</title><p>The study is cross sectional in nature and hence a causal link between socio-economic background and UMAT performance has not been established by these data. UMAT score statistics (mean and standard deviation) have varied over the years, however large numbers in the cohorts and the use of percentile ranks provide a measure independent of such statistics. The SEIFA codes are generated from a suite of summary measures in defined areas based on census information and do not apply to an individual person or dwelling. Using an individual&#x02019;s postcode is therefore only a surrogate for true socio-economic status with SEIFA codes imputing an index based on the level of socio-economic disadvantage for all people living in a defined area. It is likely a significant proportion of candidates would have been living in student dormitories or lodgings near their university or secondary school rather than their usual place of residence and this may have weakened the true underlying strength of the associations we have reported. On the other hand aggregating 21 socioeconomic indicators into a single index and then further aggregating by postcode would reduce the variance associated with each indicator and may inflate the strength of the associations reported. Finally socio-economic status linked to an area is not static over time and we have used the 2006 SEIFA codes over a period that spans 2000 to 2012 again potentially weakening the relative accuracy of imputed socio-economic status.</p></sec></sec><sec sec-type="conclusions"><title>Conclusions</title><p>We have observed a direct relationship between socio-economic background and performance in the UMAT. This observation was consistent for all sections of the UMAT and across a number of socio-demographic variables including IRSAD decile, school background and self-identification as being of ATSI origin. The association was similar to that which has already been well documented for prior academic performance. Therefore in Australia, where UMAT is often utilised alone to select candidates for interview, there may be important implications of these observations for current selection processes that may otherwise be seeking to widen medical school access to those students from a broader socio-economic base through use of an aptitude test. A prospective study of the potential impact of the UMAT on the profile of students selected to Australian medical schools is clearly warranted. The quarantining of places through affirmative action pathways to admit students from lower socio-economic backgrounds who have reached acceptable threshold scores in each section of the UMAT may be a necessary complementary approach for ensuring student diversity.</p></sec><sec><title>Competing interests</title><p>IP is the representative for the University of Western Australia on the UMAT Consortium Board of Management. AM is a member of the UMAT Test Management Committee and Chair of both the UMAT Technical Subcommittee and the UMAT Research Subcommittee.</p></sec><sec><title>Authors&#x02019; contributions</title><p>IP contributed to the conception and design of the study, acquisition, analysis and interpretation of the data; and the initial drafting and final revision of the manuscript. AM contributed to the conception and design of the study, interpretation of the data; and final revision of the manuscript for important intellectual content. Both authors read and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6920/13/155/prepub">http://www.biomedcentral.com/1472-6920/13/155/prepub</ext-link></p></sec>

Content analysis of medical students&#x02019; seminars: a unique method of analyzing clinical thinking

<sec><title>Background</title><p>The study of communication skills of Asian medical students during structured Problem-based Learning (PBL) seminars represented a unique opportunity to assess their critical thinking development. This study reports the first application of the health education technology, content analysis (CA), to a Japanese web-based seminar (webinar).</p></sec><sec><title>Methods</title><p>The authors assigned twelve randomly selected medical students from two universities and two clinical instructors to two virtual classrooms for four PBL structured tutoring sessions that were audio-video captured for CA. Both of the instructors were US-trained physicians. This analysis consisted of coding the students&#x02019; verbal comments into seven types, ranging from trivial to advanced knowledge integration comments that served as a proxy for clinical thinking.</p></sec><sec><title>Results</title><p>The most basic level of verbal simple responses accounted for a majority (85%) of the total students&#x02019; verbal comments. Only 15% of the students&#x02019; comments represented more advanced types of critical thinking. The male students responded more than the female students; male students attending University 2 responded more than male students from University 1. The total mean students&#x02019; verbal response time for the four sessions with the male instructor was 6.9%; total mean students&#x02019; verbal response time for the four sessions with the female instructor was 19% (p&#x02009;&#x0003c;&#x02009;0.05).</p></sec><sec><title>Conclusions</title><p>This report is the first to describe the application of CA to a multi-university real time audio and video PBL medical student clinical training webinar in two Japanese medical schools. These results are preliminary, mostly limited by a small sample size (n&#x02009;=&#x02009;12) and limited time frame (four sessions). CA technology has the potential to improve clinical thinking for medical students. This report may stimulate improvements for implementation.</p></sec>

<contrib contrib-type="author" id="A1"><name><surname>Takata</surname><given-names>Yukari</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>yukari.takata@gmail.com</email></contrib><contrib contrib-type="author" corresp="yes" id="A2"><name><surname>Stein</surname><given-names>Gerald H</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>jerrydoc@ufl.edu</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Endo</surname><given-names>Kuniyuki</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>ekuni@nagoya2.jrc.or.jp</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Arai</surname><given-names>Akiko</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>akikoarai@ufl.edu</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Kohsaka</surname><given-names>Shun</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>cardiotx@gmail.com</email></contrib><contrib contrib-type="author" id="A6"><name><surname>Kitano</surname><given-names>Yuka</given-names></name><xref ref-type="aff" rid="I6">6</xref><email>yukay@qd5.so-net.ne.jp</email></contrib><contrib contrib-type="author" id="A7"><name><surname>Honda</surname><given-names>Hitoshi</given-names></name><xref ref-type="aff" rid="I7">7</xref><email>user101331@aol.com</email></contrib><contrib contrib-type="author" id="A8"><name><surname>Kitazono</surname><given-names>Hidetaka</given-names></name><xref ref-type="aff" rid="I8">8</xref><email>hkitazono@gmail.com</email></contrib><contrib contrib-type="author" id="A9"><name><surname>Tokunaga</surname><given-names>Hironobu</given-names></name><xref ref-type="aff" rid="I9">9</xref><email>marutoku@mx2.fctv.ne.jp</email></contrib><contrib contrib-type="author" id="A10"><name><surname>Tokuda</surname><given-names>Yasuharu</given-names></name><xref ref-type="aff" rid="I10">10</xref><email>yasuharu.tokuda@gmail.com</email></contrib><contrib contrib-type="author" id="A11"><name><surname>Obika</surname><given-names>Mikako</given-names></name><xref ref-type="aff" rid="I11">11</xref><email>obika-m@cc.okayama-u.ac.jp</email></contrib><contrib contrib-type="author" id="A12"><name><surname>Miyoshi</surname><given-names>Tomoko</given-names></name><xref ref-type="aff" rid="I12">12</xref><email>tmiyoshi@md.okayama-u.ac.jp</email></contrib><contrib contrib-type="author" id="A13"><name><surname>Kataoka</surname><given-names>Hitomi</given-names></name><xref ref-type="aff" rid="I12">12</xref><email>hitomik@md.okayama-u.ac.jp</email></contrib><contrib contrib-type="author" id="A14"><name><surname>Terasawa</surname><given-names>Hidekazu</given-names></name><xref ref-type="aff" rid="I9">9</xref><email>qqhtera@u-fukui.ac.jp</email></contrib>

BMC Medical Education

<sec><title>Background</title><p>Learning clinical thinking is a complex task of accumulating knowledge and experience [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>]. For medical students entering clinical training, PBL and its variations offer an entry into this complex world [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>]. Small group discussions centered on a patient&#x02019;s narrative with history, physical examination and laboratory data compromise the data, summarized as the problem list, for the discussion to extract meaningful concepts leading to diagnosis and management, loosely defined as clinical thinking. An essential component of the case discussion is the verbal communication among the students and their instructor.</p><p>Instruments to measure small group speaking as evolved a technique called content analysis (CA). CA was developed for product marketing and heath education. Borg and Gall [<xref ref-type="bibr" rid="B6">6</xref>] defined CA as &#x0201c;a research technique for the objective, systematic, and quantitative description of the manifest content of communication&#x0201d;. CA includes student length of speaking, participate rates, social clues, interactions, speech content to name a few of the possible measurements [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. Very few medical educational CA applications have been reported [<xref ref-type="bibr" rid="B9">9</xref>]. A partial use of CA was explored in a recent study of the audio analysis of the &#x02018;morning report&#x02019; of new case admissions coding the interactions between supervisors and their residents [<xref ref-type="bibr" rid="B10">10</xref>].</p><p>Furthermore Asian students and by inference, Japanese medical students, are particularly unaccustomed to classroom discussion, especially when led by older faculty [<xref ref-type="bibr" rid="B11">11</xref>]. Their hindrances include dependency and respect for authority, cultural inhibition to be silent and lack of training to ask questions that broadly include development of problem solving skills. Yet PBL requires talking as a means to learn clinical thinking [<xref ref-type="bibr" rid="B12">12</xref>]. Although Japanese medical education has undergone recent structural changes, little has been reported on the outcome of these changes [<xref ref-type="bibr" rid="B13">13</xref>]. PBL has become increasing used in Japanese medical education [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. This report is the first to use CA to document medical students&#x02019; verbal responses as part of a Japanese multi-university PBL webinar developmental project.</p></sec><sec sec-type="methods"><title>Methods</title><p>Content analysis of students&#x02019; verbal responses during eight webinar PBL tutoring sessions was examined.</p><sec><title>Participants</title><p>The study involved two US-trained clinical instructors (one female, one male) and 12 fifth year Japanese medical students (four female, eight male). The instructors were selected for their three years of general internal medicine training in the United States and their Japanese-English bilingual skills. Six randomized medical students from each of two distant Japanese medical universities were randomly assigned to one of the two instructors&#x02019; two virtual &#x02018;classrooms&#x02019; (<ext-link ext-link-type="uri" xlink:href="http://www.webex.co.jp">http://www.webex.co.jp</ext-link>). The study protocol specified that each instructor led four tutoring sessions (1.5&#x000a0;hours each) over the course of four weeks. The topics and teaching materials were standardized and designed as a syllabus by a panel of Japanese &#x00026; American medical educators to promote PBL. The instructors received minimal communication skills to enhance learning; no PBL training or feedback or standardization occurred during the four tutoring sessions. All tutoring sessions had video and audio components captured for the subsequent CA. The students&#x02019; identity was masked for this analysis. All 12 students signed a detailed informed consent form.</p></sec><sec><title>Code book</title><p>A CA code book to analyze the verbal interactions during the tutoring sessions.</p><p>An underlying assumption of communication CA is that dialogues are representative of underlying cognitive processes [<xref ref-type="bibr" rid="B8">8</xref>].</p><p>The level of critical thinking expressed by students was coded using a modified version of Practical Inquiry Model (PIM), which characterizes phases of practical inquiry through descriptors and indicators [<xref ref-type="bibr" rid="B8">8</xref>]. For example, the model&#x02019;s second phase, Exploration, is characterized through &#x0201c;inquisitive&#x0201d; communication, which is often indicated through suggestions for consideration and brainstorming. Exploration is then followed by integration and resolution to complete the model&#x02019;s phases of practical inquiry.</p><p>The PIM was organized into the code book through a series of trial coding sessions, performed by health educator coders. The coding sessions comprised analysis of speech descriptors and indicators. After the code book was finalized, an inter-coder reliability test of 19 responses, or about 10%, of all comments, involving only the variables open to interpretation, resulted in a Krippendorff&#x02019;s alpha of 0.80, above the 0.75 considered acceptable [<xref ref-type="bibr" rid="B16">16</xref>]. Each students&#x02019; response for one of seven possible types, which essentially scaled the responses&#x02019; level of critical thinking, was coded independently by the health educator coders (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>). The respondent&#x02019;s identity, audience, and position (i.e. whether they were the inquisitor or respondent) were also coded. The coding authors made the type critique from watching the captured recordings of the webinars; they coded all responses and comments that the students made. They made no distinction between responses and comments.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Critical thinking response types</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Response types</bold></th><th align="left"><bold>Details of response types</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">T1<hr/></td><td align="left" valign="bottom">Simple phrase responses, based on rote memory<hr/></td></tr><tr><td align="left" valign="bottom">T2<hr/></td><td align="left" valign="bottom">Response has more depth that Type 1 but still based on rote memory, includes <italic>because, if, when, etc.</italic><hr/></td></tr><tr><td align="left" valign="bottom">T3<hr/></td><td align="left" valign="bottom">Response to &#x0201c;How would you ask a patient_______?&#x0201d;<hr/></td></tr><tr><td align="left" valign="bottom">T4<hr/></td><td align="left" valign="bottom">Response shows integration &#x02013; combines ideas to form new meanings (analytical)<hr/></td></tr><tr><td align="left" valign="bottom">T5<hr/></td><td align="left" valign="bottom">Response shows advanced integration &#x02013; combines knowledge from multiple and/or obscure sources to introduce original ideal solutions (potentially novel to instructor as well)<hr/></td></tr><tr><td align="left" valign="bottom">T6<hr/></td><td align="left" valign="bottom">Spontaneous questions directed to instructor<hr/></td></tr><tr><td align="left">T7</td><td align="left">Social commentary, greetings, appreciation, etc., unrelated to tutored topic</td></tr></tbody></table></table-wrap></sec><sec><title>Statistical analyses</title><p>Proportions and percentages between groups were compared using by chi-square test. Two-tailed p values less than 0.05 were considered as statistical significance. All statistical analysis was performed using SPSS-J version 20 (Tokyo, Japan).</p></sec></sec><sec sec-type="results"><title>Results</title><p>All students&#x02019; comments, except for those related to technical issues and social commentary at the beginning and end of each session, were included in the analysis. Descriptive statistics were primarily used to characterize interactions observed in this study.</p><p>The total mean students&#x02019; verbal response time for the four sessions with the male instructor was 6.9%; total mean students&#x02019; verbal response time for the four sessions with the female instructor was 19% (Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>). The total mean student response time for the four sessions with the male instructor was significantly lower than that with female instructor (p&#x02009;&#x0003c;&#x02009;0.05).</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Total students&#x02019; minutes response times</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left">&#x000a0;</th><th align="center"><bold>Male instructor</bold></th><th align="center"><bold>Female instructor</bold></th><th align="center"><bold>p value</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Session 1<hr/></td><td align="center" valign="bottom">5.3/91 (5.8)<hr/></td><td align="center" valign="bottom">13.4/94 (14.1%)<hr/></td><td align="center" valign="bottom">0.14<hr/></td></tr><tr><td align="left" valign="bottom">Session 2<hr/></td><td align="center" valign="bottom">8.2/93 (8.8)<hr/></td><td align="center" valign="bottom">20.0/80 (25)<hr/></td><td align="center" valign="bottom">0.02<hr/></td></tr><tr><td align="left" valign="bottom">Session 3<hr/></td><td align="center" valign="bottom">4.5/78 (5.6)<hr/></td><td align="center" valign="bottom">17.3/90 (19.2)<hr/></td><td align="center" valign="bottom">0.06<hr/></td></tr><tr><td align="left">Session 4</td><td align="center">6.6/91 (7.2)</td><td align="center">18.2/102 (17.8)</td><td align="center">0.11</td></tr></tbody></table><table-wrap-foot><p>Total students&#x02019; minutes response times divided by total session time in minutes with the percent students response times in parenthesis. The total mean student response time for the 4 sessions with the male instructor was significantly lower than that with female instructor (p&#x02009;&#x0003c;&#x02009;0.05).</p></table-wrap-foot></table-wrap><p>Total students&#x02019; comments with both instructors were 458 comments. Of the total comments, responses involving the most basic level of critical thinking (T1) accounted for over half of the comments (66%, n&#x02009;=&#x02009;302). The second level of critical thinking (T2) also accounted for the second most common response type at 15% (n&#x02009;=&#x02009;69). Notably, the more advanced levels of critical thinking (T4 and T5), which the PBL project was striving to achieve, represented just 4% of all comments (n&#x02009;=&#x02009;9) with no T5&#x02032;s being represented.</p><p>Because the numbers of advanced levels of critical thinking responses were small, we combined the type of responses as follows: &#x02018;simple responses&#x02019; type 1, 2 and 7; &#x02018;advanced thinking responses&#x02019; types 3 to 5, and spontaneous topic-related questions directed to the instructor type 6 &#x02013;two non topic related questions were deleted (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>).</p><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Combined critical thinking response types</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Response types</bold></th><th align="left"><bold>Details of response types</bold></th></tr></thead><tbody valign="top"><tr><td rowspan="3" align="left" valign="top">Simple- Types 1, 2 and 7<hr/></td><td align="left" valign="bottom">Simple phrase responses, based on rote memory<hr/></td></tr><tr><td align="left" valign="bottom">Response has more depth that Type 1 but still based on rote memory, includes <italic>because, if, when, etc.</italic><hr/></td></tr><tr><td align="left" valign="bottom">Social commentary, greetings, appreciation, etc., unrelated to tutored topic<hr/></td></tr><tr><td rowspan="3" align="left" valign="top">Advanced thinking Types 3-5<hr/></td><td align="left" valign="bottom">Response to &#x0201c;How would you ask a patient_______?&#x0201d;<hr/></td></tr><tr><td align="left" valign="bottom">Response shows integration &#x02013; combines ideas to form new meanings (analytical)<hr/></td></tr><tr><td align="left" valign="bottom">Response shows advanced integration &#x02013; combines knowledge from multiple and/or obscure sources to introduce original ideal solutions (potentially novel to instructor as well)<hr/></td></tr><tr><td align="left">Type 6</td><td align="left">Spontaneous topic-related questions directed to instructor</td></tr></tbody></table></table-wrap><p>&#x02018;Simple responses&#x02019;&#x02009;, having no comments about the syllabus topics, comprised 85% of the response types, &#x02018;advanced thinking responses&#x02019; contained 11%, and topic-related spontaneous questions were 4% for a total of 15% more advanced types of critical thinking directly related to the syllabus topics (Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref>).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Medical students&#x02019; combined critical thinking response types: 85% were simple responses, 11% advanced thinking responses and 4% topic related spontaneous questions.</p></caption><graphic xlink:href="1472-6920-13-156-1"/></fig><p>Examining the distribution of combined comments type for each session suggested that students&#x02019; comments generally decreased as time passed, giving little indication that critical analysis increased across time (Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Type and frequency of medical students&#x02019; comments across the 4 sessions suggesting their comments decreased over time.</p></caption><graphic xlink:href="1472-6920-13-156-2"/></fig><p>In another analysis we combined types 1, 2 and 7 as &#x02018;expanded simple responses&#x02019; and types 3 to 5 as &#x02018;integration thinking&#x02019;. Total comments made by students&#x02019; sex were proportionally represented with female students (n&#x02009;=&#x02009;4) accounting for 33.0% (n&#x02009;=&#x02009;142) and male students (n&#x02009;=&#x02009;8) accounting for 67.0% (n&#x02009;=&#x02009;278) (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>). There was no statistical difference between male and female students (p&#x02009;=&#x02009;0.69).</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Distribution and number of comments by males and females were not statistically different.</p></caption><graphic xlink:href="1472-6920-13-156-3"/></fig><p>Students instructed by the female instructor also commented significantly more than those under the tutorage of a male instructor, with the female instructor's group contributing 60.0% of comments (n&#x02009;=&#x02009;275).</p><p>We observed minor differences between the two medical universities, with University 2 students contributing more responses, regardless of their instructors (Figure&#x000a0;<xref ref-type="fig" rid="F4">4</xref>). There was no statistical difference between students of University 1 and those of University 2 (p&#x02009;=&#x02009;0.92).</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Distribution and number of comments by the universities were not statistically different.</p></caption><graphic xlink:href="1472-6920-13-156-4"/></fig></sec><sec sec-type="discussion"><title>Discussion</title><p>We presented the first CA of medical students&#x02019; webinars. This is a unique application of the CA technique that was developed for health educators, to medical student seminars. The most basic level of simple responses having no comments about the syllabus topics, accounted for a majority (85%) of the total students&#x02019; comments. Only 15% of the students&#x02019; comments represented more advanced types of critical thinking.</p><p>It was anticipated that data would show improved clinical thinking as measured by Types 3 to 5 changes. Such was not the case. In part the total talking time of the students was very low, much lower than anticipated, with no change over the four sessions. Conversely the dominant talking time of the instructors implied they reverted to the traditional practice of lecturing, perhaps because they lacked skills to promote discussion.</p><p>Usual communication skills training and assessment of medical students center on interactions and feedback from standardized actor patients- objective structured clinical examination (OSCE) [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B18">18</xref>]. A two-day instructional communication skills report showed 5^th grade Japanese medical students&#x02019; marginal improvement using OSCE [<xref ref-type="bibr" rid="B19">19</xref>].</p><p>However, in medical educational communities where prior educational exposure has attenuated student responses, the seminar experiences requiring verbal interactions may be necessary before adequate doctor-patient communication skills develop.</p><p>Limiting factors of this study were small sample size, short interval for discussions and small number of sessions (four). We had envisioned a larger medical student sample size. However sample size was limited by the coordination problems expressed by several Japanese medical universities to participate. The duration of the study and small number of sessions were restricted by the tutors&#x02019; already too busy daily workloads.</p><p>There are many possible reasons for the short times of students&#x02019; advanced thinking responses. For the instructors their possible reasons were: 1) the instructors were not trained to teach PBL. PBL faculty development with practice including instant feedback, is not offered in Japanese medical universities; 2) Completion of a US residency does not provide adequate PBL coaching for medical students, a major criteria for instructor selection in our study; 3) The authors provided no inter-session improvement feedback; 4) The instructors likely felt compelled to complete the seminars&#x02019; syllabus, thinking it was more important than student participation; and 5) If the total number of tutored sessions had been increased to eight sessions over eight weeks, it is possible more critical thinking responses would have been observed.</p><p>For the students their possible reasons for their short times of advanced thinking responses were: 1) The students lacked prior long term educational experiences in verbal problem solving exercises; 2) Their usual ways of classroom learning has been limited to large hall lectures, a usual cultural norm; 3) The presence of a faculty member has been an inhibiting influence since silence shows respect to one&#x02019;s elders; 4) An Asian student does not verbally challenge the instructor; 5) Speaking out has been considered culturally rude [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]; 6) The lack of familiarity with the tutors and relatively limited four sessions likely inhibited the students&#x02019; responses as suggested in a recent report [<xref ref-type="bibr" rid="B20">20</xref>].</p><p>Although the tutoring sessions did not appear to increase expression of PBL understanding over time, the students&#x02019; sex may have play a role in critical thinking. Within the scope of our very limited data, although the male students responded more, female students expressed proportionally more upper-level critical thinking than their male counterparts; the female instructor&#x02019;s group also contributed significantly more comments, suggesting that Japanese women, who traditionally have been observed to be more expressive and nurturing may find it easier to grasp PBL concepts. However, these data were limited to verbal expressions; it may not be a fair representation of true levels of cognitive activity.</p><p>Also, that University 2 students generally commented more than University 1 students, suggested local medical education styles may influence students&#x02019; respond in web-tutoring sessions. Specifically University 2 actively promotes PBL by having faculty use their PBL teaching methods acquired during PBL workshops at an American medical school, and by encouraging students to participate in their popular extra-curricular PBL club. University 1 does not offer PBL faculty development or PBL clubs. However several clinical departments at University 1 offer PBL training to their postgraduate trainees.</p><p>Nonetheless, the application of CA, from the field of health and nutrition education, has the potential to make important contributions to improving clinical thinking skills, in both Western and non-Western medical universities.</p><p>Furthermore, detailed evaluation of faculty and student performance during small group seminars, CA may be ideally suited for this task given its many year multidisciplinary history [<xref ref-type="bibr" rid="B20">20</xref>-<xref ref-type="bibr" rid="B23">23</xref>]. Our report builds on the large number of CA studies in the scientific and educational literature. For example the nursing and nutritional health literatures are enriched by many CA studies [<xref ref-type="bibr" rid="B24">24</xref>-<xref ref-type="bibr" rid="B27">27</xref>]. Recent applications of CA include marketing drugs to women [<xref ref-type="bibr" rid="B28">28</xref>], and designing nutritional educational materials [<xref ref-type="bibr" rid="B29">29</xref>]. In the current milieu of interdisciplinary education, the inclusion of health and nutritional students and faculty, the groups actively using CA, have not been highlighted [<xref ref-type="bibr" rid="B30">30</xref>,<xref ref-type="bibr" rid="B31">31</xref>]. Also recent medical educator physician critics of PBL [<xref ref-type="bibr" rid="B32">32</xref>] and PBL defenders [<xref ref-type="bibr" rid="B33">33</xref>] may find benefit from applying CA to PBL seminars.</p><p>Based upon this analysis, future studies with more adequate instruction, larger sample sizes, and with longer duration may be able to demonstrat<bold>e</bold> that CA be a universal instrument to study the impact and improvements of PBL on training medical students in clinical thinking.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>In conclusion CA was applied to a multi-university real time PBL medical student clinical training webinar. Although the results are preliminary, mostly limited by the small sample size and short duration of the study, the CA technology may have the potential to improve PBL training for medical students; the challenges presented here warrant further investigation.</p><sec><title>Ethical approval</title><p>Ethical approval for educational studies and surveys is not required according to national practice in Japan. However, the study adhered to ethical principles, and the respondents are not identifiable from the data.</p></sec></sec><sec><title>Abbreviations</title><p>PBL: Problem-based learning; CA: Content analysis; Webinar: Web-based seminar; PIM: Practical inquiry model; OSCE: Objective structured clinical examination.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors&#x02019; contributions</title><p>YT conceived of the study and designed the coding tables; GHS, KE and YTakata drafted the manuscript, KE, AA and YTokuda collected, coded and analyzed the data; SK, YK, HH, HK, HTokunaga MO, TM, HK and HTerasawa participated in the design and coordination of the medical students and technical recordings. All authors read and approved the final manuscript.</p></sec><sec><title>Authors&#x02019; information</title><p>Yukari Takata is a senior evaluation specialist at IQ Solutions in Rockville, Maryland. She recently received the PhD in health education and mass communications at the College of Journalism and Communications, University of Florida, Gainesville, Florida, USA.</p><p>Gerald H. Stein is a Clinical Assistant Professor at the Department of Medicine, School of Medicine, University of Florida, Gainesville, Florida, and a Physician Consultant at the Clinic of Jurisdiction, Veteran Affairs Medical Center, Gainesville, Florida, USA.</p><p>Kuniyuki Endo is a staff physician at the Department of Neurology, Nagoya Daini Red Cross Hospital, Nagoya, Aichi, Japan.</p><p>Akiko Arai is a candidate for the PhD at the College of Health and Human performance, University of Florida, Gainesville, Florida, USA.</p><p>Shun Kohsaka is an Assistant Professor at the Department of Cardiology, School of Medicine, Keio University, Tokyo, Japan.</p><p>Yuka Kitano is an Assistant Professor at the Department of, Emergency Medicine and Critical Care Medicine, School of Medicine, St. Marianna University, Kawasaki, Kanagawa, Japan.</p><p>Hitoshi Honda is an Attending Physician and consultant, Departments of General Internal Medicine and Infectious Diseases, Teine Keijinkai Medical Center, Sapporo, Hokkaido, Japan.</p><p>Hidetaka Kitazono is an Attending Physician and consultant, Departments of General Internal Medicine and Infectious Diseases Tokyo Bay Medical Center, Noguchi Hideyo Memorial Noguchi International Hospital, Urayasu, Chiba, Japan.</p><p>Hironobu Tokunaga MD, is an Assistant Professor at the Department of Emergency Medicine, University of Fukui Faculty of Medical Sciences, Fukui, Japan.</p><p>Yasuharu Tokuda, MD, MPH, is a Professor at the Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukub, School of Medicine, Tsukuba, Japan.</p><p>Mikako Obika, MD, is an Assistant Professor at the Center for Graduate Medical Education, Okayama, Okayama University Medical School, Japan.</p><p>Tomoko Miyoshi, MD, is a Professor at the Department of Medical Education and Primary Care, Okayama University Medical School, Okayama, Japan.</p><p>Hitomi Kataoka, MD, is a Professor at the Department of Medical Education and Primary Care, Okayama University Medical School, Okayama, Japan.</p><p>Hidekazu Terasawa, MD, is a Professor at the Department of Emergency Medicine, University of Fukui Faculty of Medical Sciences, Fukui, Japan.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6920/13/156/prepub">http://www.biomedcentral.com/1472-6920/13/156/prepub</ext-link></p></sec>

Clinical course after corticosteroid therapy in IgG4-related aortitis/periaortitis and periarteritis: a retrospective multicenter study

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<contrib contrib-type="author"><name><surname>Mizushima</surname><given-names>Ichiro</given-names></name><address><email>ichiro7753@yahoo.co.jp</email></address><xref ref-type="aff" rid="Aff26"/></contrib><contrib contrib-type="author"><name><surname>Inoue</surname><given-names>Dai</given-names></name><address><email>d-inoue@lake.ocn.ne.jp</email></address><xref ref-type="aff" rid="Aff27"/></contrib><contrib contrib-type="author"><name><surname>Yamamoto</surname><given-names>Motohisa</given-names></name><address><email>mocha0422jp@yahoo.co.jp</email></address><xref ref-type="aff" rid="Aff28"/></contrib><contrib contrib-type="author"><name><surname>Yamada</surname><given-names>Kazunori</given-names></name><address><email>kyamada50@yahoo.co.jp</email></address><xref ref-type="aff" rid="Aff26"/></contrib><contrib contrib-type="author"><name><surname>Saeki</surname><given-names>Takako</given-names></name><address><email>tsaeki04@gmail.com</email></address><xref ref-type="aff" rid="Aff29"/></contrib><contrib contrib-type="author"><name><surname>Ubara</surname><given-names>Yoshifumi</given-names></name><address><email>ubara@toranomon.gr.jp</email></address><xref ref-type="aff" rid="Aff30"/></contrib><contrib contrib-type="author"><name><surname>Matsui</surname><given-names>Shoko</given-names></name><address><email>shoko7199@gmail.com</email></address><xref ref-type="aff" rid="Aff31"/></contrib><contrib contrib-type="author"><name><surname>Masaki</surname><given-names>Yasufumi</given-names></name><address><email>yasum1macky1@gmail.com</email></address><xref ref-type="aff" rid="Aff32"/></contrib><contrib contrib-type="author"><name><surname>Wada</surname><given-names>Takashi</given-names></name><address><email>twada@m-kanazawa.jp</email></address><xref ref-type="aff" rid="Aff33"/></contrib><contrib contrib-type="author"><name><surname>Kasashima</surname><given-names>Satomi</given-names></name><address><email>sato-kasa@kinbyou.hosp.go.jp</email></address><xref ref-type="aff" rid="Aff34"/></contrib><contrib contrib-type="author"><name><surname>Harada</surname><given-names>Kenichi</given-names></name><address><email>kenichih@med.kanazawa-u.ac.jp</email></address><xref ref-type="aff" rid="Aff35"/></contrib><contrib contrib-type="author"><name><surname>Takahashi</surname><given-names>Hiroki</given-names></name><address><email>htakahas@sapmed.ac.jp</email></address><xref ref-type="aff" rid="Aff28"/></contrib><contrib contrib-type="author"><name><surname>Notohara</surname><given-names>Kenji</given-names></name><address><email>notohara@kchnet.or.jp</email></address><xref ref-type="aff" rid="Aff36"/></contrib><contrib contrib-type="author"><name><surname>Nakanuma</surname><given-names>Yasuni</given-names></name><address><email>pbcpsc@kenroku.kanazawa-u.ac.jp</email></address><xref ref-type="aff" rid="Aff35"/></contrib><contrib contrib-type="author"><name><surname>Umehara</surname><given-names>Hisanori</given-names></name><address><email>umehara606@gmail.com</email></address><xref ref-type="aff" rid="Aff32"/></contrib><contrib contrib-type="author"><name><surname>Yamagishi</surname><given-names>Masakazu</given-names></name><address><email>myamagi@med.kanazawa-u.ac.jp</email></address><xref ref-type="aff" rid="Aff37"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kawano</surname><given-names>Mitsuhiro</given-names></name><address><email>sk33166@gmail.com</email></address><xref ref-type="aff" rid="Aff26"/></contrib><aff id="Aff26"><label/>Division of Rheumatology, Department of Internal Medicine, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa Ishikawa, 920-8640 Japan </aff><aff id="Aff27"><label/>Department of Radiology, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8640 Japan </aff><aff id="Aff28"><label/>The First Department of Internal Medicine, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo, Hokkaido, 060-8543 Japan </aff><aff id="Aff29"><label/>Department of Internal Medicine, Nagaoka Red Cross Hospital, 2-297-1 Senshu, Nagaoka, Niigata, 940-2085 Japan </aff><aff id="Aff30"><label/>Nephrology Center, Toranomon Hospital, 1-3-1 Kajigaya, Takatsu, Kawasaki, Kanagawa, 212-0015 Japan </aff><aff id="Aff31"><label/>Health Administration Center, Sugitani Campus, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama, 930-0194 Japan </aff><aff id="Aff32"><label/>Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahokugun, Ishikawa, 920-0293 Japan </aff><aff id="Aff33"><label/>Division of Nephrology, Department of Laboratory Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8640 Japan </aff><aff id="Aff34"><label/>Department of Pathology and Clinical Laboratory, National Hospital Organization, Kanazawa Medical Center, 1-1 Shimoisibikimachi, Kanazawa, Ishikawa, 920-8650 Japan </aff><aff id="Aff35"><label/>Department of Human Pathology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8640 Japan </aff><aff id="Aff36"><label/>Department of Pathology, Kurashiki Central Hospital, Miwa, Kurashiki, Okayama, 710-8602 Japan </aff><aff id="Aff37"><label/>Division of Cardiology, Department of Internal Medicine, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8640 Japan </aff>

Arthritis Research &#x00026; Therapy

<sec id="Sec1" sec-type="intro"><title>Introduction</title><p>Immunoglobulin G4 (IgG4)&#x02013;related disease (IgG4-RD) is a recently recognized systemic inflammatory disease with multiorgan involvement [<xref ref-type="bibr" rid="CR1">1</xref>&#x02013;<xref ref-type="bibr" rid="CR3">3</xref>]. IgG4-RD is characterized by tumefactive lesions, a dense lymphoplasmacytic infiltration with abundant IgG4-positive plasma cells, storiform fibrosis and elevated serum IgG4 levels. Writing from a pathological viewpoint, Stone <italic>et al</italic>. [<xref ref-type="bibr" rid="CR4">4</xref>] and Kasashima <italic>et al</italic>. [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR6">6</xref>] described some patients with chronic aortitis/periaortitis or inflammatory aortic aneurysm as also having an IgG4-related condition. Microscopically, these lesions have a predilection for the adventitia and periaortic/periarterial tissue, although they also affect the media, indicating the disease have not just a periaortitis component but also an aortitis one [<xref ref-type="bibr" rid="CR4">4</xref>&#x02013;<xref ref-type="bibr" rid="CR9">9</xref>]. In addition, Inoue <italic>et al</italic>. reported characteristic computed tomography (CT) findings of 17 Japanese patients with IgG4-related periaortitis and/or periarteritis. Macroscopically, this disease represents periaortic or periarterial, circumferential or partial, thickened or masslike lesions with or without aneurysmal change [<xref ref-type="bibr" rid="CR10">10</xref>]. Presumably, IgG4-related periaortitis and periarteritis may have some overlap with IgG4-related retroperitoneal fibrosis. Inoue <italic>et al</italic>. proposed that this discrimination is dependent on the predominant location of the lesions. They deemed <italic>periaortitis</italic> appropriate to refer to lesions with predominant periaortic and concentric involvement, whereas periureteral or plaquelike lesions should be referred to as <italic>retroperitoneal fibrosis</italic>[<xref ref-type="bibr" rid="CR10">10</xref>]. In this context, the concept of IgG4-related aortitis/periaortitis and periarteritis (PAo/PA) has been proposed [<xref ref-type="bibr" rid="CR9">9</xref>]. However, the clinical characteristics and course after corticosteroid therapy in patients with IgG4-related PAo/PA have not been well-clarified. Moreover, although corticosteroid therapy has been suspected to increase the risk of aneurysm formation or rupture [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR10">10</xref>], the precise incidence of these complications and their timing in the clinical course have not been elucidated.</p><p>This state of affairs prompted us to undertake the present study to clarify the clinical characteristics and course after corticosteroid therapy in patients with IgG4-related PAo/PA.</p></sec><sec id="Sec2" sec-type="methods"><title>Methods</title><sec id="Sec3"><title>Patients</title><p>From among 333 patients with IgG4-RD at Kanazawa University Hospital, Sapporo Medical University Hospital, Nagaoka Red Cross Hospital, Toranomon Hospital, Toyama University Hospital and Kanazawa Medical University Hospital between 1 January 1995 and 30 September 2013, we identified 40 with IgG4-related PAo/PA (Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>). The diagnosis of this disease was made on the basis of the presence of consistent periaortic/periarterial radiological findings, the fulfillment of the published comprehensive diagnostic criteria (CDC) [<xref ref-type="bibr" rid="CR11">11</xref>] or each organ-specific diagnostic criteria [<xref ref-type="bibr" rid="CR12">12</xref>&#x02013;<xref ref-type="bibr" rid="CR14">14</xref>] and exclusion of other diseases. The diagnosis of extravascular lesions was made on the basis of physical examination, imaging findings and/or histopathological examination, in addition to exclusion of other conditions. According to the CDC, 25 patients (patients 2 through 5, 7, 8, 10, 11, 13 through 17, 19, 21 through 23, 26 through 28, 32, 34 through 36 and 38 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) were diagnosed with definite IgG4-RD, three (patients 1, 9 and 37) with probable IgG4-RD and 12 (patients 6, 12, 18, 20, 24, 25, 29 through 31, 33, 39 and 40) with possible IgG4-RD. Three (patients 6, 18 and 29) of these twelve patients fulfilled the revised diagnostic criteria for autoimmune pancreatitis (AIP) [<xref ref-type="bibr" rid="CR13">13</xref>]. Well-experienced physicians of this disease diagnosed the remaining nine patients with IgG4-RD on the basis of a consistent clinical picture with elevated serum IgG4 concentrations and exclusion of other diseases. Twenty-nine (80.6%) of thirty-six patients who had extravascular IgG4-related organ involvement underwent biopsy of affected organs and showed histologically typical light microscopic findings [<xref ref-type="bibr" rid="CR15">15</xref>] and copious IgG4-positive plasma cell infiltration. Histological evaluation of periaortic/periarterial lesions was performed in only one patient (patient 37) by means of incisional biopsy of the periaortic mass lesions, which did not show any vascular structures but histological findings compatible with IgG4-related retroperitoneal fibrosis. We retrospectively evaluated baseline clinical features, including subjective symptoms, laboratory data and imaging findings, in these 40 patients. Because follow-up data were absent or inadequate for seven patients (patients 1, 2, 21, 33, 37, 39 and 40), we limited the analysis of the clinical course to the remaining thirty-three patients (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>). Two patients (patients 8 and 17) had been included in earlier studies ([<xref ref-type="bibr" rid="CR16">16</xref>] and [<xref ref-type="bibr" rid="CR17">17</xref>], respectively).<table-wrap id="Tab1"><label>Table 1</label><caption><p> <bold>Baseline characteristics of 40 patients with IgG4-related aortitis/periaortitis and periarteritis</bold> </p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th/><th/><th/><th/><th/><th rowspan="3">Location of vascular lesion</th><th rowspan="3">Luminal dilatation before Tx</th><th/><th/><th rowspan="3">Risk factor of arteriosclerosis</th><th>Initial PSL Tx (mg/day)</th></tr><tr><th/><th>Follow-up</th><th>IgG</th><th>IgG4</th><th>IgE</th><th>CRP</th><th>Extravascular lesions</th><th/><th/></tr><tr><th>Patient</th><th>(mo)</th><th>(mg/dl)</th><th>(mg/dl)</th><th>(IU/ml)</th><th>(mg/dl)</th><th/><th>Symptoms</th><th/></tr></thead><tbody><tr><td>1</td><td>204</td><td>1,894</td><td>128</td><td>543</td><td>6.2</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa</td><td>abdo P, fever</td><td>(&#x02212;)</td><td>30</td></tr><tr><td>2</td><td>120</td><td>2,840</td><td>693</td><td>468</td><td>0.1</td><td>AA</td><td>(&#x02212;)</td><td>Sa</td><td>(&#x02212;)</td><td>DM, Sm</td><td>30</td></tr><tr><td>3</td><td>96</td><td>5,970</td><td>3,100</td><td>259</td><td>1.56</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa, Hy, Pa</td><td>malaise</td><td>DM, HT</td><td>40</td></tr><tr><td>4</td><td>78</td><td>2,140</td><td>557</td><td>266</td><td>&#x0003c;0.10</td><td>AA, IA</td><td>(+)</td><td>La, Sa</td><td>(&#x02212;)</td><td>DL, Sm</td><td>50</td></tr><tr><td>5</td><td>70</td><td>1,500</td><td>173</td><td>151</td><td>0.3</td><td>IA, SMA</td><td>(&#x02212;)</td><td>Pa, RF</td><td>abdo P</td><td>DM, HT, DL, Sm</td><td>20</td></tr><tr><td>6</td><td>63</td><td>2,970</td><td>1,330</td><td>419</td><td>&#x0003c;0.10</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa, Pa, Ki, RF</td><td>malaise</td><td>DM, HT, DL, Sm</td><td>40</td></tr><tr><td>7</td><td>63</td><td>2,130</td><td>715</td><td>253</td><td>&#x0003c;0.10</td><td>IA</td><td>(&#x02212;)</td><td>La, Sa</td><td>(&#x02212;)</td><td>DM, DL, Sm</td><td>40</td></tr><tr><td>8</td><td>63</td><td>2,731</td><td>269</td><td>975</td><td>0.6</td><td>AA</td><td>(&#x02212;)</td><td>Sa, RF</td><td>(&#x02212;)</td><td>DM, HT, DL, Sm</td><td>30</td></tr><tr><td>9</td><td>57</td><td>1,790^b </td><td>105^b </td><td>212^b </td><td>1.5^b </td><td>AA, IA</td><td>(+)</td><td>Hy, Lu, Pa, Ly</td><td>(&#x02212;)</td><td>DL</td><td>40</td></tr><tr><td>10</td><td>54</td><td>2,570</td><td>1,420</td><td>345</td><td>0.3</td><td>AA, IA</td><td>(+)</td><td>Pa</td><td>(&#x02212;)</td><td>DM, HT</td><td>20</td></tr><tr><td>11</td><td>48</td><td>2,950</td><td>1,540</td><td>7.9</td><td>&#x0003c;0.1</td><td>AA</td><td>(&#x02212;)</td><td>Sa, Bi, Pa, Pr</td><td>(&#x02212;)</td><td>DM</td><td>30</td></tr><tr><td>12</td><td>43</td><td>1,487</td><td>196</td><td>447</td><td>0.6</td><td>AA, IA</td><td>(&#x02212;)</td><td>RF</td><td>abdo P</td><td>DM, HT, DL, Sm</td><td>0</td></tr><tr><td>13</td><td>37</td><td>2,563</td><td>1,330</td><td>283</td><td>0.09</td><td>AA</td><td>(&#x02212;)</td><td>Sa, Ki</td><td>pollakiuria</td><td>DL, Sm</td><td>45</td></tr><tr><td>14</td><td>35</td><td>2,319</td><td>734</td><td>542</td><td>1.19</td><td>TA, AA</td><td>(&#x02212;)</td><td>Sa, Pa, Ki</td><td>(&#x02212;)</td><td>DM, DL</td><td>40</td></tr><tr><td>15</td><td>34</td><td>1,458</td><td>158</td><td>452</td><td>0.22</td><td>AA</td><td>(&#x02212;)</td><td>Sa, Ly</td><td>(&#x02212;)</td><td>DM, HT, DL, Sm</td><td>30</td></tr><tr><td>16</td><td>27</td><td>2,081</td><td>870</td><td>1,285</td><td>0.0</td><td>AA, IA</td><td>(&#x02212;)</td><td>Sa, RF, Pr</td><td>(&#x02212;)</td><td>DM, HT</td><td>20</td></tr><tr><td>17</td><td>27</td><td>1,756</td><td>408</td><td>513</td><td>0.2</td><td>AA, IA</td><td>(&#x02212;)</td><td>Sa, Pa, Ki</td><td>(&#x02212;)</td><td>DM, HT, DL, Sm</td><td>20</td></tr><tr><td>18</td><td>27</td><td>1,762</td><td>144</td><td>24</td><td>0.32</td><td>AA, IA</td><td>(+)</td><td>Pa</td><td>(&#x02212;)</td><td>DL, Sm</td><td>20</td></tr><tr><td>19</td><td>25</td><td>2,024</td><td>292</td><td>1,400</td><td>0.14</td><td>AA</td><td>(&#x02212;)</td><td>Pa, RF</td><td>(&#x02212;)</td><td>DM, HT, Sm</td><td>40</td></tr><tr><td>20</td><td>24</td><td>2,262</td><td>299</td><td>443</td><td>&#x0003c;0.05</td><td>AA</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>HT</td><td>0</td></tr><tr><td>21</td><td>24</td><td>2,184</td><td>236</td><td>365</td><td>0.3</td><td>AA</td><td>(+)</td><td>La, Sa</td><td>fever</td><td>DM, HT, DL</td><td>30</td></tr><tr><td>22</td><td>22</td><td>3,484</td><td>1,896</td><td>247</td><td>0.0</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa, Pa, Ki, Ly</td><td>(&#x02212;)</td><td>Sm</td><td>35</td></tr><tr><td>23</td><td>15</td><td>4,171</td><td>2,120</td><td>&#x0003c;20</td><td>0.32</td><td>AA, IA</td><td>(&#x02212;)</td><td>La, Sa, Ki</td><td>(&#x02212;)</td><td>DM, HT, Sm</td><td>40</td></tr><tr><td>24</td><td>13</td><td>1,837</td><td>261</td><td>687</td><td>0.06</td><td>IA</td><td>(&#x02212;)</td><td>RF</td><td>(&#x02212;)</td><td>HT, DL</td><td>30</td></tr><tr><td>25</td><td>13</td><td>1,454</td><td>196</td><td>350</td><td>0.13</td><td>AA, IA</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>abdo P</td><td>Sm</td><td>15</td></tr><tr><td>26</td><td>10</td><td>2,213</td><td>455</td><td>NA</td><td>0.56</td><td>AA, IA</td><td>(&#x02212;)</td><td>Sa, Ki</td><td>arthralgia</td><td>HT, DL, Sm</td><td>40</td></tr><tr><td>27</td><td>10</td><td>3,120</td><td>1,020</td><td>1,760</td><td>1.5</td><td>AA, IA, IMA</td><td>(&#x02212;)</td><td>La, Sa</td><td>hoarseness, fever</td><td>Sm</td><td>30</td></tr><tr><td>28</td><td>9</td><td>2,936</td><td>1,070</td><td>17</td><td>0.0</td><td>TA, AA, IA</td><td>(&#x02212;)</td><td>La, Sa, Pa, Ki</td><td>thirst</td><td>DM, DL, Sm</td><td>40</td></tr><tr><td>29</td><td>9</td><td>10,121</td><td>2,500</td><td>&#x0003c;20</td><td>0.37</td><td>IA</td><td>(+)</td><td>Pa, Ki, Ly</td><td>malaise</td><td>DM</td><td>50</td></tr><tr><td>30</td><td>9</td><td>1,200</td><td>147</td><td>NA</td><td>2.94</td><td>AA, IA</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>fever, malaise</td><td>DM, HT</td><td>30</td></tr><tr><td>31</td><td>8</td><td>1,475</td><td>210</td><td>111</td><td>0.3</td><td>IA</td><td>(&#x02212;)</td><td>Sa</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>40</td></tr><tr><td>32</td><td>4</td><td>2,938</td><td>1,520</td><td>48</td><td>0.1</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa, Ki, RF</td><td>(&#x02212;)</td><td>Sm</td><td>30</td></tr><tr><td>33</td><td>4</td><td>1,463</td><td>672</td><td>216</td><td>0.13</td><td>AA</td><td>(&#x02212;)</td><td>Sa, Pl, Ca</td><td>(&#x02212;)</td><td>Sm</td><td>0</td></tr><tr><td>34</td><td>3</td><td>2,439</td><td>782</td><td>703</td><td>0.2</td><td>AA, IA</td><td>(&#x02212;)</td><td>La, Ki, RF</td><td>(&#x02212;)</td><td>DM, DL, Sm</td><td>20</td></tr><tr><td>35</td><td>3</td><td>2,244</td><td>503</td><td>311</td><td>0.0</td><td>AA, IA</td><td>(&#x02212;)</td><td>Ki</td><td>edema</td><td>(&#x02212;)</td><td>30</td></tr><tr><td>36</td><td>2</td><td>1,950</td><td>711</td><td>737</td><td>0.0</td><td>AA</td><td>(&#x02212;)</td><td>La, Sa, Lu, Ki</td><td>(&#x02212;)</td><td>Sm</td><td>35</td></tr><tr><td>37</td><td>2</td><td>1,328</td><td>106</td><td>19</td><td>0.28</td><td>AA, IA</td><td>(+)</td><td>RF</td><td>(&#x02212;)</td><td>DL</td><td>0</td></tr><tr><td>38</td><td>1</td><td>4,420</td><td>2,680</td><td>174</td><td>0.1</td><td>IA</td><td>(&#x02212;)</td><td>La, Sa, Bi, Pa, Ki, Ne</td><td>diarrhea</td><td>Sm</td><td>50</td></tr><tr><td>39</td><td>1</td><td>2,276</td><td>835</td><td>&#x0003c;20</td><td>0.91</td><td>IA</td><td>(&#x02212;)</td><td>Sa</td><td>(&#x02212;)</td><td>HT</td><td>15</td></tr><tr><td>40</td><td>1</td><td>1,600</td><td>206</td><td>212</td><td>0.38</td><td>AA</td><td>(&#x02212;)</td><td>(&#x02212;)</td><td>abdo P, malaise</td><td>Sm</td><td>30</td></tr></tbody></table><table-wrap-foot><p> <italic>AA,</italic> Abdominal aorta; <italic>abdo,</italic> Abdominal; <italic>Bi,</italic> Bile tract; <italic>Ca,</italic> Pericarditis; <italic>CRP,</italic> C-reactive protein; <italic>DL,</italic> Dyslipidemia; <italic>DM,</italic> Diabetes mellitus; <italic>F,</italic> Female; <italic>HT,</italic> Hypertension; <italic>Hy,</italic> Hypophysitis; <italic>IA,</italic> Iliac artery; <italic>IgE,</italic> Serum immunoglobulin E at diagnosis; <italic>IgG,</italic> Serum immunoglobulin G at diagnosis; <italic>IgG4,</italic> Serum immunoglobulin G4 at diagnosis; <italic>IMA,</italic> Inferior mesenteric artery; <italic>Ki,</italic> IgG4-related kidney disease; <italic>La,</italic> Lacrimal gland, <italic>Lu,</italic> Lung; <italic>Ly,</italic> Lymph node; <italic>M,</italic> Male; M<italic>o,</italic> month; <italic>NA,</italic> Not available; <italic>Ne,</italic> Nerve; <italic>P,</italic> Pain; <italic>Pa,</italic> Pancreas; <italic>Pl,</italic> Pleuritis; <italic>Pr, P</italic>rostate; <italic>PSL,</italic> Prednisolone; <italic>RF,</italic> Retroperitoneal fibrosis; <italic>Sa,</italic> Salivary gland; <italic>Sm,</italic> Past or current smoking; <italic>SMA,</italic> Superior mesenteric artery; TA, Thoracic aorta; <italic>Tx,</italic> Treatment. ^bValue under corticosteroid therapy.</p></table-wrap-foot></table-wrap><fig id="Fig1"><label>Figure 1</label><caption><p> <bold>Flowchart of participants through the study.</bold> CS, corticosteroid; IgG4-RD, IgG4-related disease; Tbc, tuberculosis; Tx, treatment.</p></caption><graphic xlink:href="13075_2014_4377_Fig1_HTML" id="d30e2407"/></fig></p><p>This study was approved by the Medical Ethics Committee of Kanazawa University, the institutional review board of Sapporo Medical University Hospital, the Ethics Committee of Nagaoka Red Cross Hospital, the institutional review board of Toranomon Hospital, the review board of the University of Toyama and the Research Ethics Committee of Kanazawa Medical University. Informed consent for publication of all data and samples was obtained from each patient. The research was conducted in compliance with the Declaration of Helsinki.</p></sec><sec id="Sec4"><title>Imaging evaluation</title><p>All patients underwent whole-body CT examinations at the time of the initial diagnosis, and follow-up CT data were available for 33 patients, 31 of whom received corticosteroid therapy. All imaging data were reviewed by a single radiologist with extensive experience in IgG4-RD at Kanazawa University Hospital. Periaortic/periarterial lesions were described as circumferential or partial thickened wall of the affected aortas/arteries with homogeneous enhancement visualized by contrast-enhanced CT. At the time of diagnosis, we also evaluated the findings of 2-[¹⁸F]-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT) for 20 patients and of gallium scintigraphy for 12 patients.</p><p>At the time of initial diagnostic CT imaging, after noting the affected site of aortas/arteries and extravascular lesions, we measured the maximum vascular wall thickness and diameter of the lumen in both affected and adjacent sites in each lesion. These two values were then longitudinally evaluated in the 33 patients whose follow-up imaging and clinical course information were available.</p><p>Improvement and relapse of periaortic/periarterial lesions during the clinical course were defined as decrease and reincrease of vascular wall thickness, respectively, at the same site as the maximum vascular wall thickness measured at the time of initial diagnosis. Luminal dilatation of periaortic/periarterial lesions was defined as being present when the luminal diameter of the affected site was more than 3 mm larger than that of adjacent normal sites. Exacerbation of luminal dilatation was defined as being present when more than 5-mm expansion of the luminal diameter was observed at the same site as the luminal dilatation detected at the time of initial diagnosis.</p></sec><sec id="Sec5"><title>Statistical analysis</title><p>Statistical analysis was performed using SPSS version 19 software (IBM SPSS, Chicago, IL, USA). The significance of differences between groups was determined using Mann&#x02013;Whitney <italic>U</italic> test or Wilcoxon signed-rank test, and the significance of differences in frequencies was analyzed with Fisher&#x02019;s exact probability test. Data are presented as means &#x000b1; SD. Significant differences were defined as <italic>P</italic> &#x0003c; 0.05.</p></sec></sec><sec id="Sec6" sec-type="results"><title>Results</title><sec id="Sec7"><title>Baseline characteristics</title><p>The baseline clinical characteristics of 40 patients are shown in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>. Our patient group was composed of 37 men and 3 women with an average age of 66.4 &#x000b1; 7.1 years (age range, 44 to 75). One patient (patient 9 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) had been treated with prednisolone (PSL) at a dose of 5.0 mg/day for type 1 AIP. None of the other 39 patients had been treated with any immunosuppressants, including corticosteroids, before their diagnosis. Thirty-six patients (90.0%) had more than one IgG4-related extravascular lesion (average, 2.3 &#x000b1; 1.5 organs; range, 0 to 6 organs). Involvement of the salivary gland was observed in 25 patients (62.5%), lacrimal gland in 14 (35.0%), pancreas in 14 (35.0%), kidney in 13 (32.5%), retroperitoneum in 13 (32.5%), prostate in 6 (15.0%), lung in 3 (7.5%) and hepatobiliary tract and hypophysis in 2 each (5.0%). The frequency of subjective symptoms was low (fever, 10.0%; abdominal pain, 12.5%; general malaise, 12.5%). Moreover, five of seven patients with luminal dilatation of the affected lesions at the time of diagnosis complained of no subjective symptoms. With regard to the major risk factors of atherosclerosis, diabetes mellitus (DM) was present at the time of diagnosis in 20 patients (50.0%), hypertension (HT) in 17 (42.5%), dyslipidemia (DL) in 18 (45.0%), current smoking in 9 (22.5%) and past smoking in 15 (37.5%). The mean follow-up period of all 40 patients after diagnosis was 33.9 &#x000b1; 39.8 months (range, 1 to 204 months).</p><p>At diagnosis, 37 (92.5%) of 40 patients showed elevated serum IgG4 levels exceeding 135 mg/dl (average, 815 &#x000b1; 771 mg/dl; range, 105 to 3,100 mg/dl). Thirty-one (77.5%) of forty patients showed elevated serum IgG levels (average, 2,551 &#x000b1; 1,543 mg/dl; range, 1,200 to 10,121 mg/dl; normal range, 870 to 1,700 mg/dl). Twenty-three (60.5%) of thirty-eight evaluated patients showed elevated serum IgE levels (average, 403 &#x000b1; 398 IU/ml; range, 7.9 to 1,760 IU/ml; normal range, &#x0003c;250 IU/ml). Although none of the patients had leukocytosis, 16 (41.0%) of 39 evaluated patients had eosinophilia (eosinophils &#x0003e;5%). Six (15.4%) of thirty-nine evaluated patients had hypocomplementemia. Antinuclear antibodies were positive in 16 (40.0%) of 40 patients and the rheumatoid factor in only 3 (8.1%) of 37 evaluated patients. Myeloperoxidase antineutrophil cytoplasmic antibodies (ANCAs) and proteinase 3 ANCAs were not observed in any of the evaluated patients (21 and 14 patients, respectively). Only six (15.0%) of forty patients had elevated serum C-reactive protein (CRP) level (CRP &#x0003e;1 mg/dl).</p></sec><sec id="Sec8"><title>Treatment</title><p>The respective attending physicians decided the indications for treatment and the treatment regimen. Thirty-six of forty patients were treated with PSL at an average initial dose of 32.6 &#x000b1; 9.7 mg/day (range, 15 to 50 mg/day) for the lesions associated with IgG4-RD. Only one patient (patient 1 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) received cyclophosphamide in addition to PSL. Endovascular aortic repair (EVAR) was performed for the periaortic lesions with marked luminal dilatation before corticosteroid therapy in one patient (patient 18) to prevent rupture. We excluded five patients (patients 1, 2, 21, 39 and 40) from the analysis of the clinical course because their follow-up imaging data were not available. During the clinical course of the other 31 patients, the initial PSL dose was generally continued until 2 to 4 weeks after the start of therapy and then gradually tapered. The PSL dose was tapered to 5 to 10 mg/day by 12 months in 18 (94.7%) of 19 patients whose follow-up period was more than 12 months. The average PSL dose at the last review was 10.0 &#x000b1; 9.2 mg/day. Because one patient (patient 12) showed strong positivity in the tuberculin skin test and interferon &#x003b3; release assays, he was treated with antituberculosis therapy only. The other three patients (patients 20, 33 and 37) were observed without any treatment (Figure&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>).</p></sec><sec id="Sec9"><title>Radiological findings at diagnosis</title><p>CT images revealed thickened lesions surrounding the aorta/artery in all patients. The affected aorta/artery data were for two thoracic aortas (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>A), thirty-three abdominal aortas (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>C), twenty-three iliac arteries (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>E), one superior mesenteric artery (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>G) and one inferior mesenteric artery. All 33 abdominal aortic lesions affected the infrarenal abdominal aorta, and only 6 lesions also affected the suprarenal abdominal aorta. CT also revealed typical extravascular lesions, mainly in the salivary glands, lacrimal glands, pancreas and kidney. Sixteen of twenty patients who underwent FDG-PET/CT, and only four of twelve patients who underwent gallium scintigraphy, showed significant uptake of the periaortic/periarterial lesions detected by CT.<fig id="Fig2"><label>Figure 2</label><caption><p> <bold>Contrast-enhanced computed tomography findings of periaortic/periarterial lesions and changes after corticosteroid therapy.</bold> A thoracic aortic lesion <bold>(A)</bold> had slightly improved 1 month after corticosteroid therapy <bold>(B)</bold>, an abdominal aortic lesion <bold>(C)</bold> and an iliac arterial lesion <bold>(E)</bold> had almost disappeared 10 months after therapy (<bold>D</bold> and <bold>F</bold>, respectively) and a superior mesenteric arterial lesion <bold>(G)</bold> showed fair improvement 2 months after therapy <bold>(H)</bold>. Significant pre- to posttherapy decreases in maximum wall thickness of periaortic/periarterial lesions were observed <bold>(I)</bold>. Tx, Treatment.</p></caption><graphic xlink:href="13075_2014_4377_Fig2_HTML" id="d30e2516"/></fig></p></sec><sec id="Sec10"><title>Changes in radiological findings of periaortic/periarterial lesions after corticosteroid therapy</title><p>After corticosteroid therapy, reduction in the thickness of the periaortic/periarterial lesions was observed during an average follow-up period of 30.1 &#x000b1; 26.2 months (range, 1 to 96 months) in 30 (96.8%) of 31 patients whose clinical course was analyzed (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>), although 1 patient (patient 9 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) experienced relapse during PSL tapering at a dose of 7.0 mg/day. The average vascular wall thickness of the 34 periaortic/periarterial lesions of 31 patients at the time of diagnosis (7.1 &#x000b1; 3.0 mm; range, 3 to 18 mm) significantly decreased after corticosteroid therapy (2.7 &#x000b1; 2.0 mm; range, 1 to 9 mm) (Figure&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>I). Generally, obvious radiographic improvement of more than 50% reduction in thickness was observed by 2 months after the start of therapy, after which point some patients showed further improvement and others showed almost no change (Figure&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>). Eighteen of the thirty-four lesions had almost completely disappeared by the time of the last review. The rate of improvement, relapse or complete disappearance of the perivascular lesions did not differ significantly between the patients with multiple versus single vascular involvement, between those with versus without a specific other organ involvement such as AIP or between the presence or absence of any of the specific risk factors of atherosclerosis.<fig id="Fig3"><label>Figure 3</label><caption><p> <bold>Changes in maximum vascular wall thickness of periaortic/periarterial lesions after the start of corticosteroid therapy.</bold> Data for patients who underwent follow-up computed tomography within 2 months after the start of therapy are shown.</p></caption><graphic xlink:href="13075_2014_4377_Fig3_HTML" id="d30e2545"/></fig></p></sec><sec id="Sec11"><title>Luminal changes after corticosteroid therapy</title><p>Of the 31 patients whose clinical course was analyzed after corticosteroid therapy, 5 (patients 4, 9, 10, 18 and 29 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) had luminal dilatation of the periaortic/periarterial lesions at the time of the initial CT (Figures&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>A, <xref rid="Fig4" ref-type="fig">4</xref>C and <xref rid="Fig4" ref-type="fig">4</xref>E) and three (patients 10, 18 and 29) of them were diagnosed as having inflammatory aneurysm. One (patient 18) of them was treated with EVAR and PSL administration and did not have exacerbation of the luminal dilatation during the follow-up. The other four patients received PSL administration alone, in two (patients 9 and 10) of whom (50%) the luminal dilatation was exacerbated 28 and 46 months after the start of therapy, respectively (Figures&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>B, <xref rid="Fig4" ref-type="fig">4</xref>D, <xref rid="Fig4" ref-type="fig">4</xref>F and <xref rid="Fig4" ref-type="fig">4</xref>G). Throughout the clinical course, patient 9 did not have hypertension and patient 10 received antihypertensive agents, which achieved good blood pressure control (below 140/90 mmHg). The luminal diameter was stable after corticosteroid therapy in the 26 patients without luminal dilatation at diagnosis (Figure&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>H).<fig id="Fig4"><label>Figure 4</label><caption><p> <bold>Exacerbation of luminal dilatation after corticosteroid therapy.</bold> Luminal dilatation (<bold>A</bold> and <bold>C</bold>, patient 10 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>; <bold>E</bold>, patient 9) of the periaortic/periarterial lesions at the time of diagnosis was exacerbated after corticosteroid therapy (<bold>B</bold>, <bold>D</bold> and <bold>F</bold>, respectively). Red lines in each of the three paired images (<bold>A</bold> and <bold>B</bold>, <bold>C</bold> and <bold>D</bold>, and <bold>E</bold> and <bold>F</bold>, respectively) have the same length. Of five lesions from four patients with luminal dilatation, three lesions from two patients (red and yellow) in the maximum luminal diameter increased <bold>(G)</bold>, whereas no obvious increase was observed in the 28 lesions of the 26 patients without luminal dilatation <bold>(H)</bold>. Tx, Treatment.</p></caption><graphic xlink:href="13075_2014_4377_Fig4_HTML" id="d30e2637"/></fig></p></sec><sec id="Sec12"><title>Outcome of patients without corticosteroid therapy</title><p>Four patients (patients 12, 20, 33 and 37 in Table&#x000a0;<xref rid="Tab1" ref-type="table">1</xref>) did not receive corticosteroid therapy. One patient (patient 12) treated with antituberculosis therapy had gradual improvement of serum IgG4 level, periaortic/periarterial lesions and retroperitoneal fibrosis. In another untreated patient (patient 20), periaortic/periarterial lesion showed no change during the 24-month follow-up period. No new appearance of luminal dilatation was observed in these two patients. Because follow-up imaging data of the other patients were lacking, we excluded them from the analysis of the clinical course.</p></sec></sec><sec id="Sec13" sec-type="discussion"><title>Discussion</title><p>We analyzed the clinical course after corticosteroid therapy in patients with IgG4-related PAo/PA. To our knowledge, this study is the largest to evaluate corticosteroid safety and effectiveness in preventing new aneurysm formation in patients without luminal dilatation of periaortic/periarterial lesions, as well as the risk for exacerbation of luminal dilatation of such lesions in patients with it before therapy.</p><p>Biopsy of periaortic/periarterial lesions may cause massive hemorrhage. In our study, we could not perform histopathological examinations of these lesions, with the single exception of patient 37, whose specimens obtained by incisional biopsy of periaortic mass lesions showed only findings compatible with IgG4-related retroperitoneal fibrosis because of a lack of vasculature structures. To compensate for this difficulty, the presence of IgG4-related extravascular lesions, in addition to serological and typical radiological findings, was helpful in making a diagnosis of IgG4-related PAo/PA.</p><p>In contrast to our present study, the frequency of extravascular lesions was low in several previous studies. In those studies, periaortic/periarterial lesions were at an advanced stage with frequent aneurysmal formation, and the diagnosis was based mainly on the histopathological findings of the periaortic/periarterial lesions themselves because surgical treatment was selected [<xref ref-type="bibr" rid="CR4">4</xref>, <xref ref-type="bibr" rid="CR6">6</xref>, <xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR18">18</xref>, <xref ref-type="bibr" rid="CR19">19</xref>]. In contrast, our cases seem to have been at an earlier stage, attributable to the fact that the identification of extravascular lesions and the recent greater awareness of these lesions facilitated the making of an early diagnosis.</p><p>Accordingly, in vasculature-restricted cases, the correct diagnosis of IgG4-RD is much more difficult to make. To diagnose such patients early, the indications and accuracy of CT-guided biopsy of periaortic/periarterial lesions should be investigated, the risks and benefits of a diagnostic trial of corticosteroid therapy should be evaluated and a search for other valuable and less-invasive diagnostic markers should be undertaken. In such cases, it is of great importance to exclude other differential diagnoses, such as malignancy, infections, autoimmune disease and drug reactions, which can mimic IgG4-related PAo/PA [<xref ref-type="bibr" rid="CR20">20</xref>, <xref ref-type="bibr" rid="CR21">21</xref>].</p><p>A definitive therapeutic strategy for IgG4-related PAo/PA has not been established, and to date indications for treatment and the type of treatment regimen have been decided by the respective attending physician. In type 1 AIP (IgG4-related pancreatitis), the pancreatic manifestation of IgG4-RD, consensus guidelines for treatment, which are based on copious clinical experience [<xref ref-type="bibr" rid="CR22">22</xref>], have been available since 2010 [<xref ref-type="bibr" rid="CR23">23</xref>]. In AIP patients, corticosteroid administration should be employed for patients with symptoms such as obstructive jaundice and abdominal and back pain. The initial oral PSL dose of 0.6 mg/kg/day, continuation of the initial dose for 2 to 4 weeks and tapering by 5 mg every 1 to 2 weeks to a maintenance dose (2.5 to 5 mg/day) over a period of 2 to 3 months are recommended. In our study, corticosteroid therapy was started at an initial PSL dose of less than 30 mg/day for eight patients, 30 to 40 mg/day for twenty-four patients and over 40 mg/day for four patients. In most of the patients whose follow-up period was more than 12 months, the PSL dose was tapered to 5 to 10 mg/day by 12 months. In this way, the initial therapy generally following the guidelines of AIP and maintenance therapy with relatively slow tapering were performed in our study, with good efficacy attained on the whole.</p><p>The results of this study suggest that luminal dilatation of affected lesions may actually occur during corticosteroid therapy in patients with IgG4-related PAo/PA. In past studies [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR10">10</xref>], it was speculated that corticosteroid and other immunosuppressive therapies might increase the risk of aneurysm rupture. Actually, an IgG4-RD patient with multiple aneurysms who died of aneurysm rupture after high-dose corticosteroid therapy has been reported [<xref ref-type="bibr" rid="CR24">24</xref>]. In our present study, of 31 patients treated with corticosteroid, exacerbation of luminal dilatation were observed in only 2 who had already had it before therapy. Because blood pressure was maintained below 140/90 mmHg in these patients throughout their clinical course, hemodynamics seemed not to have influenced the exacerbation of luminal dilatation in any obvious fashion. In contrast, no patient without luminal dilatation showed a new appearance of it after therapy. These results suggest that more careful observation during corticosteroid therapy may be necessary to detect further luminal dilatation early in IgG4-related PAo/PA patients with preexisting luminal dilatation. However, because no patient with luminal dilatation and only one patient without luminal dilatation were observed without corticosteroid therapy, the natural course of the disease or of preexisting dilatation was not clarified. Moreover, the small number of patients with luminal dilatation precluded statistical analysis of the influence of independent risk factors on the luminal dilatation in IgG4-related PAo/PA. Therefore, whether preexisting luminal dilatation, corticosteroid therapy or some other factor is an independent risk factor for the aneurysm formation or exacerbation in IgG4-related PAo/PA patients will have to be clarified through multivariate analysis in a larger prospective study.</p><p>This study&#x02019;s results appear to support the contention that corticosteroid therapy can prevent new appearance of luminal dilatation in patients without it before therapy. In two case reports of IgG4-related aortitis/periaortitis patients with ruptured aortic aneurysms [<xref ref-type="bibr" rid="CR25">25</xref>, <xref ref-type="bibr" rid="CR26">26</xref>], immunosuppressive agents, including corticosteroids, had not been administered before aneurysm rupture. These case reports suggest that IgG4-related PAo/PA is itself a risk for aneurysm formation resulting in rupture when the lesions are left untreated. However, considering that no patient without luminal dilatation showed new appearance of it after therapy in our study, it is reasonable to surmise that corticosteroid therapy improves periaortic/periarterial lesions and prevents aneurysm formation at the affected site.</p><p>Therapeutic alternatives to corticosteroids have not been well-established in IgG4-RD. In some case reports and small case series, some oral immunosuppressive drugs, including azathioprine [<xref ref-type="bibr" rid="CR27">27</xref>], methotrexate [<xref ref-type="bibr" rid="CR28">28</xref>] and mycophenolate mofetil [<xref ref-type="bibr" rid="CR29">29</xref>], have been reported to be effective. In addition, good effectiveness of rituximab, which eliminates B cells by binding the cell-surface marker CD20, has been described [<xref ref-type="bibr" rid="CR30">30</xref>, <xref ref-type="bibr" rid="CR31">31</xref>]. However, the efficacy of these drugs remains to be evaluated with regard to their effectiveness for periaortic/periarterial lesions and their influence on luminal dilatation in IgG4-related PAo/PA.</p><p>This study has a few limitations. First, the treatment regimen and follow-up protocols were inconsistent between patients because of its retrospective and multi-institutional nature. Second, although this study included more patients than past ones, the number of patients with luminal dilatation at the time of diagnosis was small. Third, the association between histopathological findings and clinical features could not be evaluated, because biopsy specimens for histopathological analysis of the periaortic/periarterial lesions could not be procured. Fourth, no patient with luminal dilatation at the time of diagnosis was observed without corticosteroid therapy.</p></sec><sec id="Sec14" sec-type="conclusions"><title>Conclusions</title><p>The results of our study show the possibility of latent existence and progression of periaortic/periarterial lesions, the efficacy of corticosteroid therapy in preventing new aneurysm formation in patients without luminal dilatation of periaortic/periarterial lesions, and the possibility that a small proportion of patients may actually experience luminal dilatation of periaortic/periarterial lesions in IgG4-related PAo/PA. To confirm the efficacy and safety of corticosteroid therapy in patients with versus without luminal dilatation, and to devise a more useful and safe treatment strategy, including administration of other immunosuppressants, a larger-scale prospective study is required.</p></sec>

Undergraduate medical education in general practice/family medicine throughout Europe &#x02013; a descriptive study

<sec><title>Background</title><p>It is increasingly becoming evident that a strong primary health care system is more likely to provide better population health, more equity in health throughout the population, and better use of economic resources, compared to systems that are oriented towards specialty care. Developing and maintaining a strong and sustainable primary health care requires that a substantial part of graduating doctors go into primary care. This in turn requires that general practice/family medicine (GP/FM) strongly influences the curricula in medical schools. In the present paper we aim at describing the extent of GP/FM teaching in medical schools throughout Europe, checking for the presence of GP/FM curricula and clinical teaching in GP offices.</p></sec><sec><title>Methods</title><p>A brief questionnaire was e-mailed to GP/FM or other professors at European medical universities.</p></sec><sec><title>Results</title><p>259 out of 400 existing universities in 39 European countries responded to our questionnaire. Out of these, 35 (13.5%) reported to have no GP/FM curriculum. These 35 medical faculties were located in 12 different European countries. In addition, 15 of the medical schools where a GP/FM curriculum did exist, reported that this curriculum did not include any clinical component (n&#x02009;=&#x02009;5), or that the clinical part of the course was very brief - less than one week, mostly only a few hours (n&#x02009;=&#x02009;10). In total, 50 universities (19%) thus had no or a very brief GP/FM curriculum. These were mainly located in the Eastern or Southern European regions.</p></sec><sec><title>Conclusion</title><p>It is still possible to graduate from European medical universities without having been exposed to a GP/FM curriculum. The European Academy of Teachers in General Practice (EURACT) will launch efforts to change this situation.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Brekke</surname><given-names>Mette</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>mette.brekke@medisin.uio.no</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Carelli</surname><given-names>Francesco</given-names></name><xref ref-type="aff" rid="I2">2</xref><xref ref-type="aff" rid="I3">3</xref><email>francesco.carelli@alice.it</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Zarbailov</surname><given-names>Natalia</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>zarbailovnatalia@gmail.com</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Javashvili</surname><given-names>Givi</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>gjavashvili@tsmu.edu</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Wilm</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="I6">6</xref><email>stefan.wilm@med.uni-duesseldorf.de</email></contrib><contrib contrib-type="author" id="A6"><name><surname>Timonen</surname><given-names>Markku</given-names></name><xref ref-type="aff" rid="I7">7</xref><email>markku.timonen@oulu.fi</email></contrib><contrib contrib-type="author" id="A7"><name><surname>Tandeter</surname><given-names>Howard</given-names></name><xref ref-type="aff" rid="I8">8</xref><email>Howard1@bgu.ac.il</email></contrib>

BMC Medical Education

<sec><title>Background</title><p>General practice/family medicine (GP/FM) is the provision of first contact, person focused, ongoing care over time that meets the health-related needs of people, referring only patients with uncommon or serious conditions, and coordinating care when people receive services at other levels of the healthcare system [<xref ref-type="bibr" rid="B1">1</xref>]. Primary health care means GP/FM applied on a population level, and as a population strategy this requires the commitment of governments to develop and sustain such services. It is increasingly becoming evident that a strong primary health care system is more likely to provide better population health, more equity in health throughout the population, and better use of economic resources, compared to systems that are oriented towards specialty care [<xref ref-type="bibr" rid="B2">2</xref>-<xref ref-type="bibr" rid="B4">4</xref>]. The World Health Organization (WHO) identified primary health care as central to the achievement of the goal &#x0201c;Health for All&#x0201d; already in 1978 [<xref ref-type="bibr" rid="B5">5</xref>], and thirty years later encouraged all countries to orient their health care systems towards a strengthened primary care [<xref ref-type="bibr" rid="B3">3</xref>].</p><p>To develop and maintain a strong and sustainable primary health care requires that a substantial part of graduating doctors go into primary care [<xref ref-type="bibr" rid="B3">3</xref>]. This in turn requires that GP/FM strongly influences the curricula in medical schools, although institutional, legislative and market factors also play important roles [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>]. Specialty selection by medical students determines the future composition of the physician workforce. Among multiple reasons influencing a career choice either towards or away from primary care, medical school curricula may affect students in their perceptions of the role of primary care physicians. Since students are greatly influenced by the cultures of the institutions in which they are trained, the negative attitude of a university towards FM/GP may negatively affect the number of students going into this specialty [<xref ref-type="bibr" rid="B6">6</xref>]. Selection of career specialties begins in earnest during the clinical rotations with exposure to the clinical and intellectual environments of various specialties. A recent study from Israel found that as many as 62% of last year medical students considered choosing one specific specialty, while the rest considered two or more [<xref ref-type="bibr" rid="B9">9</xref>].</p><p>Still, however, undergraduate medical education seems to be out of synchrony with accelerating development and training in GP/FM [<xref ref-type="bibr" rid="B8">8</xref>]. In a former paper, our group developed a &#x0201c;minimal core curriculum&#x0201d; for undergraduate GP/FM, meant as an aid for medical schools introducing the topic for the first time, usually starting with a very brief course [<xref ref-type="bibr" rid="B10">10</xref>]. Working in a GP/FM setting requires problem solving skills, that differ highly from the disease-centered linear thinking inside university hospitals and which dominates the curricula in medical schools. In primary care the focus is upon the whole person &#x02013; body and mind &#x02013; in his/her context, and over long periods of time. Complex and poorly understood health problems, as well as clusters of diseases have to be handled, and the doctor-patient relationship is an important working tool. No student should graduate from medical school without substantial understanding of these matters [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B10">10</xref>]. In our opinion this requires &#x02013; in addition to a theoretical GP/FM curriculum - a substantial component of &#x0201c;master-apprentice&#x0201d; learning in a primary care clinic.</p><p>In the last decades many European countries have undergone dramatic changes, including democratization, economic liberalization and a redefinition of the role of the state. Health care and social services systems have been reformed, and new challenges have had to be faced [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B11">11</xref>]. In the present paper, we aim at assessing and describing the extent of GP/FM teaching in medical schools throughout Europe. Is there a GP/FM curriculum? And to which degree are the students able to participate in clinical work in a GP&#x02019;s office? Is it still possible to graduate as a doctor from a European university having learned nothing at all about GP/FM? We have not been able to find previous surveys on this matter.</p></sec><sec sec-type="methods"><title>Methods</title><p>The authors of this paper are national representatives in the Council of European Academy of Teachers in General Practice and Family Medicine (EURACT) [<xref ref-type="bibr" rid="B12">12</xref>], and are members of EURACT&#x02019;s Basic Medical Education (BME) Committee. Following a brain-storm within the BME Committee items for a questionnaire were identified. To achieve the best possible response rate the questionnaire was made as brief and simple as possible (Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>) and was accompanied by the following text: &#x0201c;The European Academy of Teachers in General Practice/Family Medicine (EURACT) is running a mapping of the presence of undergraduate FM/GP rotations/clerkships in all European Medical Schools. You are kindly requested to answer the attached short questionnaire about your own medical school&#x0201d;. The questionnaire was sent by e-mail to GP/FM professors at each medical school in the countries of interest (where there were no GP/FM professors, the dean or another relevant professor was contacted). The authors divided the countries between them, and in several countries the national EURACT representative &#x02013; not members of the BME-committee &#x02013; aided in distributing the questionnaires and collecting the answers. Generally we accepted the data as they arrived from the respondents. The data collection took place in 2011 and until the end of 2012.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p><bold>Questionnaire presented by The European Academy of Teachers in General Practice/Family Medicine (EURACT</bold>)</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Question</italic> </bold></th><th align="left"><bold> <italic>Answer</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Name of medical school<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">City<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">How many years is your medical program?<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">Does the school have a GP/FM curriculum?<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">If so, does it have a clinical component (student sits in with GP)?<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">What is the duration of this rotation in weeks?<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">During which year is the rotation presented?<hr/></td><td align="left" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left">Do you have such rotations in more than one year?</td><td align="left">&#x000a0;</td></tr></tbody></table></table-wrap><p>As the study did not collect data on human subjects, no approval from an ethics committee was needed.</p></sec><sec sec-type="results"><title>Results</title><p>We were able to obtain information from 259 European medical schools in 39 countries (Tables&#x000a0;<xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref> and <xref ref-type="table" rid="T5">5</xref>). According to the &#x0201c;Avicenna Database&#x0201d; run by the University of Copenhagen in collaboration with World Federation for Medical Education (WFME), there are about 400 medical universities in these 39 European countries (<ext-link ext-link-type="uri" xlink:href="http://avicenna.ku.dk/database/medicine">http://avicenna.ku.dk/database/medicine</ext-link>). Response rate was thus 64%.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Western Europe &#x02013; state of GP/FM curriculum in medical schools</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left" valign="bottom"><bold> <italic>Country (n&#x02009;=&#x02009;5)</italic> </bold><hr/></th><th align="center" valign="bottom"><bold> <italic>GP curriculum</italic> </bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold> <italic>Clinical component</italic> </bold><hr/></th></tr><tr><th align="left"><bold> <italic>Medical school (n&#x02009;=&#x02009;53)</italic> </bold></th><th align="center"><bold> <italic>Yes/no</italic> </bold></th><th align="center"><bold> <italic>Weeks n</italic> </bold></th><th align="center"><bold> <italic>Which year</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><italic>Austria</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Graz<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Innsbruck<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Vienna<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">5^th&#x02009;+&#x02009;6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Salzburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Belgium</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;K.U. Leuven<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">2^nd&#x02009;+&#x02009;6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Gent<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">2^nd,3^rd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;VUB Brussels<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">2^nd,5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;UA Antwerp<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">3^rd, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;UCL Brussels<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">3^rd<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;ULB Brussels<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">3^rd&#x02009;+&#x02009;6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;ULG Liege<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">2^nd&#x02009;+&#x02009;6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Netherlands</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Maastricht<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Germany</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ. Aachen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Berlin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Bochum<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Bonn<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Dresden<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;D&#x000fc;sseldorf<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Erlangen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Essen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Frankfurt<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Freiburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Giessen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;G&#x000f6;ttingen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Greifswald<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Halle<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Hamburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Hannover<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Heidelberg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">1^st or 2_nd&#x02009;+&#x02009;4_th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Homburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">No information<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Jena<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Kiel<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;K&#x000f6;ln<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th or 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Leipzig<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;L&#x000fc;beck<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">No information<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Magdeburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2-3<hr/></td><td align="center" valign="bottom">4^th&#x02009;+&#x02009;5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Mainz<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Mannheim<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No information<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Marburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;M&#x000fc;nchen (LMU)<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;M&#x000fc;nchen (TU)<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th&#x02009;+&#x02009;5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;M&#x000fc;nster<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Regensburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">?<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Rostock<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;T&#x000fc;bingen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ulm<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th&#x02009;+&#x02009;5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Witten/Herdecke<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8-10<hr/></td><td align="center" valign="bottom">1^st to 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;W&#x000fc;rzburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Switzerland</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Basel<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Bern<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 3^rd, 4^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Geneva<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">2^nd, 4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Lausanne<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 5^th, 6^th<hr/></td></tr><tr><td align="left">&#x02003;&#x02009;Univ of Z&#x000fc;rich</td><td align="center">Yes</td><td align="center">2</td><td align="center">3^rd, 4^th, 5^th</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Eastern Europe - state of GP/FM curriculum in medical schools</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left" valign="bottom"><bold> <italic>Country (n&#x02009;=&#x02009;10)</italic> </bold><hr/></th><th align="center" valign="bottom"><bold> <italic>GP curriculum</italic> </bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold> <italic>Clinical component</italic> </bold><hr/></th></tr><tr><th align="left"><bold> <italic>Medical school (n&#x02009;=&#x02009;50)</italic> </bold></th><th align="center"><bold> <italic>Yes/No</italic> </bold></th><th align="center"><bold> <italic>Weeks</italic> </bold></th><th align="center"><bold> <italic>Which year</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><italic>Belarus</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Minsk State Medical University¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Vitebsk State Medical University¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Gomel State Medical University¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;GrodNo State Medical University¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Bulgaria</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Plovdiv¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Sofia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Varna¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical University Pleven¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Medical faculty Stara Zagora¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Check Republic</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Charles Univ in Prague, first fac of med<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">4^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Charles Univ in Prague, second fac of med<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Charles Univ in Prague, third fac of med<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Charles Univ, fac of med Hradec Kralove²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Fac of med in Pilsen, Masaryk University<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th&#x02009;+&#x02009;6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Fac of med , Palacky Univ Olomouc<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Ostrava, fac med¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Georgia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Akaki Tsereteli State Univ, Caucasus<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;International Univ Tbilisi¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;David Agmashenelebi Univ of Georgia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;David Tvildiani Med Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Iv. Javakhishvili Tbilisi State Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Petre Shotadze Tbilisi Med Acad<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Shota Rustaveli State Univ¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Tbilisi Med EduUniv Hippocrates<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Tbilisi State Med Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">1^st, 2^nd<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Hungary</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Semmelweis Univ Budapest<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Szeged<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Pecs<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Moldova</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Nicolae Testemitanu Chisinau<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Poland</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Bialystok²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Wroclaw Med Univ²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Gdansk<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Silesia, School of Med in Katowice<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Lodz<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Lublin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Poznan Univ of Med Sciences²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">&#x0003c;1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Pomorski Univ of Med Stettin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Univ of Warsaw<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ludwig Rydygier CollMed Bydgoszcz<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Romania</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Gr. T. Popa, Univ of Med, Lasi<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Fac de Med Victor Papilian, Sibiu¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Transilvaia, Brasov²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Med Pharm, Victor Babes, Timisoara²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Med Pharm Iuliu Hatieganu, Cluj-Napoca<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Russia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;KrasNoyarsk²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">30&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;State Med Univ Vladivostok<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1.5<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Amurskaya State Med Acad Blagoveshensk<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;State Med Univ Kursk²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;State Med Univ Petrozavodsk²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Pavlov&#x02019;s St.Petersburg State Med Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;State North-West Med Univ St. Petersburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Slovakia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Pavol Josef Safarik Univ Kosice<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Jessenius Fac Med Martin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left">&#x02003;&#x02009;Comenius Univ Bratislava</td><td align="center">No</td><td align="center">&#x000a0;</td><td align="center">&#x000a0;</td></tr></tbody></table><table-wrap-foot><p>¹No GP/FM curriculum (n&#x02009;=&#x02009;13).</p><p>²No or&#x02009;&#x0003c;&#x02009;1&#x000a0;week clinical component (n&#x02009;=&#x02009;9).</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Northern Europe &#x02013; state of GP/FM curriculum in medical schools</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left" valign="bottom"><bold> <italic>Country (n&#x02009;=&#x02009;9)</italic> </bold><hr/></th><th align="center" valign="bottom"><bold> <italic>GP curriculum</italic> </bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold> <italic>Clinical component</italic> </bold><hr/></th></tr><tr><th align="left"><bold> <italic>Medical school (n&#x02009;=&#x02009;45)</italic> </bold></th><th align="center"><bold> <italic>Yes/no</italic> </bold></th><th align="center"><bold> <italic>Weeks n</italic> </bold></th><th align="center"><bold> <italic>Which year?</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><italic>Denmark</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Aarhus Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of south Denmark Odense<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Aalborg Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th or 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Copenhagen Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No information<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Estonia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Tartu<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">1^st, 6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Finland</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Helsinki<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4.5<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 4^th,5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Kuopio<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 3^rd, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Oulu<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Tampere<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Turku<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4.5<hr/></td><td align="center" valign="bottom">1^st, 3^rd, 5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Iceland</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med School of Iceland Reykjavik<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">2^nd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Ireland</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Limerick<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">?<hr/></td><td align="center" valign="bottom">1^st, 4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Royal College of Surgeons Med School<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">1^st, 4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Queens Univ Belfast<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Trinity College, Dublin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;NUI Galway<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">1^st,2^nd, 4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ College Cork<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">3^rd, 5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Latvia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Riga Stradins Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Latvia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Norway</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Bergen<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Oslo<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Troms&#x000f8;<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">1^st, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Norw Univ of Science and Technol, Trondheim<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Sweden</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Sahlgrenska Acedemy Gothenburg<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 3^rd, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Link&#x000f6;ping Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">years 1-6<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;&#x000d6;rebro univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">years 1-6<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ume&#x000e5; Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Karolinska Inst Stockholm<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">13<hr/></td><td align="center" valign="bottom">years 1-6<hr/></td></tr><tr><td align="left" valign="bottom"><italic>United Kingdom</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Keele Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">23<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;NewcastleMed School<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Barts and The London<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5+<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Edinburgh<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Brighton and Sussex Med School<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4++<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Cambridge<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">12+<hr/></td><td align="center" valign="bottom">4^th, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Lancaster Med School<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">15<hr/></td><td align="center" valign="bottom">years 2-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Leicester<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Nottingham<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4+<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;King&#x02019;s College, London<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Dundee Med School<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Bristol<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7-8<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of East Anglia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">19<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;St. George&#x02019;s Univ of London<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">3^rd, 5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Birmingham<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">years 1-5<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Glasgow<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">20<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 5^th<hr/></td></tr><tr><td align="left">&#x02003;&#x02009;Warwick Med School</td><td align="center">Yes</td><td align="center">8</td><td align="center">2^nd, 3^rd</td></tr></tbody></table></table-wrap><table-wrap position="float" id="T5"><label>Table 5</label><caption><p>Southern Europe &#x02013; state of GP/FM curriculum in medical schools</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left" valign="bottom"><bold> <italic>Country (n&#x02009;=&#x02009;15)</italic> </bold><hr/></th><th align="center" valign="bottom"><bold> <italic>GP curriculum</italic> </bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold> <italic>Clinical component</italic> </bold><hr/></th></tr><tr><th align="left"><bold> <italic>Medical school (n&#x02009;=&#x02009;107)</italic> </bold></th><th align="center"><bold> <italic>Yes/No</italic> </bold></th><th align="center"><bold> <italic>Weeks n</italic> </bold></th><th align="center"><bold> <italic>Which year?</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><italic>Albania</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Tirana¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Bosnia-Herzegovina</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med fak Banja Luka<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med fak Tuzla<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No information<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Croatia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Rijeka<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Zagreb<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Osijek<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Split<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Cyprus</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Nicosia¹ (only first two years of med school)<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Greece</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Athens¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Aristotle Univ of Thessaloniki<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">1^st<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Patras¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Heraklion, Crete<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ioannina<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Alexandroupoli¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Larissa¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Italy</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of L&#x02019;Aquila<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">1^st, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Fac La Sapienza<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Fac di Med et Psicol Roma²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">30&#x000a0;h<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Campus Biomedico Roma<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Udine<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Trieste¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Central Milan<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;S.Paolo Milan<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Vialba Milan<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;S.Donato Milan<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of GeNoa<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Bari et Foggia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Macedonia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ SS Cyril &#x00026; Methodius Skopje<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;State Univ Tetovo²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">15&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Goce Delcev Stip²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">15&#x000a0;h<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Malta</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ of Malta<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Montenegro</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Podgorica<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">4^th, 5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Portugal</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Coimbra<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Lisboa<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">10<hr/></td><td align="center" valign="bottom">1^sr, 2^nd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Porto<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Beira Interior<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">1^sr, 2^nd, 4^th, 5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Minho<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">16<hr/></td><td align="center" valign="bottom">5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ da Algarve<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">16<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 3^rd<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Serbia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Fak Nis<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Fak Belgrade<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Fak Kragujevac¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Med Fak Novi Sad¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Relocated Med Fak from Pristina¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Slovenia</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ljubljana<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">7<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Spain</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Cadiz<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Cordoba²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Granada²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Sevilla<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Zaragoza<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Asturias²<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;La Laguna<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Las Palmas<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Cantabria<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">?<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Salamanca<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Valladolid<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">3^rd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Albacete<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;UAB-Univ AutoNoma Barcelona<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">11<hr/></td><td align="center" valign="bottom">1^st<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ Barcelona<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Girona<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">3^rd, 4^th, 5^th 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Lleida<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Rovira I Virgili<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Extremadura<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Santiago<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">3^rd<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Univ AutoNoma de Madrid<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Complutense de Madrid<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th or 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Europ Univ Madrid<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">No information<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Alfonso<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Murcia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Navarra<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Valencia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">3^rd<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Catholic Univ Valencia<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">3^rd, 4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Miguel Hernandez, San Juan<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Pais Vasco<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Turkey</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Acibadem Istanbul<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">13<hr/></td><td align="center" valign="bottom">1^st, 2^nd, 3^rd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Cukorova Adana<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Kocatepe Afyon¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ondokuzmayis Samsun<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Osmangazi Eskisehir¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Selcuk Meram Konya¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Sutcu Imam Kahramanmaras<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Trakya Edirne<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Uludag Bursa<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Tayfur Ata SokmenHatay<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Bozok Yozgat¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Gulhane Askeri Tip Akademisi<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">Ankara¹<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ankara Univ<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;INonu Malatya<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Marmara<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8<hr/></td><td align="center" valign="bottom">3^rd,5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Pamukkale Denizli¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Mersin¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Dokuz Eyl&#x000fc;l Izmir¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Onsekiz Mart Canakkale<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">8 (elective)<hr/></td><td align="center" valign="bottom">5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Yeditepe Istanbul<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Adnan Menderes Aydin<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">5^th, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Akdeniz Antalya<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">5<hr/></td><td align="center" valign="bottom">3^rd, 6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Baskent Ankara<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Izzet Baysal Abant¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Karadeniz Techn Univ Trabzon¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">Trabzon¹<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Celal Bayar Manisa¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Yildirim Beyazit Ankara<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Capa Istanbul¹<hr/></td><td align="center" valign="bottom">No<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Gazi Osman Pasa Tokat<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">5^th<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Israel</italic><hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td><td align="center" valign="bottom">&#x000a0;<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Ben &#x02013;Gurion Univ Beer-Sheva<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Hebrew Univ Jerusalem<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">2<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Tel-Aviv Univ (6 y med school)<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">4<hr/></td><td align="center" valign="bottom">6^th<hr/></td></tr><tr><td align="left" valign="bottom">&#x02003;&#x02009;Tel-Aviv Univ (4 y med school)<hr/></td><td align="center" valign="bottom">Yes<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">4^th<hr/></td></tr><tr><td align="left">&#x02003;&#x02009;Technion Haifa</td><td align="center">Yes</td><td align="center">6</td><td align="center">6^th</td></tr></tbody></table><table-wrap-foot><p>¹No GP/FM curriculum (n&#x02009;=&#x02009;22).</p><p>²No or&#x02009;&#x0003c;&#x02009;1&#x000a0;week clinical component (n&#x02009;=&#x02009;6).</p></table-wrap-foot></table-wrap><p>Out of the 259 respondent universities, 35 (13.5%) reported to have no GP/FM curriculum (Table&#x000a0;<xref ref-type="table" rid="T6">6</xref>). These 35 medical faculties were located in eleven different European countries. In addition, 15 of the medical schools where a GP/FM curriculum did exist, reported that this curriculum did not include any clinical component (n&#x02009;=&#x02009;5), or that the clinical part of the course was very brief - less than one week, mostly only a few hours (n&#x02009;=&#x02009;10, Table&#x000a0;<xref ref-type="table" rid="T6">6</xref>). In total, 50 universities (19%) thus had no or a very brief GP/FM curriculum.</p><table-wrap position="float" id="T6"><label>Table 6</label><caption><p>Medical universities without GP/FM curriculum, or with clinical GP/FM teaching lacking or shorter than one week</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold> <italic>Country</italic> </bold></th><th align="left"><bold> <italic>Medical schools without GP/FM curriculum (n = 35)</italic> </bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom"><italic>Belarus</italic><hr/></td><td align="left" valign="bottom">Minsk State Medical University<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Vitebsk State Medical University<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Gomel State Medical University<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Grodno State Medical University<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Bulgaria</italic><hr/></td><td align="left" valign="bottom">Medical University Plovdiv<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Medical University Varna<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Medical University Pleven<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Medical faculty Stara Zagora<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Chech Republic</italic><hr/></td><td align="left" valign="bottom">Univ. Ostrava, fac med<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Georgia</italic><hr/></td><td align="left" valign="bottom">International Univ Tbilisi<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Shota Rustaveli State Univ<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Romania</italic><hr/></td><td align="left" valign="bottom">Fac de Med Victor Papilian, Sibiu<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Slovakia</italic><hr/></td><td align="left" valign="bottom">Comenius Univ Bratislava<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Albania</italic><hr/></td><td align="left" valign="bottom">University of Tirana<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Cyprus</italic><hr/></td><td align="left" valign="bottom">University of Nicosia (only first two years of med school)<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Greece</italic><hr/></td><td align="left" valign="bottom">Athens<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Patras<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Alexandroupoli<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Larissa<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Italy</italic><hr/></td><td align="left" valign="bottom">University of Trieste<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Serbia</italic><hr/></td><td align="left" valign="bottom">Med Fak Kragujevac<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Med Fak Novi Sad<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Relocated Med Fak from Pristina<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Turkey</italic><hr/></td><td align="left" valign="bottom">Kocatepe Afyon<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Osmangazi Eskisehir<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Selcuk Meram Konya<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Bozok Yozgat<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Gulhane Askeri Tip Akademisi Ankara<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Pamukkale Denizli<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Mersin<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Dokuz Eyl&#x000fc;l Izmir<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Izzet Baysal Abant<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Karadeniz Techn Univ Trabzon<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Celal Bayar Manisa<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Capa Istanbul<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom"><bold> <italic>Medical schools with no or &#x0003c;1 week clinical teaching (n = 15)</italic> </bold><hr/></td></tr><tr><td align="left" valign="bottom"><italic>Chech Republic</italic><hr/></td><td align="left" valign="bottom">Charles Univ, fac of med Hradec Kralove<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Poland</italic><hr/></td><td align="left" valign="bottom">Med Univ of Bialystok<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Wroclaw Med Univ<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Poznan Univ of Med Sciences<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Romania</italic><hr/></td><td align="left" valign="bottom">Univ Transilvaia, Brasov<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Univ Med Pharm, Victor Babes, Timisoara<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Russia</italic><hr/></td><td align="left" valign="bottom">Krasnoyarsk<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">State Med Univ Kursk<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">State Med Univ Petrozavodsk<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Italy</italic><hr/></td><td align="left" valign="bottom">Fac di Med et Psicol Roma<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Macedonia</italic><hr/></td><td align="left" valign="bottom">State Univ Tetovo<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Univ Goce Delcev Stip<hr/></td></tr><tr><td align="left" valign="bottom"><italic>Spain</italic><hr/></td><td align="left" valign="bottom">Cordoba<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Granada<hr/></td></tr><tr><td align="left">&#x000a0;</td><td align="left">Asturias</td></tr></tbody></table></table-wrap><p>Tables&#x000a0;<xref ref-type="table" rid="T2">2</xref>, <xref ref-type="table" rid="T3">3</xref>, <xref ref-type="table" rid="T4">4</xref> and <xref ref-type="table" rid="T5">5</xref> show details of the GP/FM curriculum in the medical schools in the four European regions (according to the United Nations&#x02019; Geo scheme and also including Israel). Roughly, the comprehensiveness of the GP/FM curriculum varies between the regions, as all faculties without any such curriculum are located either in Eastern or in Southern Europe, as are also the majority of schools without or with a very short clinical GP/FM component. Only few medical schools in Eastern Europe have a rotation period longer than two weeks, while the majority of schools in Northern Europe have at least five weeks, and several up to 12&#x02013;13&#x000a0;weeks.</p><p>There are substantial variations in length of the clinical component within countries and even inside the same city: for example the time spent in a GP&#x02019;s office is two weeks in one Brussels medical school and 12&#x000a0;weeks in another.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>One limitation of this study is that by labelling curricula as including or not including GP/FM, we assume the curricula to be mainly discipline based. We thus may have overlooked that a problem based or case based curriculum could include elements from GP/FM without having a proper GP/FM section. We also are aware of the fact that the mere existence of a GP/FM curriculum is not synonymous with high quality teaching. A further limitation is that we were not able to obtain data at all from some countries: Ukraine, Lithuania and France. These countries are &#x0201c;white spots on our map&#x0201d;, although we have got information from a key informant in France that all Universities have incorporated FM/GP in their BME curriculum and that all of them have a clinical component (2&#x02013;6&#x000a0;weeks). This information is in line with the rest of Western European universities, as all of them have GP/FM training with a clinical component. Also, not all universities have responded from each country. For example, we got data only from seven universities in Russia, while the number of medical schools in this country is more than 60. Similarly, for Turkey we have data from 29 universities out of more than 50. Therefore, we cannot provide statistically valid information on the situation in Europe as such. On the other hand, the information that we do have from 259 European medical schools, in itself brings new insights, although curricula are in constant evolution and data captured at a certain point of time will not reflect such a dynamic situation.</p><p>In 2010 an independent international commission published a report on the need of transforming medical education in the future [<xref ref-type="bibr" rid="B13">13</xref>]. The commission states that professional education has not kept pace with the needs of patients and populations, and that fragmented and outdated curricula produce ill-equipped graduates. Reforms are therefore needed, and a list of ten proposed reforms is given. Point number eight states the need for medical schools to achieve: &#x0201c;Expansion from academic centers to academic systems, extending&#x02026;&#x02026;.into primary care settings and communities&#x02026;&#x0201d; [<xref ref-type="bibr" rid="B13">13</xref>]. It is thus positive that the majority of medical schools throughout the European regions do have a substantial GP/FM curriculum &#x02013; 209 out of the 259 faculties assessed. Even so, there is ample room and need for improvements, as 35 schools have no GP/FM teaching whatsoever and clinical teaching is absent or very brief in several others.</p><p>Most former communist countries now let GP/FM play a key role in their health care system. A GP/FM curriculum is also increasingly being introduced into medical training at undergraduate and postgraduate level, and GP/FM is developed as an academic discipline [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B14">14</xref>]. Our study revealed that this task can not yet be seen as completed. It is especially worrying if it is possible both to graduate without any GP/FM competence and subsequetly set up a practice in a country without a mandatory vocational training program.</p><p>European primary care is currently facing high expectactations, regarding its promises to improve population health, control costs, and attribute to less socioeconomic inequality of care [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B4">4</xref>]. But: Do strong primary care systems indeed perform better? And what determines how strong primary care is? [<xref ref-type="bibr" rid="B15">15</xref>]. These important questions have recently been addressed by means of a EU-funded project: the Primary Health Care Activity Monitor for Europe (PHAMEU) [<xref ref-type="bibr" rid="B16">16</xref>]. Based on information from 31 European countries the study was able to show that strong primary care indeed was associated with better population health outcomes, lower rates of potentially avoidable hospitalization, lower socioeconomic inequality in self-rated health, a reduced growth of health care spending, but also with higher levels of total health care costs [<xref ref-type="bibr" rid="B17">17</xref>]. In total, this should support the efforts of policy makers to prioritize primary care. Development of primary care workforce is part of such efforts [<xref ref-type="bibr" rid="B18">18</xref>], and developing a comprehensive GP/FM undergraduate curriculum comprising a clinical rotation is a necessity in this process [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>].</p><p>In our study most clinical GP/FM rotations were placed in years four, five or six, but as the length of the clinical teaching period increased, it is common to spread it over several semesters. For example the 12&#x02013;13&#x000a0;weeks of rotations at three Swedish universities involve the years one to six. This probably has a positive influence on recruitment to GP/FM, as students are exposed to role models throughout their entire education [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>]. In our opinion GP/FM should be positioned as one of the main clinical topics in every European medical school, and teaching in a one-to-one situation in a GP&#x02019;s office should be offered for at least four weeks, preferably longer.</p></sec><sec sec-type="conclusions"><title>Conclusion</title><p>Although the majority of the assessed universities reported to have a theoretical GP/FM curriculum as well as a clinical rotation, it is still possible to graduate from some European medical schools without having learned about clinical work in a primary care setting. The European Academy of Teachers in General Practice (EURACT) will lance efforts to change this situation. Special efforts should be made in Eastern and Southern Europe, where a FM/GP curriculum does not exist in several universities, and where the clinical GP/FM component is generally short.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors&#x02019; contributions</title><p>The group of authors planned the study together, and all contributed to data collection. MB drafted the paper, with contribution from the group. All authors have given final approval of the version to be published.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6920/13/157/prepub">http://www.biomedcentral.com/1472-6920/13/157/prepub</ext-link></p></sec>

Comparison of tutored group with tutorless group in problem-based mixed learning sessions: a randomized cross-matched study

<sec><title>Background</title><p>Problem-based learning (PBL) involves discussions among students who resolve loosely-structured problems to facilitate learning. In the PBL curriculum, faculty tutors are employed as facilitators for small groups of students. Because of lack of time and staff shortage, the effectiveness of tutorless PBL has been discussed as an alternate option.</p></sec><sec><title>Methods</title><p>Sessions in which tutored and tutorless PBL groups are mixed were presented by 1^st-year medical students, who experienced both tutored and tutorless groups alternately in the two sessions of a year. To examine the effectiveness of tutored and tutorless PBL, written examination scores (WES) and self-contentment scores (SCS) were statistically analysed.</p></sec><sec><title>Results</title><p>WES averages did not significantly differ between the tutored and tutorless groups; however, a significantly greater variation was observed in WES in the tutorless group. SCS averages tended to be higher in the tutored PBL than in tutorless PBL groups.</p></sec><sec><title>Conclusions</title><p>Students in these tutorless PBL groups performed well in their written examinations, whereas those in the tutored PBL groups, achieved this and reported better self-contentment with their learning experience. Tutorless PBL sessions were considered to be comparable to tutored PBL sessions at least in the early stages.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Hayashi</surname><given-names>Shogo</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>sho5-884@umin.ac.jp</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Tsunekawa</surname><given-names>Koji</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>evrv@aichi-med-u.ac.jp</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Inoue</surname><given-names>Chikako</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>chicaco.inoue@gmail.com</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Fukuzawa</surname><given-names>Yoshitaka</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>yofuku@aichi-med-u.ac.jp</email></contrib>

BMC Medical Education

<sec><title>Background</title><p>In the mid-1960s, problem-based learning (PBL) was adopted as a new approach for medical education at McMaster University, Ontario, Canada. PBL has been described as 'a learning that results from the process of working towards understanding or resolution of a problem&#x02019; [<xref ref-type="bibr" rid="B1">1</xref>]. PBL not only facilitates the acquisition of knowledge but also that of other generic desirable attributes such as effective communication skills, ability to work in a team (team work), problem-solving skills, self-directed learning ability, ability to share information, appreciate other points of view and identification of personal strengths and weaknesses [<xref ref-type="bibr" rid="B2">2</xref>]. Because many of these skills are related to the tutorial process and group dynamics [<xref ref-type="bibr" rid="B3">3</xref>], the tutors&#x02019; expertise, characteristics and behaviour are believed to influence both the process perspective and learning outcomes [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>].</p><p>In the approach adopted by the McMaster University medical school, a faculty tutor was present during all group activities to monitor, assess and provide immediate input i.e. each group was tutored [<xref ref-type="bibr" rid="B6">6</xref>]. Providing a facilitator for PBL can be a problem during times of faculty/staff shortage [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B16">16</xref>]. Therefore, many schools have tried other PBL formats in an attempt to reduce the demands on faculty/staff time and resources; examples of these formats include student-tutored PBL [<xref ref-type="bibr" rid="B17">17</xref>] and tutorless PBL [<xref ref-type="bibr" rid="B6">6</xref>]. In student-tutored PBL, one student studies the problem in advance and then takes on the role as the tutor of the group instead of the faculty tutor. In tutorless PBL, neither the student tutor nor the faculty tutor is present. There have been many reports suggesting that student-tutored PBL can be just as effective as faculty-tutored PBL with regard to learning outcomes of student-tutored PBL. These sessions can be conducted by senior students [<xref ref-type="bibr" rid="B17">17</xref>-<xref ref-type="bibr" rid="B19">19</xref>] or also by peer-level students from the same class [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>]. Similarly, a meta-analysis by Leary et al. [<xref ref-type="bibr" rid="B20">20</xref>] indicated that student tutors were equally effective when compared with faculty tutors. However, there is limited information on learning outcomes of tutorless PBL [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>].</p><p>The aim of this study was to examine the effectiveness of tutorless PBL by comparing learning outcomes between tutorless and tutored groups. Roberts et al. [<xref ref-type="bibr" rid="B11">11</xref>] and Kaliyadan et al. [<xref ref-type="bibr" rid="B14">14</xref>] reported their experiences with tutorless PBL and concluded that there were no significant differences in learning outcomes between tutored and tutorless PBL. However, in the abovementioned reports, several important differences such as group member characteristics, scenarios or learning materials that were present between the tutored and tutorless PBL conditions were observed. Nicholl and Lou [<xref ref-type="bibr" rid="B15">15</xref>] recently reported on tutorless PBL using a model for a large class facilitated by one instructor; they argued that students could achieve the required learning outcomes with tutorless PBL. Moreover, these reports do not only compare learning outcomes between tutored and tutorless PBL because there are many other factors influencing the study and results. To the best of our knowledge, this is the first randomised cross-matched study comparing tutored and tutorless PBL.</p></sec><sec sec-type="methods"><title>Methods</title><p>At Aichi Medical University, the PBL course comprises the following two units: PBL1 for first year medical students and PBL2 for third and-forth year medical students. The goal of PBL1 is introduction and early exposure of clinical medicine to students, and the role of PBL2 is to train students on the art of clinical problem solving. Between 2007 and 2008, as part of the curriculum development of the existing PBL1, we conducted a randomised cross-matched study on the learning outcomes involved in PBL1 (2007, N&#x02009;=&#x02009;102; 2008, N&#x02009;=&#x02009;100). The research design is shown in Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref>. The study was performed in accordance with the provisions of the Declaration of Helsinki. The executive council of the Medical Education Center Aichi Medical University first reviewed the detail protocol, including the future plan of submission. After the review by this committee, the academic affairs department and the faculty council of our school approved this curriculum. The Institutional Review Board exempted the study from review.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Chronological list of problem-based learning (PBL) undertaken by students in this study.</bold> Group A in the left row and Group B in the right row. In the middle row, sessions experienced by all students conducted in a large classroom are chronologically displayed.</p></caption><graphic xlink:href="1472-6920-13-158-1"/></fig><p>These details were explained to all students prior to the start of PBL1, and they were advised that self-evaluation and a questionnaire were voluntary. Students were given the option of not participating in this research, but they all decided to participate. Students were randomly divided into two groups of equal numbers, Group A and Group B. Each group was randomly reorganised into seven small groups comprising seven or eight students for each session. All students attended two sessions in a year; every session covered a four day period (Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>). Small group discussions (SGDs) were conducted every day in each session about recurring scenario of chief complaints such as abdominal pain and cough, i.e. four SGDs per session. In group A, SGDs were tutored in the first session and tutorless in the second session. In group B, SGDs were tutorless in the first session and tutored in the second session. Each session was designed such that the schedule for lectures or laboratory practices did not coincide, except for a daily short lecture related to the scenario of each day. Both groups completed a daily report on every SGDs and noted details of any self-learning. On the last day, a written examination (full marks, 100 points) on the contents of each session was conducted. A questionnaire including a 5-tiered self-evaluation on self-contentment and other items shown in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref> was simultaneously distributed. An overall evaluation was conducted using a combination of the percentage of attendance and written examination scores (WES). Daily reports, tutor evaluation, self-evaluation and answers to the questionnaire were not included in the summative evaluation.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Timetable of each problem-based learning (PBL) session.</p></caption><graphic xlink:href="1472-6920-13-158-2"/></fig><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Student results on the self-evaluation in each session</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="left" valign="bottom">&#x000a0;<hr/></th><th colspan="10" align="center" valign="bottom"><bold> <italic>2007</italic> </bold><hr/></th></tr><tr><th align="left" valign="bottom">&#x000a0;<hr/></th><th colspan="5" align="center" valign="bottom"><bold> <italic>Session 1</italic> </bold><hr/></th><th colspan="5" align="center" valign="bottom"><bold> <italic>Session 2</italic> </bold><hr/></th></tr><tr><th rowspan="3" align="left" valign="bottom">&#x000a0;<hr/></th><th colspan="2" align="center" valign="bottom"><bold>Group A:</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>Group B:</bold><hr/></th><th rowspan="4" align="right" valign="bottom"><bold>p value</bold></th><th colspan="2" align="center" valign="bottom"><bold>Group B:</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>Group A:</bold><hr/></th><th rowspan="4" align="right" valign="bottom"><bold>p value</bold></th></tr><tr><th colspan="2" align="center" valign="bottom"><bold>Tutored</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>Tutorless</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>Tutored</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>Tutorless</bold><hr/></th></tr><tr><th colspan="2" align="center" valign="bottom"><bold>(n = 51)</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>(n = 50)</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>(n = 51)</bold><hr/></th><th colspan="2" align="center" valign="bottom"><bold>(n = 51)</bold><hr/></th></tr><tr><th align="left">&#x000a0;</th><th align="right"><bold>average</bold></th><th align="center"><bold>SD</bold></th><th align="right"><bold>average</bold></th><th align="center"><bold>SD</bold></th><th align="right"><bold>average</bold></th><th align="center"><bold>SD</bold></th><th align="right"><bold>average</bold></th><th align="center"><bold>SD</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">Self-directed learning<hr/></td><td align="right" valign="bottom">4.19<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">3.94<hr/></td><td align="center" valign="bottom">0.71<hr/></td><td align="right" valign="bottom">0.08<hr/></td><td align="right" valign="bottom">4.25<hr/></td><td align="center" valign="bottom">0.74<hr/></td><td align="right" valign="bottom">4.06<hr/></td><td align="center" valign="bottom">0.86<hr/></td><td align="right" valign="bottom">0.22<hr/></td></tr><tr><td align="left" valign="bottom">Activeness<hr/></td><td align="right" valign="bottom">4.13<hr/></td><td align="center" valign="bottom">0.77<hr/></td><td align="right" valign="bottom">4.06<hr/></td><td align="center" valign="bottom">0.71<hr/></td><td align="right" valign="bottom">0.60<hr/></td><td align="right" valign="bottom">4.35<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">4.20<hr/></td><td align="center" valign="bottom">0.83<hr/></td><td align="right" valign="bottom">0.31<hr/></td></tr><tr><td align="left" valign="bottom">Scientific basis<hr/></td><td align="right" valign="bottom">3.96<hr/></td><td align="center" valign="bottom">0.77<hr/></td><td align="right" valign="bottom">3.74<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">0.14<hr/></td><td align="right" valign="bottom">4.00<hr/></td><td align="center" valign="bottom">0.80<hr/></td><td align="right" valign="bottom">3.89<hr/></td><td align="center" valign="bottom">0.69<hr/></td><td align="right" valign="bottom">0.36<hr/></td></tr><tr><td align="left" valign="bottom">Group dynamics<hr/></td><td align="right" valign="bottom">4.31<hr/></td><td align="center" valign="bottom">0.79<hr/></td><td align="right" valign="bottom">3.88<hr/></td><td align="center" valign="bottom">0.96<hr/></td><td align="right" valign="bottom">0.02<hr/></td><td align="right" valign="bottom">4.57<hr/></td><td align="center" valign="bottom">0.61<hr/></td><td align="right" valign="bottom">4.27<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">0.03<hr/></td></tr><tr><td align="left" valign="bottom">Attentiveness<hr/></td><td align="right" valign="bottom">4.20<hr/></td><td align="center" valign="bottom">0.69<hr/></td><td align="right" valign="bottom">4.28<hr/></td><td align="center" valign="bottom">0.67<hr/></td><td align="right" valign="bottom">0.54<hr/></td><td align="right" valign="bottom">4.39<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">4.27<hr/></td><td align="center" valign="bottom">0.70<hr/></td><td align="right" valign="bottom">0.40<hr/></td></tr><tr><td align="center" valign="bottom">&#x000a0;<hr/></td><td colspan="10" align="center" valign="bottom"><bold> <italic>2008</italic> </bold><hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td colspan="5" align="center" valign="bottom"><bold> <italic>Session 1</italic> </bold><hr/></td><td colspan="5" align="center" valign="bottom"><bold> <italic>Session 2</italic> </bold><hr/></td></tr><tr><td rowspan="3" align="left" valign="bottom">&#x000a0;<hr/></td><td colspan="2" align="center" valign="bottom"><bold>Group A:</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Group B:</bold><hr/></td><td rowspan="4" align="right" valign="bottom"><bold>p value</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Group B:</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Group A:</bold><hr/></td><td rowspan="4" align="right" valign="bottom"><bold>p value</bold><hr/></td></tr><tr><td colspan="2" align="center" valign="bottom"><bold>Tutored</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Tutorless</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Tutored</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>Tutorless</bold><hr/></td></tr><tr><td colspan="2" align="center" valign="bottom"><bold>(n = 46)</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>(n = 45)</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>(n = 45)</bold><hr/></td><td colspan="2" align="center" valign="bottom"><bold>(n = 46)</bold><hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="right" valign="bottom"><bold>average</bold><hr/></td><td align="center" valign="bottom"><bold>SD</bold><hr/></td><td align="right" valign="bottom"><bold>average</bold><hr/></td><td align="center" valign="bottom"><bold>SD</bold><hr/></td><td align="right" valign="bottom"><bold>average</bold><hr/></td><td align="center" valign="bottom"><bold>SD</bold><hr/></td><td align="right" valign="bottom"><bold>average</bold><hr/></td><td align="center" valign="bottom"><bold>SD</bold><hr/></td></tr><tr><td align="left" valign="bottom">Self-directed Learning<hr/></td><td align="right" valign="bottom">4.41<hr/></td><td align="center" valign="bottom">0.75<hr/></td><td align="right" valign="bottom">4.42<hr/></td><td align="center" valign="bottom">0.70<hr/></td><td align="right" valign="bottom">0.96<hr/></td><td align="right" valign="bottom">4.51<hr/></td><td align="center" valign="bottom">0.59<hr/></td><td align="right" valign="bottom">4.24<hr/></td><td align="center" valign="bottom">0.66<hr/></td><td align="right" valign="bottom">0.037<hr/></td></tr><tr><td align="left" valign="bottom">Activeness<hr/></td><td align="right" valign="bottom">4.35<hr/></td><td align="center" valign="bottom">0.82<hr/></td><td align="right" valign="bottom">4.50<hr/></td><td align="center" valign="bottom">0.71<hr/></td><td align="right" valign="bottom">0.34<hr/></td><td align="right" valign="bottom">4.67<hr/></td><td align="center" valign="bottom">0.52<hr/></td><td align="right" valign="bottom">4.24<hr/></td><td align="center" valign="bottom">0.72<hr/></td><td align="right" valign="bottom">0.001<hr/></td></tr><tr><td align="left" valign="bottom">Scientific basis<hr/></td><td align="right" valign="bottom">4.20<hr/></td><td align="center" valign="bottom">0.78<hr/></td><td align="right" valign="bottom">4.34<hr/></td><td align="center" valign="bottom">0.66<hr/></td><td align="right" valign="bottom">0.33<hr/></td><td align="right" valign="bottom">4.44<hr/></td><td align="center" valign="bottom">0.62<hr/></td><td align="right" valign="bottom">4.14<hr/></td><td align="center" valign="bottom">0.70<hr/></td><td align="right" valign="bottom">0.027<hr/></td></tr><tr><td align="left" valign="bottom">Group dynamics<hr/></td><td align="right" valign="bottom">4.60<hr/></td><td align="center" valign="bottom">0.65<hr/></td><td align="right" valign="bottom">4.56<hr/></td><td align="center" valign="bottom">0.73<hr/></td><td align="right" valign="bottom">0.73<hr/></td><td align="right" valign="bottom">4.56<hr/></td><td align="center" valign="bottom">0.62<hr/></td><td align="right" valign="bottom">4.40<hr/></td><td align="center" valign="bottom">0.78<hr/></td><td align="right" valign="bottom">0.28<hr/></td></tr><tr><td align="left">Attentiveness</td><td align="right">4.50</td><td align="center">0.72</td><td align="right">4.58</td><td align="center">0.57</td><td align="right">0.55</td><td align="right">4.60</td><td align="center">0.50</td><td align="right">4.40</td><td align="center">0.67</td><td align="right">0.19</td></tr></tbody></table></table-wrap><p>The average value of WES and self-contentment scores (SCS) for each session was examined with the two-way analysis of variance (ANOVA) and uniformity of examinations was tested with the Tukey&#x02013;Kramer&#x02019;s honesty significant difference (HSD) test. The validity of grouping during each year was examined using the unpaired t-test and Bartlett&#x02019;s test by comparing the total scores of Groups A and B. With regard to WES and self-evaluation results, the average values for the t-test and dispersion of the F-test were compared between the tutored and tutorless PBL groups. The value of p&#x0003c;0.05 (two-tailed) was considered statistically significant. Furthermore, all statistical analyses were conducted using JMP 8.0.1 (SAS institute Inc., Cary, North Carolina, USA) and Prism 6.0b (Graphpad software, Inc., San Diego, CA, USA).</p></sec><sec sec-type="results"><title>Results</title><sec><title>Validity of matching and reproducibility of the result</title><p>In 2007, the average&#x02009;&#x000b1;&#x02009;standard deviation of WES in Groups A and B was 131.45&#x02009;&#x000b1;&#x02009;22.32 and 135.86&#x02009;&#x000b1;&#x02009;17.91, respectively. In 2008, WES in Groups A and B was 154.69&#x02009;&#x000b1;&#x02009;29.85 and 135.86&#x02009;&#x000b1;&#x02009;17.91, respectively. No significant difference was observed in the average value or standard deviation between both groups. Therefore, it was concluded that Groups A and B matched in each grade.</p><p>While comparing sessions, the average&#x02009;&#x000b1;&#x02009;standard deviation of WES in 2007 was 64.98&#x02009;&#x000b1;&#x02009;18.74 and 68.68&#x02009;&#x000b1;&#x02009;14.91 for sessions 1 and 2, respectively. In 2008, WES was 76.80&#x02009;&#x000b1;&#x02009;19.76 and 75.25&#x02009;&#x000b1;&#x02009;19.26 for sessions 1 and 2, respectively. The two-way ANOVA recognised a significant effect associated with the year, but no significant effects according to sessions were observed. Furthermore, no significant difference was recognised in the Tukey&#x02013;Kramer HSD test between sessions 1 and 2 in any year. The average score significantly differed between 2007 and 2008; however, because no difference was observed between the sessions in each year, results of sessions 1 and 2 for each year were considered to be reproducible. Therefore, it was decided that results for each year will be analysed by combining the results of sessions 1 and 2 in groups A and B, respectively, to form the tutored group and those of sessions 1 and 2 in groups B and A, respectively, to form the tutorless group.</p></sec><sec><title>Written examination scores</title><p>The average&#x02009;&#x000b1;&#x02009;standard deviation of WES in 2007 was 67.83&#x02009;&#x000b1;&#x02009;11.11 and 65.82&#x02009;&#x000b1;&#x02009;13.42 in the tutored and tutorless groups, respectively. In 2008, WES was 77.85&#x02009;&#x000b1;&#x02009;17.30 and 74.33&#x02009;&#x000b1;&#x02009;21.28 in the tutored and tutorless groups, respectively (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>).</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Students&#x02019; performance on the written examination score and self-contentment score during each year.</bold> The error bars represent the standard deviations.</p></caption><graphic xlink:href="1472-6920-13-158-3"/></fig><p>In the tutorless groups, a tendency towards higher average scores during both years was observed, but this difference was not significant (2007, p&#x02009;=&#x02009;0.25; 2008, p&#x02009;=&#x02009;0.20). However, in 2008, variances tended to be significantly larger than the average score in the tutorless group (2007, p&#x02009;=&#x02009;0.058; 2008, p&#x02009;=&#x02009;0.039).</p></sec><sec><title>Self-contentment scores</title><p>In 2007, the average&#x02009;&#x000b1;&#x02009;standard deviation for SCS was 4.08&#x02009;&#x000b1;&#x02009;0.78 and 3.88&#x02009;&#x000b1;&#x02009;0.88 in the tutored and tutorless groups, respectively. In 2008, SCS was 4.37&#x02009;&#x000b1;&#x02009;0.76 and 4.29&#x02009;&#x000b1;&#x02009;0.74 in the tutored and tutorless groups, respectively (Figure&#x000a0;<xref ref-type="fig" rid="F3">3</xref>). In both years, the average score tended to be lower in the tutorless group but this was not significant (2007, p&#x02009;=&#x02009;0.092; 2008, p&#x02009;=&#x02009;0.42). A tendency in the variations from the average scores was inconsistent, and no significant differences were found (2007, p&#x02009;=&#x02009;0.23; 2008, p&#x02009;=&#x02009;0.75). No correlation was observed between WES and SCS in both years (2007, r&#x02009;=&#x02009;0.023, p&#x02009;=&#x02009;0.74; 2008, r&#x02009;=&#x02009;-0.021 p&#x02009;=&#x02009;0.76).</p></sec><sec><title>Self-evaluation</title><p>Self-evaluation results, excluding SCS, for each session are shown in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>. In the tutored group of 2007, there was a tendency for high self-evaluation. In particular, self-evaluation of group dynamics were significantly different. However, inconsistent trends were recognised in 2008. In each student during both years, there was a tendency for high self-evaluation in the tutored PBL session but no significant differences were found. When the results of sessions 1 and 2 for both years were combined, no significant differences were found between the tutored and tutorless groups.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>In the present study, a mixed course comprising tutored and tutorless PBL was undertaken by first-year medical students, and the learning outcomes were analysed. The results indicate that students undertaking tutorless PBL were adequately prepared for written examinations, whereas tutored PBL prepares students for written examinations and also increases self-contentment with their learning experience.</p><p>Tutorless PBL is an efficient way to reduce demands on faculty time and resources [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. However, tutorless PBL may give rise to several problems that may impede learning [<xref ref-type="bibr" rid="B21">21</xref>], and according to Duncan-Hewitt [<xref ref-type="bibr" rid="B8">8</xref>] these are as follows: (1) students&#x02019; emotions can interfere with their willingness to participate and decrease their quality of learning, (2) misapprehensions and weak thinking, group and problem-solving skills can cause students to become engrossed in the problem-solving process, and the problem-solving skills can cause students to concentrate more on the problem-solving process and (3) there is no opportunity to directly examine students&#x02019; abilities and skills, which can be systematically improved. Despite these problems, we did not provide any special intervention for students in the tutorless group; however, sufficient guidance was provided to all students in both the tutorless and tutored groups prior to PBL. In addition, following reports regarding SGD and self-learning, we provided formative evaluation and feedback every day. With regard to the fact that WES and SCS did not significantly differ between the tutorless and tutored groups, it appears that these traditional complementary methods may have affected the learning outcomes of the tutorless group.</p><p>In contrast, variances in WES scores were considerably greater in the tutorless than tutored groups. Moreover, the average SCS in the tutored group tended to be higher than that in the tutorless group. The daily report also appeared to show that the quality and quantity of SGD and self-learning varied widely in the tutorless group. These results imply that it is unavoidable that tutorless PBL may give rise to some students who do not learn well without a structured process, thereby receiving lower scores. Although the daily short lectures directed the students to study, Lee et al. [<xref ref-type="bibr" rid="B22">22</xref>] reported that these were not correlated with perceived self directed learning ability in their case-oriented problem-stimulated mixed PBL curriculum for the 1^st and 2^nd-year medical students. Nicholl and Lou [<xref ref-type="bibr" rid="B15">15</xref>] argued that in their tutorless PBL model, it was important to provide as many opportunities as possible for formative assessment in order to monitor and adjust the development of the tutorless groups. We agree with their argument that ongoing and immediate formative assessment is valuable in tutorless PBL. Recent case reports have suggested that the use of pre-set cues, particularly pictures and videos [<xref ref-type="bibr" rid="B14">14</xref>] or e-learning resources [<xref ref-type="bibr" rid="B11">11</xref>] can help conduct effective tutorless PBL. To improve tutorless PBL, these new learning resources can positively contribute to students&#x02019; self-contentment with their learning experience.</p><p>A possible reason why WES and SCS did not significantly differ between the tutorless and tutored groups may be because students in the tutorless group communicated with those in the tutored group after every SGD, thereby allowing students in the tutorless group to get some information from those in the tutored group. This helped in decreasing the gap in learning outcomes between both groups. This may also prompt peer-assisted learning for students in both groups. Ross and Cameron [<xref ref-type="bibr" rid="B23">23</xref>] argued that peer-assisted learning is an efficient and effective way of preparing medical students for their future role as educators. Although little attention has been paid to the effects of peer-assisted learning in medical schools [<xref ref-type="bibr" rid="B24">24</xref>], the positive effects of peer-assisted learning in medical education is gaining notoriety [<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B25">25</xref>]. Although there have been no studies comparing tutorless PBL to student-tutored PBL, it appears that the effect of peer-assisted learning in student-tutored PBL is higher than that of tutored and tutorless PBL; however, a mixed course, including tutorless and tutored PBL, has unique characteristics as well as the potential to provide good learning outcomes.</p><p>There are two primary limitations to the current study. First, we were unable to develop other methods of measuring the effects of group learning and could measure only self-evaluation of group dynamics and attentiveness. The other types of evaluations such as peer evaluation may be more important in the tutorless group. Second limitation relates to the setting (four SGDs in a week session) and period (pre-clinical students), in which this study was conducted. Usually PBL tutorials are conducted in the first and second sessions with two to three days time for self study. A curriculum in which the SGD frequency is reduced during each session would be required. Moust et al. [<xref ref-type="bibr" rid="B26">26</xref>] reported that students in faculty-led PBL performed better than those in peer-facilitated groups during essay examinations designed to assess higher-order cognitive skills. Thus, we completed PBL2 for the third- and forth-year students as a tutored PBL course. More research on the cognitive effects of tutorless PBL for medical students during their clinical years is required.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Tutorless PBL can potentially produce learning outcomes that are comparable to tutored PBL; however, tutorless PBL is different from faculty/staff-tutored PBL and student-tutored PBL. Tutorless PBL has been used when PBL conducted in large classrooms [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. However, tutorless PBL should not be easily used in the same way as student-tutored PBL because of the difficulty in maintaining faculty tutors or learning rooms. An appropriate and effective curriculum can be administered in every school by combining tutored PBL and student-tutored PBL or tutorless PBL. We encourage the implementation of PBL in schools because this will potentially lead to further developments in the area of PBL.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors&#x02019; contributions</title><p>SH conducted all studies, performed statistical analyses and drafted the manuscript. KT and CI helped draft and critically appraise the manuscript. YF was involved in the conceptualisation of the study and participated in its design and coordination. All authors read and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6920/13/158/prepub">http://www.biomedcentral.com/1472-6920/13/158/prepub</ext-link></p></sec>

The impact of preparatory activities on medical school selection outcomes: a cross-sectional survey of applicants to the university of Adelaide medical school in 2007

<sec><title>Background</title><p>Selection into medical school is highly competitive with more applicants than places. Little is known about the preparation that applicants undertake for this high stakes process. The study aims to determine what preparatory activities applicants undertake and what difficulties they encounter for each stage of the application process to medical school and in particular what impact these have on the outcome.</p></sec><sec><title>Methods</title><p>A cross-sectional survey of 1097 applicants who applied for a place in the University of Adelaide Medical School in 2007 and participated in the UMAT (Undergraduate Medicine and Health Sciences Admission Test) and oral assessment components of the selection process. The main outcome measures were an offer of an interview and offer of a place in the medical school and were analysed using logistic regression.</p></sec><sec><title>Results</title><p>The odds of a successful outcome increased with each additional preparatory activity undertaken for the UMAT (odds ratio 1.22, 95% confidence interval 1.11 to 1.33; P&#x02009;&#x0003c;&#x02009;0.001) and the oral assessment (1.36, 1.19 to 1.55; P&#x02009;&#x0003c;&#x02009;0.001) stage of selection. The UMAT preparatory activities associated with the offer of an interview were attendance of a training course by a private organisation (1.75, 1.35 to 2.27: P&#x02009;&#x0003c;&#x02009;0.001), use of online services of a private organisation (1.58, 1.23 to 2.04; P&#x02009;&#x0003c;&#x02009;0.001), and familiarising oneself with the process (1.52, 1.15 to 2.00; p&#x02009;=&#x02009;0.021). The oral assessment activities associated with an offer of a place included refining and learning a personal resume (9.73, 2.97 to 31.88; P&#x02009;&#x0003c;&#x02009;0.001) and learning about the course structure (2.05, 1.29 to 3.26; P&#x02009;=&#x02009;0.022).</p><p>For the UMAT, applicants who found difficulties with learning for this type of test (0.47, 0.35 to 0.63: P&#x02009;&#x0003c;&#x02009;0.001), with the timing of UMAT in terms of school exams (0.48, 0.5 to 0.66; P&#x02009;&#x0003c;&#x02009;0.001) and with the inability to convey personal skills with the UMAT (0.67, 0.52 to 0.86; P&#x02009;=&#x02009;0.026) were significantly less likely to be offered an interview.</p></sec><sec><title>Conclusions</title><p>Medical schools make an enormous effort to undertake a selection process that is fair and equitable and which selects students most appropriate for medical school and the course they provide. Our results indicate that performance in the selection processes can be improved by training. However, if these preparatory activities may be limited to those who can access them, the playing field is not even and increasing equity of access to medical schools will not be achieved.</p></sec>

<contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Laurence</surname><given-names>Caroline O</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>caroline.laurence@adelaide.edu.au</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Zajac</surname><given-names>Ian T</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>ian.zajac@csiro.au</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Lorimer</surname><given-names>Michelle</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>michelle.lorimer@adelaide.edu.au</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Turnbull</surname><given-names>Deborah A</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>deborah.turnbull@adelaide.edu.au</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Sumner</surname><given-names>Karen E</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>Karen.Sumner@ruraldoc.com.au</email></contrib>

BMC Medical Education

<sec><title>Background</title><p>The selection of students into medical courses is a major issue for university medical schools and it is often embroiled in controversy [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>]. With more applicants than places, the selection process is very competitive and often scrutinised to ensure that it selects applicants with suitable characteristics for medicine and no systemic biases exist within the process. In countries such as Australia [<xref ref-type="bibr" rid="B4">4</xref>], NZ [<xref ref-type="bibr" rid="B5">5</xref>], Canada [<xref ref-type="bibr" rid="B6">6</xref>], US [<xref ref-type="bibr" rid="B6">6</xref>] and the UK [<xref ref-type="bibr" rid="B7">7</xref>] the selection process to medical school consists of an aptitude/cognitive test, non-cognitive measures such as interviews and ratings of academic achievement such as matriculation score or grade point average.</p><p>While a variation on these processes is widely used, the evidence base for selection is small [<xref ref-type="bibr" rid="B8">8</xref>]. Most research has focused on the predictive validity of different parts of the selection process, such as the aptitude/cognitive tests like the MCAT [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>], UKCAT [<xref ref-type="bibr" rid="B11">11</xref>-<xref ref-type="bibr" rid="B13">13</xref>], or UMAT [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>] or ratings of academic achievement such as grade point average [<xref ref-type="bibr" rid="B16">16</xref>].</p><p>Another small body of research has focused on demographic and socio-economic factors that may influence or predict the outcome of the process and if the process biases against disadvantaged groups [<xref ref-type="bibr" rid="B17">17</xref>-<xref ref-type="bibr" rid="B20">20</xref>].</p><p>There is little research regarding the lengths applicants go to in order to maximise their chance of receiving an offer or the difficulties they encounter during this process [<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B23">23</xref>]. Moreover, there is little evidence on of the impact of the preparatory activities undertaken or difficulties encountered on the outcome [<xref ref-type="bibr" rid="B24">24</xref>]. The aims of this study are: to determine what preparation applicants undertake for each stage of the application process at the University of Adelaide Medical School (UMAT and oral assessment); to determine what difficulties they encounter at each stage of the application process; and if the preparatory activities or difficulties encountered impact on the outcome of the selection process.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Survey</title><p>We conducted a questionnaire-based survey of all applicants who applied to the University of Adelaide for entry into the undergraduate medicine course in 2007. A summary of the selection process at the University of Adelaide Medical School is provided in Table&#x000a0;<xref ref-type="table" rid="T1">1</xref>. The selection process has three parts. Firstly, applicants sit the Undergraduate Medical and Health Sciences Admission Test (UMAT) and their score for this test determines who proceeds on to the next stage of the process, the oral assessment. Those who undertake the oral assessment receive a score. An offer of a place is then based on a composite score of the UMAT, oral assessment score and an applicant&#x02019;s Australian Tertiary Admissions Rank. A student who receives an offer can either accept the place or not.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>The selection process at the University of Adelaide at time of study</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Application stage</bold></th><th align="left"><bold>Description</bold></th><th align="left"><bold>Assessing</bold></th><th align="left"><bold>Applicant no.s</bold></th></tr></thead><tbody><tr><td rowspan="4" align="left" valign="bottom">1. UMAT (Undergraduate medicine &#x00026; health sciences admission test)<hr/></td><td align="left" valign="bottom">Managed nationally by Australian Council for Educational Research (ACER)<hr/></td><td align="left" valign="bottom">&#x025cf; Logical reasoning and problem solving<hr/></td><td rowspan="4" align="left" valign="bottom">&#x0003e;2500<hr/></td></tr><tr><td align="left" valign="bottom">Test held mid year, prior to the December selection process<hr/></td><td align="left" valign="bottom">&#x025cf; Understanding<hr/></td></tr><tr><td align="left" valign="bottom">Written examination<hr/></td><td align="left" valign="bottom">&#x025cf; people<hr/></td></tr><tr><td align="left" valign="bottom">No preparation required Used as a screening tool<hr/></td><td align="left" valign="bottom">&#x025cf; Non-verbal reasoning<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">2. Oral assessment<hr/></td><td align="left" valign="bottom">Structured interview (35&#x000a0;minutes)<hr/></td><td align="left" valign="bottom">&#x025cf; Non-cognitive qualities<hr/></td><td rowspan="2" align="left" valign="bottom">~350<hr/></td></tr><tr><td align="left" valign="bottom">Two assessors &#x02013; Faculty &#x00026; community<hr/></td><td align="left" valign="bottom">&#x025cf; Humanistic qualities<hr/></td></tr><tr><td align="left">3. ATAR (Australian Tertiary Admissions Rank)</td><td align="left">Score of 90 or above (max score 100)</td><td align="left">&#x025cf; Academic ability</td><td align="left">190</td></tr></tbody></table></table-wrap><p>International students applying for fee-paying places were excluded as the selection process varied slightly for this group. The survey consisted of four questionnaires which corresponded with the various stages of the application process &#x02013; applicants who participated in the UMAT, applicants who were offered an oral assessment, applicants who were offered a place but did not accept and applicants who were offered a place and accepted. For each successive group an additional section was included that pertained to the particular stage in the application process. The questionnaires were developed following a focus group with first year medical school students who had experienced the application process approximately six months earlier and a review of the literature. The questionnaires covered a number of areas including socio-demographics, interest in medicine, preparatory activities and difficulties encountered during the various stages of the application process. A copy of the key components of the questionnaire used in this analysis are provided Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>.</p><p>The questionnaires were mailed to applicants between March and June 2007, following the completion of the medical school application process. In order to maximise the response rate the project team adopted Dillman&#x02019;s Total Design Method [<xref ref-type="bibr" rid="B25">25</xref>]. This involves mailing out an initial questionnaire, which is followed by a reminder and after that a final questionnaire for those who do not respond.</p></sec><sec><title>Statistical analysis</title><p>Chi-squared tests and two sample t-tests (where appropriate) were used to determine if there were differences between responders and non-responders in terms of age, sex and location of residence.</p><p>For the responders, three outcome groups were determined &#x02013; those who completed the UMAT, those who attended the oral assessment and those who were offered a place, irrespective of whether they accepted the offer or not. The analysis was undertaken in two parts reflecting the different stages of the application process. Firstly, those offered an oral assessment interview following the UMAT were compared with those who were not offered an oral assessment interview. Then for those who progressed to the oral assessment, comparisons were then made between those who were offered a place with those who were not offered a place in the medical school.</p><p>The number of preparatory activities was calculated and logistic regression used to determine if there was an association between the number of preparatory activities undertaken and a successful outcome (offer of an interview or offer of a place).</p><p>To determine what preparatory activities (or difficulties) were undertaken, separate logistic regression analyses were performed for each outcome.</p><p>Statistical significance was set at 5%. To account for multiple testing, Bonferroni Correction was applied for each preparatory activity and difficulty encountered, resulting in a corrected P value. The corrected and uncorrected P values are reported in the tables, along with odds ratios and 95% confidence intervals. All analysis was performed using SAS 9.2 (SAS Institute, Cary NC, USA).</p><p>This study was approved by the University of Adelaide Human Research Ethics Committee.</p></sec></sec><sec sec-type="results"><title>Results</title><p>A total of 2150 questionnaires were distributed and an overall response rate of 51% (1097/2150) was achieved. Response rates varied with the application stage. For applicants who did not receive an offer of an interview following the UMAT, the response rate was 46% (739/1610). For those applicants who participated in the oral interviews but did not receive and offer, the response rate was 55% (108/201), while a response rate of 74% (250/339) was obtained from those applicants who received an offer of a place.</p><p>A comparison of age and sex of responders to non-responders showed there were no statistical differences in the groups in terms of age with both groups having the same mean age (17.7&#x000a0;years, p&#x02009;=&#x02009;0.68). However, significantly more females and fewer males were in the response group (60% and 40% respectively) than the non-response group (52%, 48% respectively, p &#x0003c;0.001).</p><sec><title>Participant characteristics</title><p>The socio-demographic characteristics of the respondents are summarised in Table&#x000a0;<xref ref-type="table" rid="T2">2</xref>. Of all applicants in the study, there were a greater proportion of female applicants (60%) and applicants who attended a non-government school (57%). Over a third of respondents were from South Australia (37%) and 78% spoke English as their main language. Over half (56%) of the respondents had a family member who worked in the health profession, with the most common family member being their mother (57%). 15% of respondents had lived rurally for at least eight or more years.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Demographic characteristics of respondents by outcome</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th colspan="2" align="left"><bold>Characteristics</bold></th><th align="left"><bold>Completed UMAT but not offered oral assessment (</bold><bold><italic>N</italic></bold>&#x02009;<bold>=&#x02009;738)</bold></th><th align="left"><bold>Completed, UMAT, offered oral assessment but were not offered a place (</bold><bold><italic>N</italic></bold>&#x02009;<bold>=&#x02009;109)</bold></th><th align="left"><bold>Completed UMAT and oral assessment and were offered a Place (</bold><bold><italic>N</italic></bold>&#x02009;<bold>=&#x02009;250)</bold></th><th align="left"><bold>All applicants in the study (</bold><bold><italic>N</italic></bold>&#x02009;<bold>=&#x02009;1097)</bold></th></tr></thead><tbody><tr><td rowspan="4" align="left" valign="bottom">Age in Years<hr/></td><td align="left" valign="bottom">Mean<hr/></td><td align="left" valign="bottom">18.0<hr/></td><td align="left" valign="bottom">17.9<hr/></td><td align="left" valign="bottom">17.9<hr/></td><td align="left" valign="bottom">18.0<hr/></td></tr><tr><td align="left" valign="bottom"><italic>(SD)</italic><hr/></td><td align="left" valign="bottom"><italic>(1.7)</italic><hr/></td><td align="left" valign="bottom"><italic>(0.7)</italic><hr/></td><td align="left" valign="bottom"><italic>(0.9)</italic><hr/></td><td align="left" valign="bottom"><italic>(1.4)</italic><hr/></td></tr><tr><td align="left" valign="bottom">Median<hr/></td><td align="left" valign="bottom"><italic>18.0</italic><hr/></td><td align="left" valign="bottom"><italic>18.0</italic><hr/></td><td align="left" valign="bottom"><italic>18.0</italic><hr/></td><td align="left" valign="bottom"><italic>18.0</italic><hr/></td></tr><tr><td align="left" valign="bottom">Range<hr/></td><td align="left" valign="bottom">15-49<hr/></td><td align="left" valign="bottom">16-19<hr/></td><td align="left" valign="bottom">17-23<hr/></td><td align="left" valign="bottom">15-49<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Sex (%)<hr/></td><td align="left" valign="bottom">Male<hr/></td><td align="left" valign="bottom">295 (40.0)<hr/></td><td align="left" valign="bottom">45 (41.3)<hr/></td><td align="left" valign="bottom">102 (40.8)<hr/></td><td align="left" valign="bottom">442 (40.3)<hr/></td></tr><tr><td align="left" valign="bottom">Female<hr/></td><td align="left" valign="bottom">443 (60.0)<hr/></td><td align="left" valign="bottom">64 (58.7)<hr/></td><td align="left" valign="bottom">148 (59.2)<hr/></td><td align="left" valign="bottom">655 (59.7)<hr/></td></tr><tr><td rowspan="4" align="left" valign="bottom">Schooling (%)<hr/></td><td align="left" valign="bottom">Non-Government<hr/></td><td align="left" valign="bottom">407 (55.2)<hr/></td><td align="left" valign="bottom">63 (57.8)<hr/></td><td align="left" valign="bottom">154 (61.6)<hr/></td><td align="left" valign="bottom">624 (56.9)<hr/></td></tr><tr><td align="left" valign="bottom">Government<hr/></td><td align="left" valign="bottom">268 (36.3)<hr/></td><td align="left" valign="bottom">43 (39.5)<hr/></td><td align="left" valign="bottom">86 (34.4)<hr/></td><td align="left" valign="bottom">397 (36.2)<hr/></td></tr><tr><td align="left" valign="bottom">Other (eg overseas school)<hr/></td><td align="left" valign="bottom">22 (3.0)<hr/></td><td align="left" valign="bottom">2 (1.8)<hr/></td><td align="left" valign="bottom">5 (2.0)<hr/></td><td align="left" valign="bottom">29 (2.6)<hr/></td></tr><tr><td align="left" valign="bottom">Combination of non-Governement and Government<hr/></td><td align="left" valign="bottom">36 (4.9)<hr/></td><td align="left" valign="bottom">1 (0.9)<hr/></td><td align="left" valign="bottom">5 (2.0)<hr/></td><td align="left" valign="bottom">42 (3.8)<hr/></td></tr><tr><td rowspan="3" align="left" valign="bottom">Main language (%)<hr/></td><td align="left" valign="bottom">English<hr/></td><td align="left" valign="bottom">555 (75.2)<hr/></td><td align="left" valign="bottom">89 (81.7)<hr/></td><td align="left" valign="bottom">212 (84.8)<hr/></td><td align="left" valign="bottom">856 (78.0)<hr/></td></tr><tr><td align="left" valign="bottom">Other<hr/></td><td align="left" valign="bottom">172 (23.3)<hr/></td><td align="left" valign="bottom">20 (18.4)<hr/></td><td align="left" valign="bottom">37 (9.7)<hr/></td><td align="left" valign="bottom">229 (20.9)<hr/></td></tr><tr><td align="left" valign="bottom">Missing<hr/></td><td align="left" valign="bottom">11 (1.5)<hr/></td><td align="left" valign="bottom">0<hr/></td><td align="left" valign="bottom">1 (0.7)<hr/></td><td align="left" valign="bottom">12 (1.1)<hr/></td></tr><tr><td rowspan="2" align="left" valign="bottom">Lived rurally (%)*<hr/></td><td align="left" valign="bottom">Yes<hr/></td><td align="left" valign="bottom">115 (15.6)<hr/></td><td align="left" valign="bottom">18 (16.5)<hr/></td><td align="left" valign="bottom">27 (10.8)<hr/></td><td align="left" valign="bottom">160 (14.6)<hr/></td></tr><tr><td align="left" valign="bottom">No<hr/></td><td align="left" valign="bottom">623 (84.4)<hr/></td><td align="left" valign="bottom">91 (83.5)<hr/></td><td align="left" valign="bottom">223 (58.2)<hr/></td><td align="left" valign="bottom">937 (85.4)<hr/></td></tr><tr><td rowspan="8" align="left" valign="bottom">Home state (%)**<hr/></td><td align="left" valign="bottom">South Australia<hr/></td><td align="left" valign="bottom">281 (38.1)<hr/></td><td align="left" valign="bottom">38 (34.9)<hr/></td><td align="left" valign="bottom">85 (34.0)<hr/></td><td align="left" valign="bottom">404 (36.8)<hr/></td></tr><tr><td align="left" valign="bottom">Victoria<hr/></td><td align="left" valign="bottom">209 (28.3)<hr/></td><td align="left" valign="bottom">34 (31.2)<hr/></td><td align="left" valign="bottom">79 (31.6)<hr/></td><td align="left" valign="bottom">322 (29.4)<hr/></td></tr><tr><td align="left" valign="bottom">New South Wales<hr/></td><td align="left" valign="bottom">102 (13.8)<hr/></td><td align="left" valign="bottom">15 (13.8)<hr/></td><td align="left" valign="bottom">53 (21.2)<hr/></td><td align="left" valign="bottom">170 (15.5)<hr/></td></tr><tr><td align="left" valign="bottom">Queensland<hr/></td><td align="left" valign="bottom">42 (5.7)<hr/></td><td align="left" valign="bottom">10 (9.2)<hr/></td><td align="left" valign="bottom">12 (4.8)<hr/></td><td align="left" valign="bottom">64 (5.8)<hr/></td></tr><tr><td align="left" valign="bottom">Western Australia<hr/></td><td align="left" valign="bottom">48 (6.5)<hr/></td><td align="left" valign="bottom">5 (4.6)<hr/></td><td align="left" valign="bottom">6 (2.4)<hr/></td><td align="left" valign="bottom">59 (5.4)<hr/></td></tr><tr><td align="left" valign="bottom">Australian Capital Territory<hr/></td><td align="left" valign="bottom">18 (2.4)<hr/></td><td align="left" valign="bottom">2 (1.8)<hr/></td><td align="left" valign="bottom">7 (2.8)<hr/></td><td align="left" valign="bottom">27 (2.5)<hr/></td></tr><tr><td align="left" valign="bottom">Tasmania<hr/></td><td align="left" valign="bottom">14 (1.9)<hr/></td><td align="left" valign="bottom">2 (1.8)<hr/></td><td align="left" valign="bottom">3 (1.2)<hr/></td><td align="left" valign="bottom">19 (1.7)<hr/></td></tr><tr><td align="left" valign="bottom">Northern Territory<hr/></td><td align="left" valign="bottom">5 (0.7)<hr/></td><td align="left" valign="bottom">2 (1.8)<hr/></td><td align="left" valign="bottom">0 (0)<hr/></td><td align="left" valign="bottom">7 (0.6)<hr/></td></tr><tr><td colspan="2" align="left" valign="bottom">Family member worked in health profession<hr/></td><td align="left" valign="bottom">422 (57.2)<hr/></td><td align="left" valign="bottom">63 (57.8)<hr/></td><td align="left" valign="bottom">133 (53.2)<hr/></td><td align="left" valign="bottom">618 (56.3)<hr/></td></tr><tr><td rowspan="3" align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom">Mother***<hr/></td><td align="left" valign="bottom">225 (53.3)<hr/></td><td align="left" valign="bottom">39 (61.9)<hr/></td><td align="left" valign="bottom">87 (65.4)<hr/></td><td align="left" valign="bottom">351 (56.8)<hr/></td></tr><tr><td align="left" valign="bottom">Father***<hr/></td><td align="left" valign="bottom">144 (34.1)<hr/></td><td align="left" valign="bottom">28 (44.4)<hr/></td><td align="left" valign="bottom">51 (38.3)<hr/></td><td align="left" valign="bottom">223 (36.1)<hr/></td></tr><tr><td align="left" valign="bottom">Other relative eg Aunt, Uncle***<hr/></td><td align="left" valign="bottom">126 (29.9)<hr/></td><td align="left" valign="bottom">17 (27.0)<hr/></td><td align="left" valign="bottom">44 (33.1)<hr/></td><td align="left" valign="bottom">187 (30.3)<hr/></td></tr><tr><td align="left">&#x000a0;</td><td align="left">Sibling***</td><td align="left">75 (17.8)</td><td align="left">13 (20.6)</td><td align="left">18 (13.5)</td><td align="left">106 (17.2)</td></tr></tbody></table><table-wrap-foot><p>* Rurality is defined as having lived rurally for &#x02265;8&#x000a0;years.</p><p>** Home State based on address on initial application.</p><p>***Includes only those who selected yes to primary question and is multiple response.</p></table-wrap-foot></table-wrap></sec><sec><title>Amount of preparation</title><p>The number of preparatory activities undertaken for the UMAT and oral assessment component of the application process by the outcome (offered or not offered an oral assessment interview or offered or not offered a place) is shown in Figure&#x000a0;<xref ref-type="fig" rid="F1">1</xref> and Figure&#x000a0;<xref ref-type="fig" rid="F2">2</xref>, respectively. The mean number of activities for the UMAT was 2.9 (2.8 for those who did not receive an offer of oral assessment interview and 3.2 activities for those who received an offer of an oral assessment interview). Regarding the UMAT, for every one additional activity that applicants undertook, the odds of being offered an interview increased by 1.22 (95% confidence interval [CI] 1.11 - 1.33; P&#x02009;&#x0003c;&#x02009;0.001). The mean number of activities undertaken for the oral assessment was 3.1 ( 2.4 for those who did not receive an offer of a place and 3.4 for those who did receive an offer of a place). For the oral assessment, for every one additional activity that applicants undertook, they increased their odds of being offered a place by 1.36 (95% CI 1.19 - 1.55; P&#x02009;&#x0003c;&#x02009;0.001).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Number of preparation activities for UMAT by outcome.</p></caption><graphic xlink:href="1472-6920-13-159-1"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Number of preparation activities for oral assessment by outcome.</p></caption><graphic xlink:href="1472-6920-13-159-2"/></fig></sec><sec><title>Type of preparation</title><sec><title>UMAT</title><p>The 1097 applicants undertook a range of preparatory activities for the UMAT and the most common activity was the completion of example questions (83%) (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>). This was followed by familiarisation with the process (67%), speaking with others who had completed the UMAT previously (57%) and use of online services of private organisations (48%) (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>). Only a very small proportion of applicants indicated that they undertook no preparation for the UMAT (3%).</p><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>UMAT and Oral assessment preparatory activities which resulted in the offer of an interview or a place in the medical school</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Stage of application process</bold></th><th align="left"><bold>Activity</bold></th><th align="left"><bold>Frequency of responses (%)</bold></th><th align="left"><bold>Odds ratio of being offered an interview or place given they did activity (95% CIs)</bold></th><th align="left"><bold>Uncorrected P value</bold></th><th align="left"><bold>Corrected P value</bold></th></tr></thead><tbody><tr><td rowspan="7" align="left" valign="bottom">UMAT (n&#x02009;=&#x02009;1097)<hr/></td><td align="left" valign="bottom">Completed example questions<hr/></td><td align="left" valign="bottom">912 (83.1)<hr/></td><td align="left" valign="bottom">1.15 (0.81-1.61)<hr/></td><td align="left" valign="bottom">0.435<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Familiarised myself with the process<hr/></td><td align="left" valign="bottom">734 (66.9)<hr/></td><td align="left" valign="bottom">1.52 (1.15-2.00)<hr/></td><td align="left" valign="bottom">0.003<hr/></td><td align="left" valign="bottom">0.021*<hr/></td></tr><tr><td align="left" valign="bottom">Spoke with people who had completed it before<hr/></td><td align="left" valign="bottom">621 (56.6)<hr/></td><td align="left" valign="bottom">1.16 (0.90-1.50)<hr/></td><td align="left" valign="bottom">0.254<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Utilised online services of private organisation<hr/></td><td align="left" valign="bottom">524 (47.8)<hr/></td><td align="left" valign="bottom">1.58 (1.23-2.04)<hr/></td><td align="left" valign="bottom">&#x0003c;0.001<hr/></td><td align="left" valign="bottom">&#x0003c;0.001*<hr/></td></tr><tr><td align="left" valign="bottom">Attended training course offered by private organisation<hr/></td><td align="left" valign="bottom">395 (36.0)<hr/></td><td align="left" valign="bottom">1.75 (1.35-2.27)<hr/></td><td align="left" valign="bottom">&#x0003c;0.001<hr/></td><td align="left" valign="bottom">&#x0003c;0.001*<hr/></td></tr><tr><td align="left" valign="bottom">Other (eg books, seminars)<hr/></td><td align="left" valign="bottom">45 (4.1)<hr/></td><td align="left" valign="bottom">1.14 (0.61&#x02013;2.13)<hr/></td><td align="left" valign="bottom">0.680<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Nothing<hr/></td><td align="left" valign="bottom">37 (3.4)<hr/></td><td align="left" valign="bottom">0.75 (0.36-1.58)<hr/></td><td align="left" valign="bottom">0.454<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">&#x000a0;<hr/></td><td align="left" valign="bottom"><bold>Activity</bold><hr/></td><td align="left" valign="bottom"><bold>Frequency of responses (%)</bold><hr/></td><td align="left" valign="bottom"><bold>Odds ratio of being offered an interview or place given they did activity (95% CIs</bold>)<hr/></td><td align="left" valign="bottom"><bold>Uncorrected P value</bold><hr/></td><td align="left" valign="bottom"><bold>Corrected P value</bold><hr/></td></tr><tr><td rowspan="10" align="left" valign="bottom">Oral assessment (n&#x02009;=&#x02009;37)<hr/></td><td align="left" valign="bottom">Learnt about the course structure<hr/></td><td align="left" valign="bottom">233 (64.9)<hr/></td><td align="left" valign="bottom">2.05 (1.29-3.26)<hr/></td><td align="left" valign="bottom">0.002<hr/></td><td align="left" valign="bottom">0.022**<hr/></td></tr><tr><td align="left" valign="bottom">Discussed the oral assessment with previous applicants<hr/></td><td align="left" valign="bottom">211 (58.8)<hr/></td><td align="left" valign="bottom">1.72 (1.09-2.71)<hr/></td><td align="left" valign="bottom">0.019<hr/></td><td align="left" valign="bottom">0.209<hr/></td></tr><tr><td align="left" valign="bottom">Prepared and learnt answers to possible questions<hr/></td><td align="left" valign="bottom">184 (51.3)<hr/></td><td align="left" valign="bottom">1.60 (1.02-2.52)<hr/></td><td align="left" valign="bottom">0.042<hr/></td><td align="left" valign="bottom">0.462<hr/></td></tr><tr><td align="left" valign="bottom">Practiced interviews with family and friends<hr/></td><td align="left" valign="bottom">175 (48.8)<hr/></td><td align="left" valign="bottom">1.31 (0.83-2.06)<hr/></td><td align="left" valign="bottom">0.238<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Used the online services of a private company<hr/></td><td align="left" valign="bottom">86 (24.0)<hr/></td><td align="left" valign="bottom">2.06 (1.15-3.71)<hr/></td><td align="left" valign="bottom">0.014<hr/></td><td align="left" valign="bottom">0.154<hr/></td></tr><tr><td align="left" valign="bottom">Attended a training course offered by a private company<hr/></td><td align="left" valign="bottom">60 (16.7)<hr/></td><td align="left" valign="bottom">1.24 (0.67-2.31)<hr/></td><td align="left" valign="bottom">0.493<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Refined and learnt a personal resume<hr/></td><td align="left" valign="bottom">57 (15.9)<hr/></td><td align="left" valign="bottom">9.73 (2.97-31.88)<hr/></td><td align="left" valign="bottom">&#x0003c;0.001<hr/></td><td align="left" valign="bottom">&#x0003c;0.001**<hr/></td></tr><tr><td align="left" valign="bottom">School organised practice interviews<hr/></td><td align="left" valign="bottom">39 (10.9)<hr/></td><td align="left" valign="bottom">1.52 (0.69-3.31)<hr/></td><td align="left" valign="bottom">0.294<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Old scholars who studied/study medicine came to my school to talk<hr/></td><td align="left" valign="bottom">37 (10.3)<hr/></td><td align="left" valign="bottom">3.99 (1.38-11.56)<hr/></td><td align="left" valign="bottom">0.006<hr/></td><td align="left" valign="bottom">0.066<hr/></td></tr><tr><td align="left" valign="bottom">Other (eg career advisor, self-reflection)<hr/></td><td align="left" valign="bottom">5858 (16.2)<hr/></td><td align="left" valign="bottom">1.17 (0.63-2.19)<hr/></td><td align="left" valign="bottom">0.617<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left">&#x000a0;</td><td align="left">Nothing</td><td align="left">27 (7.5)</td><td align="left">1.04 (0.44-2.45)</td><td align="left">0.920</td><td align="left">1</td></tr></tbody></table><table-wrap-foot><p>*Statistically significant P values after Bonferroni correction for 7 comparisons.</p><p>** Statistically significant P values after Bonferroni correction for 11 comparisons.</p></table-wrap-foot></table-wrap><p>After adjustment for multiple comparisons, several preparatory activities undertaken for the UMAT were significantly associated with obtaining an offer of an oral assessment interview. Those applicants who attended training courses by private organisations (OR 1.75, 95% CI 1.35 - 2.27; P&#x02009;&#x0003c;&#x02009;0.001 corrected), used the online services of private organisation (OR 1.58, 95% CI 1.23 - 2.04; P&#x02009;&#x0003c;&#x02009;0.001 corrected) or who familiarised themselves with the process (OR 1.52, 95% CI 1.15 - 2.00; P&#x02009;=&#x02009;0.021 corrected) were significantly more likely to receive an offer of an interview than those who did not undertake the activity (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>).</p></sec><sec><title>Oral assessment</title><p>For the 359 applicants who were awarded an oral assessment interview, the most common preparatory activities were learning about the course structure (65%), discussing the process with previous applicants (59%), preparing answers for possible questions (51%) and having practice interviews with family and friends (49%) (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>). A small number of applicants (8%) did no preparation for the interview.</p><p>Two preparatory activities for the oral assessment interview were significantly associated with being offered a place in medical school after adjustment for multiple comparisons (Table&#x000a0;<xref ref-type="table" rid="T3">3</xref>). These activities were refining and learning a personal resume (OR 9.73, 95% CI 2.97 - 31.88; P&#x02009;&#x0003c;&#x02009;0.001 corrected) and learning the course structure (OR 2.05, 95% CI 1.29 - 3.26; P&#x02009;=&#x02009;0.022 corrected).</p></sec></sec><sec><title>Difficulties encountered</title><sec><title>UMAT</title><p>Applicants (n&#x02009;=&#x02009;1097) reported a number of difficulties with the UMAT. Time limit of the test (56%), inability to convey personal skills (50%) and the inability to determine if answers were correct (43%) were the most common difficulties encountered (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>).</p><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Difficulties encountered with the UMAT and Oral assessment and their association with either the offer of an interview or a place in the medical school</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Stage of application process</bold></th><th align="left"><bold>Difficulty</bold></th><th align="left"><bold>Frequency of responses (%)</bold></th><th align="left"><bold>Odds ratio of being offered an interview or place given they found the variable difficult (95% CIs)</bold></th><th align="left"><bold>Uncorrected P value</bold></th><th align="left"><bold>Corrected P value</bold></th></tr></thead><tbody><tr><td rowspan="13" align="left" valign="bottom">UMAT (n&#x02009;=&#x02009;1097)<hr/></td><td align="left" valign="bottom">Time limit<hr/></td><td align="left" valign="bottom">619 (56.4)<hr/></td><td align="left" valign="bottom">0.88 (0.68-1.14)<hr/></td><td align="left" valign="bottom">0.325<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Can&#x02019;t convey personal skills<hr/></td><td align="left" valign="bottom">547 (49.9)<hr/></td><td align="left" valign="bottom">0.67 (0.52-0.86)<hr/></td><td align="left" valign="bottom">0.002<hr/></td><td align="left" valign="bottom">0.026<hr/></td></tr><tr><td align="left" valign="bottom">No idea if answers are correct<hr/></td><td align="left" valign="bottom">474 (43.2)<hr/></td><td align="left" valign="bottom">0.95 (0.74-1.22)<hr/></td><td align="left" valign="bottom">0.689<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Inability to prepare/learn for this type of test<hr/></td><td align="left" valign="bottom">364 (33.2)<hr/></td><td align="left" valign="bottom">0.47 (0.35-0.63)<hr/></td><td align="left" valign="bottom">&#x0003c;0.001<hr/></td><td align="left" valign="bottom">&#x0003c;0.001**<hr/></td></tr><tr><td align="left" valign="bottom">Preparation costs<hr/></td><td align="left" valign="bottom">313 (28.5)<hr/></td><td align="left" valign="bottom">0.76 (0.57-1.01)<hr/></td><td align="left" valign="bottom">0.056<hr/></td><td align="left" valign="bottom">0.728<hr/></td></tr><tr><td align="left" valign="bottom">Timing of UMAT in terms of school exams<hr/></td><td align="left" valign="bottom">293 (26.7)<hr/></td><td align="left" valign="bottom">0.48 (0.35-0.66)<hr/></td><td align="left" valign="bottom">&#x0003c;0.001<hr/></td><td align="left" valign="bottom">&#x0003c;0.001**<hr/></td></tr><tr><td align="left" valign="bottom">Absence of breaks<hr/></td><td align="left" valign="bottom">194 (17.7)<hr/></td><td align="left" valign="bottom">1.10 (0.80-1.53)<hr/></td><td align="left" valign="bottom">0.554<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Attendance costs<hr/></td><td align="left" valign="bottom">200 (18.2)<hr/></td><td align="left" valign="bottom">0.68 (0.48-0.95)<hr/></td><td align="left" valign="bottom">0.025<hr/></td><td align="left" valign="bottom">0.325<hr/></td></tr><tr><td align="left" valign="bottom">Exam environment<hr/></td><td align="left" valign="bottom">171 (15.6)<hr/></td><td align="left" valign="bottom">0.91 (0.64-1.29)<hr/></td><td align="left" valign="bottom">0.596<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Timing of results in terms of school exams<hr/></td><td align="left" valign="bottom">153 (14.0)<hr/></td><td align="left" valign="bottom">0.69 (0.47-1.02)<hr/></td><td align="left" valign="bottom">0.061<hr/></td><td align="left" valign="bottom">0.793<hr/></td></tr><tr><td align="left" valign="bottom">Getting to the exam<hr/></td><td align="left" valign="bottom">94 (8.6)<hr/></td><td align="left" valign="bottom">0.86 (0.54-1.37)<hr/></td><td align="left" valign="bottom">0.527<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Other (eg stress, organisation)<hr/></td><td align="left" valign="bottom">124 (11.3)<hr/></td><td align="left" valign="bottom">0.93 (0.63-1.40)<hr/></td><td align="left" valign="bottom">0.752<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">No difficulties<hr/></td><td align="left" valign="bottom">40 (3.7)<hr/></td><td align="left" valign="bottom">2.35 (1.25-4.43)<hr/></td><td align="left" valign="bottom">0.007<hr/></td><td align="left" valign="bottom">0.091<hr/></td></tr><tr><td align="left" valign="bottom"><bold>Stage of application process</bold><hr/></td><td align="left" valign="bottom"><bold>Difficulty</bold><hr/></td><td align="left" valign="bottom"><bold>Frequency of responses (%)</bold><hr/></td><td align="left" valign="bottom"><bold>Odds ratio of being offered an interview or place given they found the variable difficult (95% CIs)</bold><hr/></td><td align="left" valign="bottom"><bold>Uncorrected P value</bold><hr/></td><td align="left" valign="bottom"><bold>Corrected P value</bold><hr/></td></tr><tr><td rowspan="16" align="left" valign="bottom">Oral Assessment (n&#x02009;=&#x02009;359)<hr/></td><td align="left" valign="bottom">Hard to judge my performance<hr/></td><td align="left" valign="bottom">236 (65.7)<hr/></td><td align="left" valign="bottom">0.87 (0.54-1.41)<hr/></td><td align="left" valign="bottom">0.571<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Cost of attending interview<hr/></td><td align="left" valign="bottom">124 (34.5)<hr/></td><td align="left" valign="bottom">0.78 (0.49-1.24)<hr/></td><td align="left" valign="bottom">0.294<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">It was difficult to structure answers<hr/></td><td align="left" valign="bottom">92 (25.6)<hr/></td><td align="left" valign="bottom">0.67 (0.40-1.10)<hr/></td><td align="left" valign="bottom">0.111<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Getting to the interview<hr/></td><td align="left" valign="bottom">62 (17.3)<hr/></td><td align="left" valign="bottom">0.75 (0.42-1.34)<hr/></td><td align="left" valign="bottom">0.334<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Questions were baffling<hr/></td><td align="left" valign="bottom">59 (16.4)<hr/></td><td align="left" valign="bottom">0.58 (0.32-1.03)<hr/></td><td align="left" valign="bottom">0.060<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Inability to learn/prepare for the interview<hr/></td><td align="left" valign="bottom">59 (16.4)<hr/></td><td align="left" valign="bottom">0.49 (0.27-0.86)<hr/></td><td align="left" valign="bottom">0.012<hr/></td><td align="left" valign="bottom">0.204<hr/></td></tr><tr><td align="left" valign="bottom">Situational cards<hr/></td><td align="left" valign="bottom">53 (14.8)<hr/></td><td align="left" valign="bottom">1.59 (0.80-3.15)<hr/></td><td align="left" valign="bottom">0.186<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Absence of questions concerning personal achievements<hr/></td><td align="left" valign="bottom">51 (14.2)<hr/></td><td align="left" valign="bottom">0.95 (0.50-1.79)<hr/></td><td align="left" valign="bottom">0.862<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Interview environment<hr/></td><td align="left" valign="bottom">38 (10.6)<hr/></td><td align="left" valign="bottom">0.94 (0.45-1.94)<hr/></td><td align="left" valign="bottom">0.862<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Time pressure<hr/></td><td align="left" valign="bottom">24 (6.7)<hr/></td><td align="left" valign="bottom">1.33 (0.51-3.45)<hr/></td><td align="left" valign="bottom">0.554<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Timing of the interview<hr/></td><td align="left" valign="bottom">21 (5.9)<hr/></td><td align="left" valign="bottom">0.56 (0.23-1.37)<hr/></td><td align="left" valign="bottom">0.199<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">The interviewer tried to distract me<hr/></td><td align="left" valign="bottom">20 (5.6)<hr/></td><td align="left" valign="bottom">1.79 (0.59-5.55)<hr/></td><td align="left" valign="bottom">0.299<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Received short notice of interview<hr/></td><td align="left" valign="bottom">15 (4.2)<hr/></td><td align="left" valign="bottom">0.48 (0.17-1.36)<hr/></td><td align="left" valign="bottom">0.249*<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Preparation costs<hr/></td><td align="left" valign="bottom">14 (3.9)<hr/></td><td align="left" valign="bottom">0.57 (0.19-1.68)<hr/></td><td align="left" valign="bottom">0.374*<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Presence of other applicants&#x02019; parents<hr/></td><td align="left" valign="bottom">11 (3.1)<hr/></td><td align="left" valign="bottom">0.76 (0.22-2.64)<hr/></td><td align="left" valign="bottom">0.741*<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left" valign="bottom">Other (eg anxiety, intimidating interviewers)<hr/></td><td align="left" valign="bottom">58 (16.2)<hr/></td><td align="left" valign="bottom">1.17 (0.63-2.19)<hr/></td><td align="left" valign="bottom">0.617<hr/></td><td align="left" valign="bottom">1<hr/></td></tr><tr><td align="left">&#x000a0;</td><td align="left">No difficulties</td><td align="left">27 (7.5)</td><td align="left">1.04 (0.44-2.45)</td><td align="left">0.920</td><td align="left">&#x000a0;</td></tr></tbody></table><table-wrap-foot><p>*Fisher&#x02019;s exact test was used.</p><p>** Statistically significant P values after Bonferroni correction for 13 comparisons.</p></table-wrap-foot></table-wrap><p>The reported difficulties which resulted in a statistically significant decrease in the likelihood of being offered an oral assessment interview included difficulties with the timing of the UMAT in terms of school exams (OR 0.48, 95% CI 0.35 - 0.66; P&#x02009;&#x0003c;&#x02009;0.001 corrected), the perceived inability to prepare or learn for this type of test (OR 0.47, 95% CI 0.35 - 0.63; P&#x02009;&#x0003c;&#x02009;0.001 corrected) or feeling the test did not allow applicants to convey personal skills (OR 0.67, 95% CI 0.52 - 0.86; P&#x02009;=&#x02009;0.026 corrected) (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>).</p></sec><sec><title>Oral assessment</title><p>Those who participated in the oral assessment interview (n&#x02009;=&#x02009;359) also encountered several difficulties, the most common being their inability to judge their performance (66%), the cost of attending the interview (36%) and difficulty in structuring answers (26%) (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>). None of the difficulties identified with the oral assessment interview significantly decreased the likelihood of being offered a place in the program (Table&#x000a0;<xref ref-type="table" rid="T4">4</xref>).</p></sec></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>This study is one of the first to investigate the impact of preparatory activities undertaken by applicants on the outcome of an application to medical school. Our results show that the more preparatory activities an applicant undertakes for the various stages of the selection process the more likely they are to be offered an interview or place in the medical school. We also found that certain distinct preparatory activities increased the odds of being offered an interview or a place. Some of the difficulties encountered during the process reduced the odds of being offered an interview.</p><p>There is limited research on the preparatory activities undertaken for medical school selection and there are even fewer studies that have investigated the impact of these activities on outcome (ie selection). Most of the literature on the role of preparation and coaching on performance in medical school exams comes from the USA. This research focuses on exams that occur once the student is in medical school, such as the USMLE step 1 exam [<xref ref-type="bibr" rid="B26">26</xref>,<xref ref-type="bibr" rid="B27">27</xref>], and often involve relatively small samples.[<xref ref-type="bibr" rid="B27">27</xref>] While there is some research on applicant preferences for new processes such as the Multiple Mini Interview (MMI) over traditional approaches [<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B29">29</xref>], we found only one other study that had investigated student attitudes to parts of the selection process [<xref ref-type="bibr" rid="B30">30</xref>]. Their results correspond with the difficulties outlined in our study such as cost and perceived level of difficulty but only included those who had been successful in the medical school selection process. One small Australian study of 287 applicants assessed the impact of coaching on the UMAT and coaching and repeat testing on the MMI [<xref ref-type="bibr" rid="B21">21</xref>]. They found that coaching had a small effect on the parts of the UMAT test but was ineffective in improving the scores of the MMI. A small New Zealand study [<xref ref-type="bibr" rid="B24">24</xref>] investigated the impact of preparatory course and tutoring on the UMAT score and found these had no significant impact on the score. This contrasts with our results, although this study looked at the UMAT score result not success. However, they found that students undertaking the preparatory courses and spending more money on UMAT preparation had greater confidence in gaining an approved UMAT score.</p><p>Preparation for the various stages of the application process seems to contribute to a successful outcome and these results raise some important issues that have implications for the selection processes used by medical schools. We provide evidence that when there is a highly desired outcome [<xref ref-type="bibr" rid="B31">31</xref>], such as entry to medical school, applicants are likely highly motivated and consequently undertake a range of activities to ensure they achieve the desired outcome.</p><p>Of particular importance is the influence that preparatory activities have on this outcome. This particularly relates to the aptitude test such as the UMAT and UKCAT which are designed to measure innate aptitude and be less amenable to coaching. Messick and Jungeblut [<xref ref-type="bibr" rid="B32">32</xref>] defined &#x02018;coaching&#x02019; as encompassing activities such as test familiarisation, practice with feedback, training strategies for specific formats, general test taking and skill development exercises. The majority of the preparatory activities identified by applicants in our study fall under this definition and for the UMAT, coaching does in fact seem to make a difference to the outcome. Research from the US, where coaching for the MCAT is long established, provides some evidence of practice effects, but the results are mixed and may be influenced by applicant motivation [<xref ref-type="bibr" rid="B33">33</xref>].</p><p>While the above issues are important, the most critical concern that this study raises is around equity. This is of particular importance in a climate where medical schools are increasingly seen as having a social accountability mandate. Prideaux et al [<xref ref-type="bibr" rid="B8">8</xref>] argue that widening access to unrepresented groups such as those from a rural, ethnic or low socio-economic background, is a values issue and not a &#x02018;a technical question of choosing one selection method over another&#x02019; (page 219). We believe our results challenge this argument. If coaching in its broadest definition is associated with successfully applying to medicine, it has equity implications. The activities identified by the applicants in this study may not be accessible to all applicants due to cost, geography or opportunities. Training programs provided by commercial companies for medical school selection are costly, with basic packages start at $AU395 and range up to more than $AU1500. There can also be travel costs associated with attending workshops or courses. Some applicants may not be able to attend training if they live in rural or remote areas. The opportunity to discuss the application process with previous applicants may also favour applicants from medical families or those who attended private schools where access to old scholars to discuss the process or practice techniques is provided. Applicants from disadvantaged groups may not have access to such resources. These issues are of particular concern in a time when many medical schools/governments want to broaden the diversity of medical students, particularly from underrepresented groups [<xref ref-type="bibr" rid="B34">34</xref>]. This aspect may explain why, even with advent of strategies to increase underrepresented groups, widening access has not been totally successful [<xref ref-type="bibr" rid="B34">34</xref>]. Research has indicated that certain demographic characteristics are associated with a successful outcome to medical school [<xref ref-type="bibr" rid="B19">19</xref>]. Our results suggest that investigating the impact of socio-demographic characteristics on preparatory activities would be worthwhile in order to understand the role of these activities on outcome.</p><p>What can be done to address this additional variable in the selection process? Options to level the playing field may be the provision of coaching activities to disadvantaged groups, using processes shown to be less influenced by coaching such as the MMI, scores on such tests be weighted accordingly to account for level of disadvantage [<xref ref-type="bibr" rid="B35">35</xref>] or new selection processes such as the use of personality testing and testing emotional intelligence. However, changing the selection process is complicated and costly and it is likely that if a new process is developed, applicants will again adapt and find ways to increase their likelihood of being selected.</p><sec><title>Limitations</title><p>A key strength of the study is that data were collected as soon as the selection process had been completed when the applicants&#x02019; experiences were relatively fresh and it included applicants who were successful and unsuccessful, providing a full perspective on the selection processes. However, there are several limitations. The response rate achieved was 51% of the total applicants for 2007 and together with the fact that participants were drawn from one cohort may limit the generalisation of the results. It is likely that the results are biased to successful applicants, with the response rate lowest for those who were unsuccessful and highest for those who were successful.</p><p>While it was possible to identify individual preparatory activities or difficulties encountered that impacted on the outcome, we could not determine which combination of activities or difficulties that had the most influence on the outcome. This resulted from the large number of combinations possible as well as multiple combinations. Increasing the sample size or limiting the variables available for the respondents could address this issue in the future. Finally, while the results showed that certain preparatory activities were associated with a successful outcome, this does not indicate that the applicant is more suitable for the medical program or a career in medicine.</p></sec></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Medical schools make an enormous effort to undertake a selection process that is fair and equitable and which selects students most appropriate for medical school and the course they provide. Our results indicate that performance in the selection processes can be improved by training. However, if these preparatory activities may be limited to those who can access them, the playing field is not even and increasing equity of access to medical schools will not be achieved.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors&#x02019; contributions</title><p>CL conceived the idea, developed the design, coordinated the study, contributed to the analysis and interpretation of the results and drafted the manuscript. DT and IZ contributed to planning the research, analysis and the interpretation of the results and critically revised the paper. ML performed the analysis, contributed to the interpretation of the results and critically revised the paper. KS contributed to the planning of the research and critically revised the paper. All authors approved the final version of the manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1472-6920/13/159/prepub">http://www.biomedcentral.com/1472-6920/13/159/prepub</ext-link></p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional file 1</title><p>Survey questions relating to the preparatory activities and difficulties with the UMAT and Oral Assessment.</p></caption><media xlink:href="1472-6920-13-159-S1.docx"><caption><p>Click here for file</p></caption></media></supplementary-material></sec>