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recently , magnetic thin films used in magnetic components , such as inductive devices and magnetic heads , have attracted more and more attention because of the increasing demand for improvement and miniaturization of electromagnetic devices [ 1 - 3 ] . the basic requirements for these films operated in the ghz range are high resistivity , high permeability , high saturation magnetization ms and appropriate large anisotropy field hk so as to effectively suppress eddy current loss and to possess high ferromagnetic resonance ( fmr ) frequency fr ( which determines the cut - off frequency for high frequency application ) . insulator granular films ( migf ) consisting of magnetic metal nano - granules uniformly distributed in an insulator matrix are one of the best candidates for satisfying above demands , because these films have the advantages of high ms and high of magnetic metals and high of insulator . up to now , soft magnetic properties of granular films composed of magnetic metals ( and their alloys ) and various x-(oxide , nitride and fluoride ) where x is nonmagnetic elements such as hf , al , si , zr , rare earth , etc . have been investigated [ 4 - 8 ] . the mechanism of the soft magnetic properties of migfs has been confirmed to be the exchange coupling between granules through the intergranular regions . however , the microscopic origin of exchange coupling has not been totally understood , such as the medium of the exchange coupling for different constitution nano - granular films . by considering the high resistivity of semiconductor oxides , metal semiconductor granular films ( msgf ) consisting of magnetic metal nano - granules uniformly distributed in semiconductor matrix , such as the feco zno systems , may possess good high frequency properties . as one kind of wide used semiconductor oxides due to the excellent electrical and optical properties , the current carriers of zno may mediate the exchange coupling between magnetic granules and hence favor good soft magnetic properties . so far , there is no work studying systematically the soft magnetic properties of the feco zno granule films reported . in order to test whether zno can mediate the exchange coupling , the structure , magnetic properties and electronic transport properties of feco zno msgfs were systematically studied in this work . ( fe65co35)x(zno)1x granular thin films were deposited on water - cooled glass substrates by radio frequency ( rf ) magnetron sputtering at room temperature with a background pressure lower than 4 10 pa . a three - inch fe65co35 alloy disk covered with zno chips ( 5 5 1 mm ) the sputtering power was 100 w , and sputtering ar gas was kept at 0.40 pa . an external magnetic field obtained by a pair of permanent magnets located on both sides of the substrates was applied during deposition to induce an in - plane uniaxial anisotropy for the samples , and the intensity of external magnetic field is 500 oe . the saturation magnetization 4ms , coercivity hc and in - plane magnetic anisotropy field hk were determined by a vibrating sample magnetometer ( vsm , lakeshore 7304 model ) . the composition of the samples was measured by energy dispersive x - ray spectroscopy ( eds ) . the structure and microstructure were analyzed by x - ray diffraction ( xrd ) ( philips xpert model ) with cu - k radiation and high resolution transmission electronic microscopy ( hrtem ) , respectively . the permeability spectra were carried out with a pna e8363b vector network analyzer using the shorted microstrip transmission - line perturbation method , which works from 100 mhz to 5 ghz . all the above measurements were performed for as - deposited samples without any post - heat treatment . figure 1 shows the bright - field hrtem image ( a ) and the corresponding electron diffraction ( ed ) pattern ( b ) , as well as the xrd spectrum ( c ) for a typical sample of x = 0.67 . it can be seen that the sample consists of bcc fe65co35 particles embedded uniformly in the insulating zno matrix from the image ( a ) . the average size of fe65co35 particles and the distance between them are about 2 and 4.5 nm , respectively . figure 1b exhibits the diffraction peaks from ( 110 ) and ( 200 ) planes of bcc fe65co35 alloys , ( 111 ) and ( 103 ) planes of zno , respectively , which are totally consistent with those of xrd shown in fig . the average diameter of fe65co35 grains calculated by the full width at half maximum ( fwhm ) of the diffraction peak in xrd spectrum through scherrer s equation is about 2.1 nm , which is close to that of fe65co35 granules . hrtem image ( a ) and the corresponding ed pattern ( b ) as well as the xrd spectrum ( c ) for a typical sample of x= 0.67 figure 2 expresses the x dependence of hc , hk and . it is seen that increases slowly with decreasing x and then increases rapidly after x < 0.64 . hc is less than 15 oe , and hkis over 43 oe in the range 0.63 < x < 0.72 , which means good soft magnetic properties are obtained in this range . especially , for the sample with x = 0.67 , its in - plane hysteresis loop shown in fig . the coercivities in hard and easy axes are 1.43 and 7.08 oe , respectively , 4ms = 9.85 kg , and reaches 2.06 m cm , which is three orders higher than that of pure feco alloy . these good soft magnetic properties and the high imply that the films are promising for high frequency applications . h curves for ( fe65co35)0.67(zno)0.33 film figure 4 shows the dependence of complex permeability = j on frequency f for the film of x = 0.67 , where and represent the real and imaginary parts of complex permeability , respectively . it can be seen that is more than 100 below 1.83 ghz and then gradually decreases with frequency , while the imaginary part gradually increases to a maximum at f = 2.31 ghz , which can be ascribed to the ferromagnetic resonance ( fmr ) . lifshitz equations taking account of the coherent spin procession , as shown by the solid lines in fig . the fmr frequency obtained by this fitting is around 2.31 ghz , consistent with the experimental result . the high fmr frequency also implies that the granular films are promising for applications in the high frequency range . permeability spectra ( ~ f ) for the sample of x = 0.67 considering the mechanism of the good soft magnetic properties , we turn to the exchange coupling model . according to the herzer s statement , if the average size d of magnetic particles and the distance s between particles are reduced to be smaller than a characteristic length , namely , the exchange length lex , the exchange interaction between particles may take place , which tends to align the magnetic moments of neighboring particles parallel and so overcome the magneto - crystalline anisotropy and the demagnetization effect of individual particles . as a result , the effective magnetic anisotropy of materials is reduced significantly , which leads to the decrease in coercivity and hence the increase in permeability , and then good soft magnetic properties are obtained . for fe65co35 , the characteristic length lex is about 26 nm , while the d and s are 2.3 and 4.5 nm , respectively , based on the results of hrtem spectrum . thus , ( d + s ) lex holds , exchange coupling can be realized in this film , which is responsible for the good soft magnetic properties . in order to study the microscopic origin of the exchange coupling between granules , the temperature dependence of resistivity figure 5a depicts thecurve , which exhibits a negative temperature coefficient of resistivity ( tcr ) . 5b , which indicates that the mechanism of electronic transportation of the sample is weak localized electron electron interaction . these weak localized electrons may also be the medium of exchange coupling between magnetic granules . resistivity as a function of temperature ( a ) and fitting result ( b ) for a typical sample of x = 0.67 ( fe65co35)x(zno)1x granular films were fabricated by magnetron sputtering , and good soft magnetic properties have been obtained in x range from 0.63 to 0.72 with small hc and appropriate large hk . for the typical sample of x = 0.67 , the coercivity of 1.43 oe in the hard axis and 7.08 oe in the easy axis are obtained , and the resistivity reaches 2.06 m cm . at a frequency lower than 1.83 ghz , the real part of the complex permeability of this sample is more than 100 , and the fmr frequency reaches 2.31 ghz , which implies that the film is promising for high frequency applications . the good soft magnetic properties origin from the exchange coupling between magnetic granules and the weak localized electrons existing in films may be the medium of this exchange coupling . this work was supported by national natural science foundation of china ( grant no.50571041 ) . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

a series of nano - granular films composed of magnetic metal ( fe65co35 ) granules with a few nanometers in size and semiconductor oxide ( zno ) have been fabricated by a magnetron sputtering method , and excellent soft magnetic properties have been achieved in a wide metal volume fraction ( x ) range for as - deposited samples due to the exchange coupling between feco granules ( a ferromagnetic interaction in nano - scale ) . in a wide range ( 0.53 < x < 0.71 ) , the films exhibit coercivity hc not exceeding 15 oe , along with high resistivity . especially for the sample with x = 0.67 , coercivities in hard and easy axes are 1.43 and 7.08 oe , respectively , 4ms = 9.85 kg , and reaches 2.06 103 cm . the dependence of complex permeability = j on frequency shows that the real part is more than 100 below 1.83 ghz and that the ferromagnetic resonance frequency reaches 2.31 ghz , implying the promising for high frequency application . the measured negative temperature coefficient of resistivity reveals that may be the weak localized electrons existing in samples mediate the exchange coupling .

juxtafacet cysts ( jfcs ) are uncommon intraspinal , extradural lesions that arise from the facet joints . jfcs can be further distinguished as either synovial cysts , if histology confirms the presence of a synovial lining membrane , or alternatively as a ganglion cyst , if no such membrane is present . however , few differences exist between the two in terms of diagnosis and management.1 2 3 4 5 6 7 their pathogenesis remains unclear , with most authors citing excessive joint mobility leading to synovial fluid herniation through joint capsule defects.2 4 7 calcification in the cyst walls is a common finding on imaging for jfcs , appearing as peripheral areas of high attenuation on computed tomography ( ct ) and peripheral hypointensity on t1- and t2-weighted magnetic resonance imaging ( mri).8 9 10 but although calcification of the cyst walls are commonly reported findings,9 10 11 complete calcification of the cyst is exceedingly rare.11 12 13 we present here an unusual case of a jfc presenting as a giant , multilobular , completely calcified mass with significant extraforaminal extension . the patient was a 57-year - old woman who presented with a 2-year history of gradually worsening lower back and left leg pain . she also reported numbness in her left heel and foot and subjective weakness in the left foot . her past medical history was significant for hypothyroidism , hypertension , mild levoscoliosis , rheumatoid arthritis , and scleroderma managed on weekly methotrexate injection . her past surgical history was negative for any previous lumbar surgeries , but the patient had received multiple cervical surgeries resulting in c3c7 fusion . on neurologic exam , the patient was found to have decreased strength in plantarflexion and dorsiflexion of the left lower extremity but was otherwise nonfocal . the patient 's imaging was most notable for a large , multilobular mass , measuring 54 31 30 mm , arising from the left l5s1 facet joint that had caused destruction of the joint itself and that showed severe encroachment of the adjoining foramen , with coexisting grade i anterolisthesis of l5 on s1 . the lesion was hyperdense on plain radiograph ( fig . 1 ) and on ct imaging ( fig . 2a , 2b , and 2c ) and appeared hypointense on t1- and t2-weighted mri sequences ( fig . preoperative plain film of the lumbar spine showing a large , completely calcified lesion extending out from the l5s1 intervertebral space . ( a ) axial computed tomography ( ct ) image taken preoperatively , showing the origin of the lesion from the left l5s1 facet joint . ( b ) a preoperative midsagittal ct image demonstrating grade 1 anterolisthesis of l5 on s1 . ( c ) a preoperative sagittal ct image demonstrating foraminal stenosis at l5s1 caused by the lesion . ( a ) t1-weighted magnetic resonance image ( mri ) of the same lesion , which appears uniformly hypointense . ( b ) the patient underwent an l5 laminectomy for excision of the mass along with an l5 to s1 fixation and fusion . intraoperatively , the lesion was noted to consist of a pastelike material that was compressing the thecal sac and that extended ventrally out of the foramen . histologic analysis identified the lesion as a mineralized simple pseudocyst , containing mineralized proteinaceous material that did not refract plane polarized light after ethanol fixation ( fig . 4a and 4b ) . ( a ) hematoxylin and eosin stained sections of the resection reveal dense membranous fibrous connective tissue separating spaces associated with dense calcifications as well as smaller loculated spaces filled with calcium ( 20 magnification ) . ( b ) higher magnification ( 40 ) reveals the wall of the spaces to be associated with granular calcifications and no obvious cellular lining . the patient experienced no intraoperative or postoperative complications , and her postoperative hospital course was unremarkable . by postoperative day 2 , the patient had recovered full strength in her extremities but still had some residual numbness , and she was discharged to home in stable condition on postoperative day 3 . at 2-year follow - up , the patient continued to be free of her lower extremity pain and weakness and had not experienced any subsequent postsurgical complications ( fig . 5a and 5b ) . ( a ) postoperative plain film ( anterior - posterior ) showing removal of the mass and placement of fixation hardware . mineralized , extradural lesions of the lumbar spine arising from the facet joint are uncommon causes of spinal pathology . the differential diagnosis consists of several uncommon disorders , including synovial osteochondromatosis,14 tumoral calcinosis,15 tumoral calcium pyrophosphate dehydrate crystal deposition disease ( pseudogout),16 and lumbar presentation of ossification of the ligamentum flavum.17 the case we present here represents another potential consideration in that differential diagnosis . completely calcified jfcs are rare , with only a handful of instances existing in the literature . almefty et al presented four patients with multiple - level , bilateral , consistently calcified thoracic spinal cysts.12 these lesions were atypical due to their number , location , and mineralization . kasliwal and deutsch presented on the case of a unilateral lumbar jfc at the l4l5 level.13 similar to our case , the patient presented with radicular symptoms in the setting of a lumbar lesion that appeared hypointense on t1- and t2-weighted mri . it should be noted that calcification of jfcs can result from treatment as well , as mtellus et al noted a case of a cyst undergoing complete calcification after intracystic steroid injection therapy.11 the case presented here was remarkable not just for its mineralization but also for its size . jfcs have typically been recorded to be between 10 and 20 mm in diameter at their widest dimension on imaging.10 18 the case we report here , however , measured 54 mm at its widest dimension , showing significant extension into nearby structures . moreover , although jfcs frequently extend into the spinal canal and cause compression of the thecal sac,2 the majority of the lesion in our case existed outside the spinal canal . this may explain how the cyst grew to be so large before the patient presented for evaluation . it should also be noted that our patient 's medical history included rheumatoid arthritis and scleroderma , and systemic inflammatory diseases have been associated with jfcs in the past.1 19 moreover , inflammatory arthritides are known to contribute to the pathogenesis of synovial cysts outside of the spine.20 however , no studies to date have established systemic inflammation as a risk factor for jfc development , and the role of inflammatory joint disease in the pathogenesis jfcs remains unclear . many methods of treating jfcs have been described , including simple laminotomy with cystectomy and the potential addition of a laminectomy , hemilaminectomy , mesial facetectomy , or foraminotomy.3 4 minimally invasive approaches with tubular retractors have also been described.19 21 in terms of treatment of this patient , it was presumed that the symptoms of back pain were secondary to facet dysfunction and that the left - sided plantar flexion weakness was due to foraminal stenosis . due to the size and mineralization of the lesion , it was felt that a complete facetectomy was needed to adequately address the underlying pathology , necessitating a concurrent fusion . our case highlights the significant variation that exists in the size , location , and content of jfcs . although uncommon , jfcs should be considered in the differential diagnosis of lesions arising from the facet joint , even if they appear calcified and noncystic in nature .

study design case report . objective to report the case of one patient who developed a giant , completely calcified , juxtafacet cyst . methods a 57-year - old woman presented with a 2-year history of progressively worsening lower back pain , left leg pain , weakness , and paresthesias . imaging showed a giant , completely calcified mass arising from the left l5s1 facet joint , with coexisting grade i l5 on s1 anterolisthesis . the patient was treated with laminectomy , excision of the mass , and l5s1 fixation and fusion . results the patient had an uncomplicated postoperative course and had complete resolution of her symptoms as of 1-year follow - up . conclusions when presented with a solid - appearing , calcified mass arising from the facet joint , a completely calcified juxtafacet cyst should be considered as part of the differential diagnosis .

panax ginseng ( ginseng ) has been used traditionally in eastern asia over thousands of years . it has been used orally to treat various diseases including hypertension , diabetes mellitus , liver and kidney dysfunction , mental disorders , and postmenopausal disorders . in addition , topical applications have also been used to heal wounds and reduce skin inflammation . in the past few decades , it has been proved that ginseng extracts actually show a wide range of effects against human diseases . their potential therapeutic effects have been mainly attributed to its immunomodulatory , neuroprotective , antioxidative , antitumor , and hepatoprotective activities . ginseng contains a number of active ingredients including ginsenosides , polysaccharides , phytosterols , peptides , polyacetylenes , fatty acids , and polyacetylenic alcohols , which have different effects on carbohydrate and lipid metabolism , cognition , angiogenesis , and the neuroendocrine , immune , cardiovascular , and central nervous systems . among the active constituents of ginseng , ginsenosides are known to be the major biologically active components of ginseng and the most widely studied . several studies have shown that ginsenosides play important roles in the pharmacological effects of ginseng . . however , ginseng contains other constituents , including ginsenoyne , phenolic compounds , polyacetylenes , sesquiterpenes , methoxypyrazine , alkylpyrazine derivatives , sesquiterpene alcohols , panasinsanols , and -carboline . white ginseng is peeled , dried , ginseng root and red ginseng is produced by steaming fresh ginseng root at 98100c for 23 h , and then drying until the moisture content is < 15% . red and white ginseng have both been shown to have immunomodulatory , anti - inflammatory , antioxidant , and antiatopic activities . moreover , red ginseng has been reported to have more potent pharmacological activities than white ginseng in some respects [ 2022 ] . the differences in biological activities between red and white ginseng are caused by the chemical changes of ginsenosides after the steaming process . steaming partially converts the original ginsenosides to deglycoslated derivatives . as a result , the species and amounts of ginsenosides are quite different based on the processing method used . chu et al showed that a total of 53 and 43 compounds were tentatively identified in white ginseng and red ginseng samples , respectively . the featured compounds are mainly malonyl ginsenosides in white ginseng , and decarboxyl products of mal - ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in red ginseng . it is interesting that ginsenosides show a wide variety of biological activities , although the absorption rates from orally administered intact ginsenosides are very low . in the human intestinal tract , thus , the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . in this context several studies showed that the transformation of ginsenosides into deglycosylated ginsenosides is needed to increase ginseng 's effectiveness in vivo . abnormal changes in skin color induce significant cosmetic problems with a negative effect on quality of life . there are two groups of pigmentary disorders : disorders of the quantitative and qualitative distribution of normal pigment and the abnormal presence of exogenous or endogenous pigments in the skin . hyperpigmentation - related diseases include melasma , lentigines , nevus , ephelis , freckles , postinflammatory hyperpigmentation , and age spots . postinflammatory hyperpigmentation appears in many skin conditions , including acne , eczema , and contact dermatitis . skin color is determined by various factors including melanin content , oxygenation state of hemoglobin in capillary vessels , carotenoid content , water content , and organization of collagen fibers in the dermis . among these factors , melanin is the major determinant of skin color . in this context , melanogenesis is a biochemical pathway responsible for melanin synthesis that is controlled by complex regulatory mechanisms . melanogenesis occurs in melanocytes confined in separate cytoplasmic organelles called melanosomes , which contain key enzymes of melanogenesis . differences in skin color are related to the size , number , shape , and distribution of melanosomes , whereas melanocyte density typically remains relatively constant . although tyrosinase is the key regulatory enzyme of melanogenesis , tyrosinase - related protein ( trp)-1 , dopachrome tautomerase ( dct / trp2 ) , and melanosomal matrix proteins ( pmel17 , mart-1 ) carry out important roles in regulating melanogenesis . the genes of tyrosinase , trp-1 , and dct contain common transcription starting sites , the microphthalmia - associated transcription factor ( mitf ) binding sites . the intracellular signal transduction pathways of protein kinase c , cyclic amp ( camp ) , and nitrogen oxide are involved in the regulation of melanogenesis . various endogenous and exogenous factors , such as estrogen and ultraviolet ( uv ) radiation , affect melanogenesis via signal transduction pathways . these endogenous / exogenous factors exert their actions directly on melanocytes or indirectly via surrounding skin cells . melanocytes , keratinocytes , dermal fibroblasts , and other skin cells communicate with each other by factors that are secreted and cell cell contacts . it has been shown that the interactions between keratinocytes and melanocytes are critical in the regulation of melanogenesis . in addition , dermal factors have been found to be involved in the regulation of melanogenesis . at the same time , stimulated melanocytes secret a number of signal molecules targeting not only keratinocytes but also skin immune cells . soluble factors released by melanocytes include proinflammatory cytokines and chemokines such as interleukin ( il)-1/1 , il-6 , il-8 il-10 , tumor necrosis factor ( tnf)- , transforming growth factor ( tgf)- , catecholamines , eicosanoids , serotonin , -melanocyte stimulating factor ( -msh ) , and nitric oxide ( no ) . a variety of hypopigmenting agents including hydroquinone , arbutin , tretinoin , kojic acid , azelaic acid , vitamin c , n - acetylglucosamine , niacinamide , linoleic acid , ellagic acid , methimazole , dioic acid , soy extract , licorice extract , rucinol , and glycolic acid have been used alone or in combination to treat abnormal hyperpigmentation . these agents can interfere with the pigmentation process at several different steps of skin pigmentation . however , the treatment of hyperpigmented conditions still remains challenging and the results are often discouraging . thus there is a need for novel skin - whitening agents that are highly effective and tolerable . in this article , we review recent reports investigating the skin - whitening effect of ginseng and its components and the underlying mechanisms of action , and then discuss their potential as candidates for novel skin - whitening agents . p. ginseng is one of the most widely used medicinal plants in traditional oriental medicine . over thousands of years , it has been used to improve the overall condition of skin , as well as to treat a wide variety of diseases . however , genuine scientific approaches to clarify the efficacy of ginseng in skin have only been made in recent years . several reports have shown that ginseng extract , powder , or some other constituents could inhibit melanogenesis in vitro and in vivo . table 1 summarizes the direct effects of ginseng and its components on skin color and key enzymes involved in melanogenesis . song et al reported that red ginseng powder improved melasma in a human clinical trial . they orally administered korean red ginseng powder for 24 weeks to female patients with melasma . after 24 weeks , the melasma area and severity index score decreased and melasma quality of life scale showed improvement in 91% of patients . in addition , 74% of the patients showed some improvement on the patient- and investigator - rated global improvement scales . most of reports investigating the antimelanogenic effect of ginseng were conducted in vitro used purified tyrosinase or melanocyte cell lines . in melan - a cells treated with ethanol extract of ginseng seeds , melanin content and in addition to the crude extract or powder , several studies tested the effects of specific constituents of ginseng . the phenol compounds inhibited tyrosinase activity while ginsenoside prevents uvb - induced intracellular increase of reactive oxygen species [ 4648 ] . in some reports , ginsenosides alone exerted antimelanogenic effects . aglycone of ginsenoside rh4 inhibited melanin synthesis in b16 melanoma cells , possibly by involvement of protein kinase a pathway . it significantly reduced melanin content and tyrosinase activity in -msh and forskolin - stimulated b16 melanoma cells . it reduced the camp level and camp response - element binding protein level in b16 melanoma cells , and this might be responsible for the downregulation of mitf and tyrosinase . in addition , ginsenoside rb1 inhibited melanogenesis through the inhibition of tyrosinase activity in -msh - stimulated b16 cells in a dose - dependent manner . the crude methanol extract of the fresh leaves of p. ginseng showed inhibitory activity on mushroom tyrosinase , and p - coumaric acid was characterized as the principal tyrosinase inhibitor in the extract . p - coumaric acid inhibited melanogenesis in b16f10 melanoma cells stimulated by -msh , and was suggested to interfere with melanogenesis by its structural similarity with tyrosine . interestingly , p - coumaric acid showed weaker inhibition against mushroom tyrosinase but more strongly inhibited human or murine tyrosinase in comparison with kojic acid and arbutin . enzyme kinetics analysis indicate that p - coumaric acid is a mixed type ( for tyrosine ) or competitive inhibitor ( for dopa ) of human tyrosinase . in addition , p - coumaric acid potently inhibits melanogenesis in human epidermal melanocytes exposed to uvb . cinnamic acid , one of the major components of cinnamomum cassia blume , is found in the root and seed of p. ginseng . cinnamic acid significantly reduced melanin production , tyrosinase activity , and tyrosinase expression in the melan - a cells . in addition , cinnamic acid showed depigmenting activity on the uvb - tanned skin of brown guinea pigs . it is already known that the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . although the contents of total ginsenosides in red ginseng and fermented red ginseng using lactobacillus brevis were not significantly different , the ginsenoside metabolite content was higher in fermented red ginseng compared to red ginseng . the tyrosinase inhibitory activity of fermented red ginseng extract was more potent compared with red ginseng extract in a test using mushroom tyrosinase . as reviewed above , crude extract or some components of ginseng and its components showed antimelanogenic activities by direct inhibition on key enzymes of melanogenesis , such as tyrosinase . moreover , ginseng and its components could exert antimelanogenic activity via action on the several factors related in melanocyte physiology . among a large number of soluble factors produced from melanocytes , keratinocytes , fibroblasts , and immune cells in skin , adrenocorticotropic hormone ( acth ) , -msh , endothelin-1 , prostaglandin e2 , prostaglandin f2 , no , and histamine are well - known stimulators of melanogenesis [ 37,5963 ] . il-1/1 and granulocyte - macrophage colony - stimulating factor ( gm - csf ) stimulate melanogenesis , while il-6 , tgf-1 , and tnf- downregulate melanin production . gm - csf has been reported to be involved in regulating the proliferation and differentiation of epidermal melanocytes . treatment of melan - a cells with conditioned media from uv - irradiated sp-1 keratinocytes increased melanocyte proliferation , and the proliferative effect of the conditioned media was blocked by anti - gm - csf antibody treatment . when uv - irradiated sp-1 keratinocytes were treated with red ginseng extract or saponin of red ginseng , the increased melanocyte proliferation by the conditioned media was blocked . in that report , red ginseng extract or saponin of red ginseng treatment decreased the expression of gm - csf induced by uv - b irradiation in sp-1 keratinocytes . as mentioned above , inflammatory cytokines such as il-1 and tnf- take part in the regulation of melanogenesis . ginseng extracts and ginsenosides have been reported to have anti - inflammatory activities in several different studies . ginsenosides inhibit different inducer - activated signaling protein kinases and transcription factor nuclear factor ( nf)-b , and then decrease the production of proinflammatory cytokines and mediators of inflammation . korean red ginseng extracts decreased tnf- and il-8 production in lipopolysaccharide ( lps)-stimulated hacat keratinocytes and show radical scavenging and antioxidant activity in human dermal fibroblasts . these findings suggest that ginseng extracts and ginsenosides might affect melanogenesis through their anti - inflammatory activities . the effect of ginseng on no production is still questionable . sun ginseng , a new processed ginseng prepared by steaming at high temperature , reduced uv - b - induced cell damage and decreased no production by inhibition of inducible no synthase mrna synthesis in hacat keratinocytes and human dermal fibroblasts . red ginseng marc oil inhibited inducible no synthase and cyclooxygenase-2 via nf-b and p38 pathways in lps - stimulated raw264.7 cells . in addition , ginsenjilinol , a protopanaxatriol - type saponin obtained from the roots of p. ginseng , shows inhibitory activity on no production in lps - stimulated raw264.7 cells . by contrast , there are some controversial reports that ginseng extract enhanced no production or no signaling . hong et al reported that ginseng extract administration stimulated nongenomic endothelial no synthase activation and enhanced no production in spontaneously hypertensive rats . in another report , water extract of korean red ginseng exerted vasoprotective effects through augmentation of no production by inhibiting arginase . therefore , the effect of ginseng on melanogenesis via no signaling remains to be clarified by further study . considerable numbers of immune cells including langerhans cells , macrophages , mast cells , and t cells are working actively in skin tissue . because the immunostimulatory activities of many ginsenosides are known , it is not surprising that ginsenosides could enhance the reactivity of skin immune cells . in a recent paper , a cream containing 0.1% ginsenoside f1 ( a metabolite of ginsenoside rg1 ) showed a significant whitening effect on artificially tanned human skin . however , ginsenoside f1 did not directly inhibit mrna expression of tyrosinase or dct in normal human epidermal melanocytes . instead , ginsenoside f1 enhanced production of il-13 from human epidermal t cells , and il-13 significantly reduced the mrna expression and protein amount of both tyrosinase and dct resulting in visible brightening of normal human epidermal melanocyte pellets . these results suggest that ginsenosides might be able to regulate melanogenesis via their effect on skin immune cells . recently , several reports have shown that extract , powder , or some constituents of ginseng could inhibit melanogenesis in vivo or in vitro . the underlying mechanisms of the antimelanogenic effect of ginseng or its components included the direct inhibition of key enzymes of melanogenesis ( tyrosinase and dct ) , inhibition of transcription factors ( mitf , nf-b ) or signaling pathways ( protein kinase a pathway and protein kinase c pathway ) involved in melanogenesis , decreasing the production of inducers of melanogenesis ( camp , gm - csf ) , and enhancing production of antimelanogenic factor ( il-13 ) . fig . although issues surrounding the antimelanogenic activity of ginseng still remain controversial , especially in its effect on the production of proinflammatory cytokines and no , these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin - whitening agents .

abnormal changes in skin color induce significant cosmetic problems and affect quality of life . there are two groups of abnormal change in skin color ; hyperpigmentation and hypopigmentation . hyperpigmentation , darkening skin color by excessive pigmentation , is a major concern for asian people with yellow brown skin . a variety of hypopigmenting agents have been used , but treating the hyperpigmented condition is still challenging and the results are often discouraging . panax ginseng has been used traditionally in eastern asia to treat various diseases , due to its immunomodulatory , neuroprotective , antioxidative , and antitumor activities . recently , several reports have shown that extract , powder , or some constituents of ginseng could inhibit melanogenesis in vivo or in vitro . the underlying mechanisms of antimelanogenic properties in ginseng or its components include the direct inhibition of key enzymes of melanogenesis , inhibition of transcription factors or signaling pathways involved in melanogenesis , decreasing production of inducers of melanogenesis , and enhancing production of antimelanogenic factor . although there still remain some controversial issues surrounding the antimelanogenic activity of ginseng , especially in its effect on production of proinflammatory cytokines and nitric oxide , these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin whitening agents .

aneuploid strains were generated by sporulation of the above polyploid strains , followed by karyotype stability tests and determination as described in fig . qpcr assays were designed with primers in non - coding regions on each chromosome arm ( supplementary table 1 lists primer sequences ) . dna samples were prepared by alkaline lysis , and qpcr reactions were performed in 384-well plates using a biomek fx ( beckman coulter ) to assemble 10 l reactions and an abi 7900ht ( applied biosystems ) for cycling . equal amounts ( od600 ) of aneuploid and euploid control cultures were spotted , using the biomek fx robot , onto omnitrays containing various solid media and grown under conditions listed in supplementary table 2 . omnitrays representing three biological replicates of each tested condition were scanned on an hp scanjet 4070 desktop scanner . whole - cell lysates were prepared from 50 ml cycling yeast cultures by bead - beating . the ms / ms datasets were searched using sequest23 against a database of 11,986 sequences , consisting of 5,816 s. cerevisiae non - redundant proteins ( ncbi ) , 177 contaminants and 5,993 decoy sequences . all statistical analyses were performed in the r environment25 using standard packages and custom scripts . aneuploid strains were generated by sporulation of the above polyploid strains , followed by karyotype stability tests and determination as described in fig . qpcr assays were designed with primers in non - coding regions on each chromosome arm ( supplementary table 1 lists primer sequences ) . dna samples were prepared by alkaline lysis , and qpcr reactions were performed in 384-well plates using a biomek fx ( beckman coulter ) to assemble 10 l reactions and an abi 7900ht ( applied biosystems ) for cycling . equal amounts ( od600 ) of aneuploid and euploid control cultures were spotted , using the biomek fx robot , onto omnitrays containing various solid media and grown under conditions listed in supplementary table 2 . omnitrays representing three biological replicates of each tested condition were scanned on an hp scanjet 4070 desktop scanner . whole - cell lysates were prepared from 50 ml cycling yeast cultures by bead - beating . the ms / ms datasets were searched using sequest23 against a database of 11,986 sequences , consisting of 5,816 s. cerevisiae non - redundant proteins ( ncbi ) , 177 contaminants and 5,993 decoy sequences . all statistical analyses were performed in the r environment25 using standard packages and custom scripts .

aneuploidy , referring here to genome contents characterized by abnormal numbers of chromosomes , has been associated with developmental defects , cancer , and adaptive evolution in experimental organisms19 . however , it remains unresolved how aneuploidy impacts gene expression and whether aneuploidy could directly bring phenotypic variation and improved fitness over that of euploid counterparts . in this work , we designed a novel scheme to generate , through random meiotic segregation , 38 stable and fully isogenic aneuploid yeast strains with distinct karyotypes and genome contents between 1n and 3n without involving any genetic selection . through phenotypic profiling under various growth conditions or in the presence of a panel of chemotherapeutic or antifungal drugs , we found that aneuploid strains exhibited diverse growth phenotypes , and some aneuploid strains grew better than euploid control strains under conditions suboptimal for the latter . using quantitative mass spectrometry - based proteomics , we show that the levels of protein expression largely scale with chromosome copy numbers , following the same trend observed for the transcriptome . these results provide strong evidence that aneuploidy directly impacts gene expression at both the transcriptome and proteome levels and can generate significant phenotypic variation that could bring about fitness gains under diverse conditions . our findings suggest that the fitness ranking between euploid and aneuploid cells is context- and karyotype - dependent , providing the basis for the notion that aneuploidy can directly underlie phenotypic evolution and cellular adaptation .

the chemistry of metallocenes rapidly expanded in the 1970s with organozircocene emerging as one of the most useful classes of transition metal derivatives for use in organic synthesis . one important reagent belonging to this class of organometallic compounds is the chlorobis(cyclopentadienyl)hydrido - zirconium 1 , also called zirconocene hydrochloride but better known as schwartz 's reagent . wailes and weigold were the first to prepare 1 and , subsequently , schwartz and many other scientists exploited its potential in the functionalization of organic compounds . selected examples of the utility of this reagent in organic synthesis are depicted in figure 1 . schwartz 's reagent is most often used in the hydrozirconation of triple and double bonds ( figure 1 , reaction a ) . however , its use in this reaction is sometimes hampered by the functional group compatibility of the process that is limited by the oxophilic , hard lewis - acid character of the schwartz reagent . this characteristic has been therefore exploited in other useful synthetic organic transformations such as the chemoselective reduction of tertiary amides to aldehydes ( figure 1 , reaction b ) , and the reduction of secondary amides to n - substituted imines ( figure 1 , reaction c ) . the latter transformation , when performed with most other metal hydride reagents , results either in over - reduction of the imine to give the corresponding amine or in the reductive cleavage of the amide . interestingly , 1 has also been employed as a reducing agent for phosphine oxides to the corresponding phosphines ( figure 1 , reaction d ) . recently , bhat and co - workers reported a very mild and useful deacetylation procedure of acetamides involving reagent 1 . the methodology proved to be very efficient for aromatic , heteroaromatic , and aliphatic amides and moreover no epimerization was observed during the n - deacetylation of chiral acetamides . the amine functionality is present in many compounds of biological interest , for instance natural products and pharmaceuticals . nucleoside and nucleotide chemistry frequently involves protecting procedures at the amine and hydroxyl groups and benefit of selective deprotection methods , in order to obtain the target compound efficiently without any side reactions . n - acetyl protection is one of the most widely used in nucleoside and nucleotide chemistry . however , the requirement of strong base or acid and/or high reaction temperatures during the deprotection step ( n - deacetylation ) is often not compatible with some nucleosidic substrates and especially their prodrugs . harsh conditions are also likely to yield fully deprotected products , an outcome often undesired . zinc bromide and hydrazine hydrate were initially reported as selective and mild n - deacetylating agents for nucleosides , although their compatibility with other protecting groups was not explored broadly . our goal was the identification of a selective n - deacetylation methodology involving mild reaction conditions that could be applied to a broad spectrum of differently protected nucleosides . therefore , we became immediately interested in bhat 's methodology and in its possible application in nucleoside chemistry . to the best of our knowledge , no attempt to use schwartz 's reagent in the n - deacetylation of nucleosides and their prodrugs has been previously reported . herein , we would like to report our efforts directed at deacetylation of n - acetyl purine and pyrimidine nucleoside analogues bearing a variety of protecting groups ( pgs ) at the hydroxyl moiety , commonly used in nucleoside chemistry , such as o - acetyl , o - tert - butyloxycarbonyl , o - tert - butyldimethylsilyl , o - tetrahydropyranyl , o - benzoyl , and o - isopropylidene groups , with the aim of evaluating the ability of reagent 1 to selectively remove the acetyl group from the amino moiety , leaving the other pgs untouched . our investigation began with the preparation , according to literature procedures , of differently substituted n - acetyl-2-deoxycytidine ( 2a - c ) and n - acetylcytidine ( 2d - g ) , as indicated in table 1 . with all the o - protected nucleosides in hand , we proceeded to test the deacetylation reaction . first , compound 2a was subjected to the action of three equivalents of reagent 1 in a thf solution , under a nitrogen atmosphere , for 30 minutes ( table 1 , entry 1 ) . pleasingly , we were able to isolate after aqueous work - up and column chromatography compound 3a in 57% yield , where only the n - acetyl group was removed . application of these conditions to 2b , once again , led to the selective removal of the acetyl group from the amino moiety without affecting the silicon protected hydroxyl groups and yielding 3b in 49% yield ( table 1 , entry 2 ) . table 1 deacetylation reaction of differentially protected n - acetyl-2-deoxycytidines and n - acetylcytidines , promoted by schwartz reagententrycpdr1r2r31 ( eq)temp ( c)time ( h)prod.yield ( % ) recovered sm ( % ) 12ahacac3rt0.53a574022bhtbdmstbdms3rt0.53b493332chthpthp3rt33c682042dohbocboc3rt0.53d395552eoacacac3rt33e702362fobzbzbz3rt33f534172gcme2 h3rt33g553882ahacac6rt33a554292ahacac370123a4839 similar results were obtained testing the compatibility of schwartz 's reagent with other protecting groups such as tetrahydropyranyl , tertbutoxycarbonyl , benzoyl , and isopropylidene ( table 1 , entries 37 ) . in all examples , the o - protecting groups were unaffected by this procedure , and only the acetyl group on the nucleobase was removed , affording the desired compounds in moderate to good yields ( 3970% ) , along with the recovery of unreacted starting material . attempts to increase the yield of the desired n - deacetylated nucleoside either by increasing the equivalents of 1 ( up to six ) or by adding it portionwise over a period of three hours , proved to be unsuccessful and some amount of starting material was always recovered ( table 1 , entry 8) . incrementing the temperature up to 70c did not enhance the yield ( table 1 , entry 9 ) . subsequently , we decided to broaden the scope of the methodology by testing it on examples of other pyrimidine and purine nucleoside derivatives bearing different pgs ( table 2 ) . lamivudine was acetylated on both oxygen and amine groups and the resulting n , o - diacetylated compound 2h was then submitted to the deacetylating protocol ( table 2 , entry 1 ) . as expected , the acetyl group was removed only from the nitrogen on the nucleobase , leaving the o - acetyl group untouched . table 2 n - deacetylation of protected pyrimidine and purine nucleosides , promoted by schwartz 's reagententrycompoundproductyield ( % ) 1 56 5 53 2 25 3 47 4 76 n - acetyl - o - trityl - lamivudine 2i was found to be a poor substrate for this protocol ( table 2 , entry 2 ) . the desired product 3i was obtained only in 25% isolated yield , most probably due to the partial trityl group instability to the reaction conditions . no starting material was isolated in this case . when the protocol was tested on purine nucleoside analogues , reagent 1 proved efficient in removing the acetyl group selectively from the amine group on both the guanine ring of peracetylated acyclovir 2j and the adenine ring of n - acetyl-5-o - acetyl-2,3-o - isopropylidene adenosine 2k , affording 3j and 3k respectively in 47% and 76% isolated yields ( table 2 , entries 3,4 ) . finally , n , n - diacetyl-2,3-isopropylidene adenosine 2l was submitted to the optimized protocol . in this case schwartz 's reagent was able to remove both n - acetyl groups , affording compound 3k in 53% yield , with only traces of mono - n - acetylated derivative 2k . . reported a practical method to generate schwartz 's reagent in situ using li(ot - bu)3alh , demonstrating the advantage of this procedure both in reducing tertiary amides to aldehydes and in hydrozirconation reactions , in comparison to the use of the commercial schwartz 's reagent . we , therefore , decided to assess this efficient procedure in the deacetylation of nucleoside 2 g in order to be able to compare the in situ methods with the use of the preformed 1 ( scheme 1 ) . therefore , to a thf solution of 2 g and 1.5 equivalents of cp2zrcl2 in thf , solid li(ot - bu)3alh was added in one portion and the mixture was stirred for 40 minutes . aqueous workup , followed by column chromatography , afforded compound 3 g in 80% isolated yield , demonstrating a yield improvement from the previous 55% , possibly due to the partial air , light , and moisture sensitivity of 1 , which are reported to be responsible for the reduction of its effectiveness with time . figure 1 n - deacetylation of n - acetyl-2,3-o - isopropylidene cytidine 2 g with in situ generated schwartz 's reagent 1 . nucleoside analogues ( nas ) are an important class of molecules accounting for half of all antiviral drugs currently on the market and a number of anticancer agents that are widely used . unfortunately , many nucleoside analogues are not phosphorylated effectively in vivo , and thus their therapeutic potential is often quite limited . using various approaches , the ionizable phosphate group can be masked by derivatization , generating prodrugs with increased biological activity . among different nucleoside monophosphate prodrug strategies is the phosphoroamidate prodrug approach ( protide ) , consisting of an amino acid ester promoiety linked via p gilead has just launched on the market its anti - hcv protide , sofosbuvir ( psi-7977 ) , whereas nucana biomed has taken to trial a gemcitabine protide ( nuc-1031 ) , for patients with advanced solid tumours . considering the importance that the protide technology is assuming in the antiviral and anticancer scenarios , we were interested in checking if schwartz 's reagent is compatible with the phosphoramidate motif and whether this protocol can be used at later stage in the synthesis of this class of prodrugs . figure 2 synthesis and deacetylation of n - acetyl-2,3-o - isopropylidene- cytidine-5-o-[napthyl ( cyclohexyl - l - alanyl ) ] phosphate ( 5 ) promoted by schwartz reagent . phosphoroamidate 5 was synthesized from 2 g and ( 2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate 4 in 40% yield ( scheme 2 ) , according to a previously described method . phosphoroamidate 5 was then treated with three equivalents of the schwartz 's reagent , yielding the corresponding deacetylated prodrug 6 in 65% isolated yield . at the same time we were conducting our investigations , snieckus et al . reported a practical method to generate schwartz 's reagent in situ using li(ot - bu)3alh , demonstrating the advantage of this procedure both in reducing tertiary amides to aldehydes and in hydrozirconation reactions , in comparison to the use of the commercial schwartz 's reagent . we , therefore , decided to assess this efficient procedure in the deacetylation of nucleoside 2 g in order to be able to compare the in situ methods with the use of the preformed 1 ( scheme 1 ) . therefore , to a thf solution of 2 g and 1.5 equivalents of cp2zrcl2 in thf , solid li(ot - bu)3alh was added in one portion and the mixture was stirred for 40 minutes . aqueous workup , followed by column chromatography , afforded compound 3 g in 80% isolated yield , demonstrating a yield improvement from the previous 55% , possibly due to the partial air , light , and moisture sensitivity of 1 , which are reported to be responsible for the reduction of its effectiveness with time . figure 1 n - deacetylation of n - acetyl-2,3-o - isopropylidene cytidine 2 g with in situ generated schwartz 's reagent 1 . nucleoside analogues ( nas ) are an important class of molecules accounting for half of all antiviral drugs currently on the market and a number of anticancer agents that are widely used . nas mostly require phosphorylation to be active . unfortunately , many nucleoside analogues are not phosphorylated effectively in vivo , and thus their therapeutic potential is often quite limited . using various approaches , the ionizable phosphate group can be masked by derivatization , generating prodrugs with increased biological activity . among different nucleoside monophosphate prodrug strategies is the phosphoroamidate prodrug approach ( protide ) , consisting of an amino acid ester promoiety linked via p gilead has just launched on the market its anti - hcv protide , sofosbuvir ( psi-7977 ) , whereas nucana biomed has taken to trial a gemcitabine protide ( nuc-1031 ) , for patients with advanced solid tumours . considering the importance that the protide technology is assuming in the antiviral and anticancer scenarios , we were interested in checking if schwartz 's reagent is compatible with the phosphoramidate motif and whether this protocol can be used at later stage in the synthesis of this class of prodrugs . figure 2 synthesis and deacetylation of n - acetyl-2,3-o - isopropylidene- cytidine-5-o-[napthyl ( cyclohexyl - l - alanyl ) ] phosphate ( 5 ) promoted by schwartz reagent . phosphoroamidate 5 was synthesized from 2 g and ( 2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate 4 in 40% yield ( scheme 2 ) , according to a previously described method . phosphoroamidate 5 was then treated with three equivalents of the schwartz 's reagent , yielding the corresponding deacetylated prodrug 6 in 65% isolated yield . in conclusion , we have demonstrated that the general and mild method for the deacetylation of n - acetyl group developed by bhat et al . can be successfully applied to purine and pyrimidine nucleosides , with diverse o - protection . moreover , we have shown that schwartz 's reagent is compatible with the phosphoramidate moiety and can be used for n - deacetylation reaction in the later stages of the synthesis of this class of nucleoside monophosphate prodrugs . lastly , we have proved that modification of the reported procedure involving in situ generation of schwartz 's reagent leads to yield improvement and is promising of similar results on other nucleoside substrates . schwartz reagent was purchased from sigma - aldrich or prepared in situ as described and all nucleosides used as starting materials were purchased from carbosynth . h nmr ( 500 mhz ) , c nmr ( 125 mhz ) , and p nmr ( 202 mhz ) spectra were recorded on a bruker avance 500 mhz spectrometer at 25c . chemical shifts ( ) are quoted in parts per million ( ppm ) relative to internal cd3od ( 3.34 h nmr , 49.86 c nmr ) and cdcl3 ( 7.26 h nmr , 77.36 c nmr ) or external 85% h3po4 ( 0.00 p nmr ) . the following abbreviations are used in the assignment of nmr signals : singlet ( s ) , doublet ( d ) , triplet ( t ) , quartet ( q ) , quintet ( qn ) , multiplet ( m ) , broad singlet ( bs ) , doublet of doublet ( dd ) , and doublet of triplet ( dt ) . high and low - resolution mass spectrometry analyses were performed on a bruker microtof - lc system . to a stirred solution of n - acetyl nucleoside ( 100 mg ) in anhydrous thf ( 2 ml ) , schwartz 's reagent ( 36 eq . ) is added at room temperature and the reaction mixture is stirred for 0.53 hours . the combined organic layer is washed with brine solution and dried over anhydrous na2so4 , filtered and evaporated to afford the crude product , which is purified by silica gel column chromatography to finally afford pure deacetylated nucleoside . n - deacetylation reaction via in situ generation of schwartz 's . to a solution of n - acetyl nucleoside ( 100 mg ) and cp2zrcl2 ( 1.5 equiv ) in thf ( 2 ml ) at rt , solid lialh(otbu)3 ( 1.5 equiv ) was rapidly added . the resulting solution was stirred at rt for 40 minutes and the reaction was monitored by tlc analysis . the combined organic layer is washed with brine solution and dried over anhydrous na2so4 , filtered and evaporated to afford the crude product , which is purified by silica gel column chromatography to finally afford pure deacetylated nucleoside . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3a as a white solid ( 0.050 g , 57% yield ) . h nmr ( 500 mhz ) cdcl3 h 2.08 ( 3h , s , ococh3 ) , 2.09 ( 3h , s , ococh3 ) , 2.192.12 ( 1h , m , h-2a ) , 2.58 ( 1h , dd , j = 4.0 , 14.0 hz , h-2b ) , 4.244.26 ( 1h , m , h-4 ) , 4.32 ( 2h , d , j = 12.5 hz , h-5 ) , 5.185.19 ( 1h , m , h-3 ) , 6.11 ( 1h , d , j = 6.5 hz , h-5 ) , 6.22 ( 1h , t j = 6.5 hz , h-1 ) , 7.57 ( 1h , d , j = 6.5 hz , h-6 ) . c nmr ( 125 mhz ) cdcl3 c 20.90 , 20.98 ( ch3 ) , 38.24 ( ch2 - 2 ) , 63.82 ( ch2 - 5 ) , 74.12 ( c-3 ) , 82.63 ( c-4 ) , 88.18 ( c-1 ) , 95.87 ( c-5 ) , 140.21 ( c-6 ) , 170.32 ( c - o ) , 155.15 ( c-2 ) , 164.90 ( c-4 ) ; ms ( es+ ) m / z ( 312 ) [ m+1 ] ; hrms ( es ) m / z found 312.1192 [ calcd for c13h18n3o6 ( m+h ) 312.1190 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3b as a white solid ( 0.044 g , 49% ) . h nmr ( 500 mhz ) cdcl3 h 0.12 ( 6h , s , 2 ch3 ) , 0.15 ( 6h , s , 2 ch3 ) , 0.91 ( 9h , s , c(ch3)3 ) , 0.98 ( 9h , s , c(ch3)3 ) , 2.092.13 ( 1h , m , h-2a ) , 2.352.40 ( 1h , m , h-2b ) , 3.83 ( 1h , dd , j = 2.5 , 11.5 hz , h-5a ) , 3.92 ( 1h , dd , j = 3.0 , 11.0 hz , h-5b ) , 3.933.96 ( 1h , m , h-4 ) , 4.474.49 ( 1h , m , h-3 ) , 5.89 ( 1h , d , j = 7.5 hz , h-5 ) , 6.24 ( 1h , t j = 6.0 hz , h-1 ) , 7.97 ( 1h , d , j = 7.5 hz , h-6 ) ; c nmr ( 125 mhz ) cdcl3 c 4.92 , 4.58 ( sich3 ) , 17.93 , 18.94 ( c(ch3)3 ) 26.04 , 26.22 ( c(ch3)3 ) , 42.13 ( c-2 ) , 62.04 ( c-5 ) , 70.44 ( c-3 ) , 85.80 ( c-1 ) , 87.25 ( c-4 ) , 94.26 ( c-5 ) , 141 ( c-6 ) , 155.94 ( c-2 ) , 165.87 ( c-4 ) ; ms ( es+ ) m / z 456 [ m+1 ] ; hrms ( es ) m / z found 456.2710 [ calcd for c21h42n3o4si2 ( m+h ) 456.2708 ] . 3,5-o - bis(tetrahydropyranyl)-2-deoxycytidine ( 3c ) . prepared from 2c according to standard procedure for the n - deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3c as a white solid ( 0.061 mg , 68% ) . h nmr ( 500 mhz ) cd3od h 1.441.48 ( 8h , m , ch2 py ) , 1.611.76 ( 4h , m , ch2 py ) , 2.342.50 ( 1h , m , h-2b ) , 1.952.12 ( 1h , m , h-2a ) , 3.433.47 ( 2h , m , ch2o py ) , 3.51 ( 0.25h , dd , j = 3.0 , 11.0 hz , h-5b ) , 3.53 ( 0.25h , dd , j = 3.0 , 11.5 hz , h-5b ) , 3.543.61 ( 0.5h , dt j = 3.9 , 16.5 hz , h-5b ) , 3.713.82 ( 2h , m , ch2o py ) , 3.803.86 ( 0.5 , m , h-5a ) , 3.903.95 ( 0.5h , m , h-5a ) , 4.074.11 ( 0.5h , m , h-4 ) , 4.134.15 ( 0.25h , m , h-4 ) , 4.174.19 ( 0.25h , m , h-4 ) , 4.294.31(0.25h , m , h-3 ) , 4.344.36 ( 0.5h , m h-3 ) , 4.404.41 ( 0.25h , m , h-3 ) , 4.554.67 ( 2h , m , cho py ) , 5.775.80 ( 1h , m , h-5 ) , 6.116.16 ( 1h , m , h-1 ) , 7.88 ( 0.25h , d , j = 7.5 hz , h-6 ) , 7.89 ( 0.25h , d , j = 7.5 hz , h-6 ) , 7.94 ( 0.25h , d , j = 7.5 hz , h-6 ) , 7.97 ( 0.25h , d , j = 7.0 hz , h-6 ) ; c nmr ( 125 mhz ) cd3od c 20.47 , 20.50 , 20.68 , 20.77 ( ch2 py ) , 26.51 , 26.55 ( ch2 py ) , 31.69 , 31.75 , 31.94 , 32.01 ( ch2 py ) , 39.54 , 39.71 , 40.82 , 40.98 ( c-2 ) , 63.69 , 63.73 , 63.75 , 63.92 ( ch2o ) , 68.30 , 68.42 , 68.49 , 68.80 ( c-5 ) , 77.39 , 77.82 , 78.25 ( c-3 ) , 85.41 , 85.47 , 85.97 , 85.18 ( c-4 ) , 87.65 , 87.83 , 87.87 , 87.97 ( c-1 ) , 95.80 , 95.90 ( c-5),99.29 , 99.36 , 99.79 , 99.88 , 100.14 , 100.33 , 100.97,101.03 ( ch py ) , 142.38 , 142.81 ( c-6 ) , 158.17 ( c-2 ) , 167.63 ( c-4 ) ; ms ( es+ ) m / z = 396 [ m+1 ] . hrms ( es ) m / z found 396.2127 [ calcd for c19h30n3o6 ( m+h ) 396.2129 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3d as a white solid ( 0.035 g , 39% yield ) . h nmr ( 500 mhz ) cd3od h 1.47 ( 9h , s , c(ch3)3 ) , 1.50 ( 9h , s , c(ch3)3 ) , 3.77 ( 1h , dd , j = 3.0 , 12.5 hz , h-5 ) , 3.88 ( 1h , dd , j = 2.5 , 12.5 hz , h-5 ) , 4.224.20 ( 1h , m , h-4 ) , 5.27 ( 1h , dd , j = 5.5 hz , j = 5.5 hz , h-3 ) , 5.32 ( 1h , dd , j = 4.0 , 4.0 hz , h-2 ) , 5.90 ( 1h , d , j = 7.5 hz , h-5 ) , 6.10 ( 1h , d , j = 4.5 hz , h-1 ) , 8.0 ( 1h , d , j = 7.5 hz , h-6 ) ; c nmr ( 125 mhz ) cd3od c 27.96 , 28.02 ( c(ch3)3 ) , 31.70 ( c(ch3)3 ) , 61.96 ( c-5 ) , 74.17 ( c-3 ) , 77.25 ( c-2 ) , 83.93 ( c-4 ) , 89.94 ( c-1 ) , 96.64 ( c-5 ) , 143.07 ( c-6 ) , 153.61 , 153.89 ( coc(ch3)3 ) , 158.01(c-2 ) , 167.56 ( c-4 ) ; ms ( es+ ) m / z = 444 [ m+1 ] . hrms ( es ) m / z found 444.1973 [ calcd for c19h30n3o9 ( m+h ) 444.1977 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3e as a white solid ( 0.063 g , 70% yield ) . h nmr ( 500 mhz ) cd3od h 2.10 ( 3h , s , coch3 ) , 2.11 ( 3h , s , coch3 ) , 2.12 ( 3h , s , coch3 ) , 4.344.40 ( 3h , m , h4 , h5 ) , 5.41 ( dd , j = 5.0 , 5.9 hz , 1h , h3 ) , 5.48 ( dd , j = 4.6 , 6.0 hz , 1h , h3 ) , 5.96 ( 1h , d , j = 7.5 hz , h5 ) , 5.98 ( 1h , d , j = 4.6 hz , h1 ) , 7.69 ( 1h , d , j = 7.5 hz , h6 ) ; c nmr ( 125 mhz ) cd3od c 20.40 ( coch3 ) , 20.45 ( coch3 ) , 20.69 ( coch3 ) , 64.32 ( c5 ) , 71.68 ( c3 ) , 74.69 ( c2 ) , 80.99 ( c4 ) , 91.09 ( c1 ) , 96.76 ( c5 ) , 143.23 ( c6 ) , 157.91 ( c4 ) , 167.82 ( c2 ) , 171.35 ( coch3 ) , 171.39 ( coch3 ) , 172.19 ( coch3 ) ; ms ( es+ ) . hrms ( es ) m / z found 370.1243 [ calcd for c15h20n3o8 ( m+h ) 370.1245 ] . cytidine 2 , 3 , 5-tribenzoate ( 3f ) . prepared from 2f according to standard procedure for the n - deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 96/4 to give 3f as a white solid ( 0.049 g , 53% yield ) . h nmr ( 500 mhz ) cd3cl h 2.12 ( 3h , s , coch3 ) , 4.63 ( 1h , dd , j = 3.9 , 12.2 hz , h5 ) , 4.724.68 ( 1h , m , h4 ) , 4.76 ( 1h , dd , j = 2.8 , 12.2 hz , h5 ) , 5.745.78 ( 1h , m , h2 ) , 5.795.84 ( 1h , m , h3 ) , 6.31 ( 1h , d , j = 4.2 hz , h1 ) , 7.317.24 ( 5h , m , ph , h5 ) , 7.377.42 ( 2h , m , ph ) , 7.437.49 ( 2h , m , ph ) , 7.507.55 ( 1h , m , ph ) , 7.817.89 ( 5h , m , ph , h6 ) , 7.988.08 ( 2h , m , ph ) , 9.58 ( 1h , br s , nhcoch3 ) . c nmr ( 125 mhz ) cd3cl c 63.92 ( c5 ) , 71.20 ( c3 ) , 74.45 ( c2 ) , 80.15 ( c4 ) , 89.25 ( c1 ) , 95.45 ( c5 ) , 128.46 ( ch ar ) , 128.49 ( ch - ar ) , 128.68 ( ch - ar ) , 128.75 ( c - ar ) , 129.40 ( c - ar ) , 129.71 ( ch - ar ) , 129.85 ( ch - ar ) , 129.97 ( ch - ar ) , 133.49 ( ch - ar ) , 133.61 ( ch - ar ) , 133.62 ( ch - ar ) , 141.36 ( c6 ) , 155.28 ( c5 ) , 165.33 ( coph ) , 165.37 ( coph ) , 165.39 ( c4 ) , 166.14 ( coph ) ; ms ( es+ ) m / z ( 556 ) [ m+1 ] ; hrms ( es ) m / z found 556.1711 [ calcd for c30h26n3o8 ( m+h ) 556.1714 ] . 2 , 3-o - isopropylidene - cytidine ( 3 g ) . prepared from 2 g according to standard procedure for the n - deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3 g as a white solid ( 0.043 g , 49% yield ) . prepared according to the in situ schwartz 's reagent generation procedure for the deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3 g as a white solid ( 0.070 g , 80% yield ) . h nmr ( 500 mhz ) cd3od h 1.25 ( 3h , s , ch3 ) , 1.45 ( 3h , s , ch3 ) , 3.62 ( 1h , dd , j = 4.5 , 12.0 hz , h-5a ) , 3.69 ( 1h , dd , j = 3.5 , 12.0 hz , h-5b ) , 4.114.13 ( 1h , m , h-4 ) , 4.724.74 ( 1h , m , h-3 ) , 4.784.80 ( 1h , m , h-2 ) , 5.75 ( 1h , s , h-1 ) , 5.81 ( 1h , d , j = 7.5 hz , h-5 ) , 7.73 ( 1h , d , j = 7.5hz , h-6 ) ; c nmr ( 125 mhz ) cd3od c 25.55 ( cch3)2 ) , 27.55 ( cch3)2 ) , 63.19 ( c5 ) , 82.25 ( c3 ) , 86.40 ( c2 ) , 88.69 ( c4 ) , 95.45 ( c1 ) , 96.20 ( c5 ) , 115.03 ( cch3)2 ) , 144.42 ( c6 ) , 154.90 ( c2 ) , 167.82 ( c4).ms ( esi+ ) m / z = ( 284 ) [ m+1].hrms ( es ) m / z found 284.1243 [ calcd for c12h18n3o5 ( m+h ) 284.1241 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3h as a white solid ( 0.048 g , 56% yield ) . h nmr ( 500 mhz ) cd3od h 1.90 ( 3h , s , coch3 ) , 2.95 ( 1h , dd , j = 3.0 , 12.5 hz , h-4 ) , 3.35 ( 1h , dd , j = 5.5 , 12.5 hz , h-4 ) , 4.20 ( 1h , dd , j = 3.0 , 12.5 hz , ch2oh ) , 4.35 ( 1h , dd , j = 5.0 , 12.5 hz , ch2oh ) , 5.20 ( 1h , dd , j = 3.5 , 5.0 hz , h-2 ) , 5.70 ( 1h , d , j = 7.5 hz , h-5 ) , 6.10 ( 1h , dd , j = 3.5 , 5.5 hz , h-5 ) , 7.68 ( 1h , d , j = 7.5 hz , h-6 ) ; c nmr ( 125 mhz ) cd3od c 20.62 ( ch3 ) , 38.15 ( c-4 ) , 65.64 ( ch2oh ) , 84.44 ( c-2 ) , 89.11 ( c-5 ) , 96.03 ( c-6 ) , 142.49 ( c-5 ) , 157.83 ( c-2 ) , 167.72 ( c-4 ) , 172.08 ( coch3 ) ; ms ( esi+ ) m / z = 272 [ m+1 ] . hrms ( es ) m / z found 272.0701 [ calcd for c10h14n3o4s ( m+h ) 272.0700 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3i as a white solid ( 0.023 g , 25% yield ) . h nmr ( 500 mhz ) cdcl3 h 3.09 ( 1h , dd , j = 2.5 , 12.5 hz , h-4 ) , 3.44 ( 1h , dd , j = 5.5 , 12.5 hz , h-4 ) , 3.49 ( 2h , d , j = 3.5 hz , ch2oh ) , 5.19 ( 1h , t , j = 3.5 hz , h-2 ) , 5.38 ( 1h , d , j = 7.0 hz , h-5 ) , 6.27 ( 1h , dd , j = 3.0 , 5.5 hz , h-5 ) , 7.267.15 ( 10h , m , ph ) , 7.397.37 ( 5h , m , ph ) , 7.93 ( 1h , d , j = 7.0 hz , h-6 ) ; c nmr ( 125 mhz ) cdcl3 c 36.72 ( c-4 ) , 61.31 ( ch2oh ) , 83.90 ( c-2 ) , 84.75 ( c-5 ) , 84.88 ( c - ph ) , 91.12 ( c-5 ) , 124.39 , 125.59 , 126.23 ( ch - ph ) , 139.06 ( c-6 ) , 140.08 ( c - ph ) , 153.01 ( c-2 ) , 163.16 ( c-4 ) ; ms ( esi+ ) m / z = 472 [ m+1 ] . hrms ( es+ ) m / z found 472.1685 [ calcd for c27h26n3o3s ( m+h ) 472.1689 ] . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3j as a white solid ( 0.041 g , 47% ) . h nmr ( 500 mhz ) ( cd3)2so h 1.95 ( 3h , s , coch3 ) , 3.65 ( 2h , dd , j = 7.0 , 7.5 hz , ch2o ) , 4.10 ( 2h , dd , j = 5.0 , 6.0 hz , ch2o ) , 6.55 ( 2h , s , nch2o ) , 7.80 ( 1h , s , h-8 ) , 10.65 ( 1h , brs , nh ) ; c nmr ( 125 mhz ) ( cd3)2so c 20.53 ( ch3 ) , 62.70 ( nch2 ) , 66.48 ( ch2o ) , 71.80 ( nch2o ) , 116.45 ( c-5 ) , 137.62 ( c-8 ) , 151.39 ( c-4 ) , 153.92 ( c-2 ) , 156.71(c-6 ) , 170.22 ( coch3 ) ; ms ( esi+ ) m / z = 268 [ m+1 ] . hrms ( es ) m / z found 268.1044 [ calcd for c10h14n5o4 ( m+h ) 268.1040 ] . prepared from 2k or 2l according to standard procedure for the n - deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3k as a white solid ( 0.068 g , 76% from 2k ; 0.034 g , 53% from 2l ) . h nmr ( 500 mhz ) cd3cl h 1.33 ( 3h , s , ch3).1.55 ( 3h , s , ch3 ) , 1.92 ( 3h , s , coch3 ) , 4.15 ( 1h , dd , j = 6.2 hz , 11.7 hz , h5 ) , 4.29 ( 1h , dd , j = 4.4 hz , 11.7 hz , h5 ) , 4.434.38 ( 1h , m , h4 ) , 4.99 ( 1h , dd , j = 3.4 hz , 6.3 hz , h3 ) , 5.41 ( 1h , dd , j = 2.0 hz , 6.3 hz , h2 ) , 5.94 ( 2h , br s , nh2 ) , 6.04 ( 1h , d , j = 2.0 hz , h1 ) , 7.82 ( 1h , s , h8 ) , 8.28 ( 1h , s , h2 ) ; c nmr ( 125 mhz ) cdcl3 c 20.68 ( coch3 ) , 25.41 ( cch3 ) , 27.14 ( cch3 ) , 64.09 ( c5 ) , 81.73 ( c4 ) , 84.23 ( c3 ) , 85.04 ( c2 ) , 91.06 ( c1 ) , 114.59 ( c5 ) , 120.35 ( cch3 ) , 139.71 ( c8 ) , 149.27 ( c4 ) , 153.22 ( c2 ) , 155.71 ( c6 ) , 170.41 ( coch3 ) . ms ( es+ ) m / z ( 350 ) [ m+1 ] ; hrms ( es ) m / z found 350.1451 [ calcd for c15h20n5o5 ( m+h ) 350.1459 ] . n - acetyl-2,3-o - isopropylidene-5-o-[napthyl ( cyclohexyl - l - alanyl ) ] pho - sphate cytidine ( 5 ) . to a solution of 2 g ( 0.5 g , 1.53 mmol ) and ( 2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propa - noate ( 4 , 1.02 g , 3.073 mmol ) in anhydrous thf ( 10ml ) , 1 m bumgcl ( 3.08 ml , 3.073 mmol ) is added dropwise and the reaction mixture is stirred at room temperature overnight . after this period the crude is purified by column chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 . the compound 5 is recovered as a white solid ( 0.420 , 40% yield ) . h nmr ( 500 mhz ) cd3od h 1.281.40 ( 11h , m , chch3 , ch3 , 2 ch2-chex ) , 1.521.58 ( 4h , m , ch3 , ch2-chex ) , 1.691.78 ( 4h , m , 2 ch2-chex ) , 2.15 , 2.18 ( 3h , s , coch3 ) , 3.994.02 ( 1h , m , chch3 ) , 4.404.52 ( 3.5h , m , ch-2 , h-4 , h-5 ) , 4.684.72 ( 2h , m , h-2 , h-3 ) , 4.90 ( 0.5h , m , h-3 ) , 5.80 ( 1h , d , j = 2.5 hz , h1 ) , 7.25 ( 0.5h , d , j = 7.5 hz , h-5 ) , 7.35 ( 0.5h , d , j = 7.5 hz , h-6 ) , 7.397.42 ( 1h , m , naph ) , 7.477.49 ( 1h , m , naph ) , 7.527.58 ( 2h , m , naph ) , 7.707.72 ( 1h , m , naph ) , 7.877.89 ( 1.5h , m , naph , h-5 ) , 7,96 ( 0.5h , d , j = 7.5 hz , h-5 ) , 8.088.10 ( 0.5h , m , naph ) , 8.138.15 ( 0.5h , m , naph ) ; c nmr ( 125 mhz ) cd3od c 20.45 ( d , jcp = 7.5 hz , chch3 ) , 20.62 ( d , jcp = 7.5 hz , chch3 ) , 24.57 ( coch3 ) , 24.60 , 24.62 ( ch2-chex ) , 25.42 , 25.52 , ( ch3 ) , 26.38 ( ch2-chex ) , 27.37 , 27.43 ( ch3 ) , 32.35 , 32.45 ( ch2-chex ) , 51.89 , 51.97 ( chch3 ) , 68.01 , 68.02 ( d , jcp = 7.5 hz , c-5 ) , 75.00 ( cho ) , 82.22 , 82.24 ( ch3 ) , 86.57 ( ch2 ) , 87.51 ( d , jcp = 2.5 hz , c-4 ) , 87.57 ( d , jcp = 2.5 hz , c-4 ) , 96.58 , 96.65 ( c-1 ) , 98.02 , 98.12 ( c-6 ) , 115.06 , 115.21 ( c - naph ) , 116.22 ( d , jcp = 3.7 hz , ch - naph ) , 116 . 49 ( d , jcp = 3.7 hz , ch - naph ) , 122.60 , 122.68 , 126.15 , 126.53 ( ch - naph ) , 126.59 , 127.60 , 127.70 , 127.80 , 127.85 , 127.93 , 128.97 ( ch - naph ) , 136.28 ( c - naph ) , 147.20 , 147.35 ( c5 ) , 147.85 ( d , jcp = 7.5 hz , ipsoc - naph ) , 148.00 ( d , jcp = 7.5 hz , ipsoc - naph ) , 157.55 , 157.59 ( c-2 ) , 164.57 ( c-4 ) , 172.85 , 172.91 ( coch3 ) , 174.32 ( d , jcp = 3.7 hz , co2 ) , 174.63 ( d , jcp = 3.7 hz , co2 ) ; p nmr ( 500 mhz ) meod p 3.92 , 4.15 . ms ( esi+ ) m / z = 685 [ m+1 ] . hrms ( es+ ) m / z found 685.2631 [ calcd for c33h42n4o10p ( m+h ) 685.2633 ] . 2,3-o - isopropylidene-5-o-[napthyl ( cyclohexyl - l - alanyl ) ] phosphate cytidine ( 6 ) . prepared from 5 according to standard procedure for the n - deacetylation reaction . the crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 6 as a white solid ( 0.061 g , 65% yield ) . h nmr ( 500 mhz ) cd3od h 1.321.41 ( 12h , m , chch 3 , ch3 , 3 ch2- chex ) , 1.54 ( 3h , s , ch3 ) , 1.551.84 ( 4h , m , ch2-chexyl ) , 4.004.05 ( 1h , m , chch3 ) , 4.344.44 ( 3h , m , h-4 , h-5 ) , 4.644.77 ( 2h , m , h-2 , h-3 ) , 4.814.84 ( m , 1h , cho ) , 5.74 ( 0.5h , d , j = 7.5 hz , h-6 ) , 5.785.81 ( 1.5h , m , h-1 , h-6 ) , 7.437.48 ( 1.5h , m , ch - naph . ) , 7.507.53 ( 1.5h , m , ch - naph ) , 7.557.59 ( 2h , m , ch - naph , h-5 ) , 7.73 ( 1h , d , j = 5.0 hz , ch - naph ) , 7.907.92 ( 1h , m , ch - naph ) , 8.168.20 ( 1h , m , naph ) ; c nmr ( 125 mhz ) cd3od c 20.45 ( d , jcp = 7.5 hz , chch3 ) , 20.60 ( d , jcp = 7.5 hz , ch3 ) , 24.61 ( ch2-chex ) , 25.50 , 25.56 , ( ch3 ) , 26.39 ( ch2-chex ) , 27.45 , 27.47 ( ch3 ) , 32.36 , 32.45 ( ch2-chex ) , 51.91 , 51.96 ( chch3 ) , 68.11 , 68.13 ( d , jcp = 5.0 hz , c-5 ) , 74.98 ( cho ) , 82.23 , 82.27 ( c-3 ) , 86.04 , 86.09 ( c-2 ) , 86.63 ( d , jcp = 2.5 hz , c-4 ) , 86.70 ( d , jcp = 2.5 hz , c-4 ) , 95.65 ( c-1 ) , 96.24 ( c-6 ) , 115.24 , 115.31 ( c - naph ) , 116.31 ( d , jcp = 3.7 hz , ch - naph ) , 116 . 48 ( d , jcp = 3.7 hz , ch - naph ) , 122.74 , 126.08 , 126.54 , 126.58 , 127.55 , 127.60 , 127.84 127.87 , 128.93 ( ch - naph ) , 136.33 ( c - naph ) , 144.19 ( c-5 ) , 147.85 ( d , jcp = 7.5 hz , ipsoc - naph ) , 148.00 ( d , jcp = 7.5 hz , ipsoc - naph ) , 157.84 ( c-2 ) , 167.84 ( c-4 ) , 174.32 ( d , jcp = 3.7 hz , co2 ) , 174.63 ( d , jcp = 3.7 hz , co2 ) ; p nmr ( 500 mhz ) cd3od p 3.96 , 4.11 ; ms ( es+ ) m / z = 643 [ m+1 ] . hrms ( es+ ) m / z found 643.2521 [ calcd for c31h40n4o9p ( m+h ) 643.2527 ] . supplementary file includes experimental procedures for the synthesis of protected nucleosides and analytical data for all new compounds . go to the publisher 's online edition of nucleosides , nucleotides and nucleic acids to view the free supplementary files .

protection and deprotection strategies involving the n - acetyl group are widely utilized in nucleoside and nucleotide chemistry . herein , we present a mild and selective n - deacetylation methodology , applicable to purine and pyrimidine nucleosides , by means of schwartz 's reagent , compatible with most of the common protecting groups used in nucleoside chemistry .

in order to assess sexual well - being and provide treatment or education for iranian women 's sexuality , it is necessary to understand their sexuality and the meanings they give to sexual behaviors . sexual behavior is a complex concept that is difficult to measure . according to webster 's online dictionary , the manner of conducting oneself and sexual simply means whatever relates to sex , the sexes or sexual reproduction. thus , sexual behavior would be an act by someone that expresses their sexuality . in the archive for sexology , erwin j. haeberle introduces sexual as a word with double meaning , referring to human anatomy as well as to human behavior . he defines sexual behavior as erotic behavior . therefore , in contrast with webster , the critical dictionary in the archive for sexology defines human reproduction as asexual behavior because it concentrates on the biological facts without reference to the erotic feelings of the man and the woman involved . the functional analysis of sexual behaviors has led researchers to develop instruments to gather observable data . in order to change subjective meanings to observable data , the researchers need to extract meanings from the contexts and populations they seek to measure . many sexually related measures and instruments have been published ; they measure perceptions , attitudes , beliefs and so on . however , the contexts from which the concepts and meanings were extracted to develop these instruments vary . doubtless , their reliability in measuring sexual behaviors as socially constructed , complex and dynamic phenomena can be questioned . in order to determine a research approach for the context of iranian women 's sexuality , review and assessment of existing instruments was essential . after a brief overview of the existing instruments measuring non - risky sexual behaviors among heterosexual women , we look at their cultural appropriateness to measure sexual behaviors within an iranian context . finally , because we find these instruments insufficient , we suggest criteria for cultural - specific sexually related measures in the iranian settings . for the purpose of this paper , we searched literature using medline , pub - med , psychinfo and cinahl from june 2006 to june 2011 . the mesh terms used to search title and abstract included sexuality , sexual behavior , questionnaire and sexual behavior and questionnaire or sexually related measure , sexually related tools limited to human , female , english and reproductive age . focused exclusively on the concept of sexual behavior , we screened titles and abstracts . considering that not all abstracts highlighted the specified population and name of the applied tools , these articles were found in full , and methods sections were reviewed to identify populations and the tools used . retrieved articles were included if they used a structured instrument(s ) to measure sexual behaviors of the female population . we excluded those studies whose outcome of interest included risky sexual behaviors or sexual behaviors measured among same sex relationships . we excluded same sex relationships because this form of relationships is illegal and deniable in iran . if the original paper was unavailable , we selected the article that used the given tool to measure the study outcomes . in this stage , tools in languages other than english , those employed just for menopausal women and those not related to sexual behaviors were excluded . using gagnon and simon sexual script theory as the theoretical framework , cultural appropriateness ( i.e. understanding and language of sexuality , ethics , and morality ) was our selection criteria for the instruments . evaluation of the 143 articles revealed that a majority of them ( 65% ) had utilized the female sexual function index ( fsfi ) in their research . the second most used scale was the pelvic organ prolapsed / urinary incontinence sexual function questionnaire ( pisq-12 ) , utilized in 10.4% of the final articles . the golombok - rust inventory of sexual satisfaction ( griss ) and the arizona sexual experience scale ( asex ) were the next most used instruments by 6.9% and 5.6% of articles , respectively . we reviewed all instruments and determined the subscales as well as measurement types [ table 1 ] . we categorized these instruments into three groups based on their working definitions of sexual behavior and theoretical frameworks . these instruments were reviewed by the authors , who are iranian experts in sexology , reproductive health , and epidemiology . of 50 instruments , we found 19 tools applicable in the iranian culture [ table 2 ] . the third group included those which were focused on a specific sexual problem rather than looking at sexual behaviors overall [ table 4 ] . the multidimensional derogatis sexual functioning inventory ( dsfi ) did not fit into any of those three categories . with this overview , it appears that there are significant challenges to using these instruments in iranian contexts . instruments features including the subscales compatible scales for iranian culture instruments incompatible with iranian culture problem - focused instruments inspiring gagnon and simon 's description of sexual behaviors defined with social scripts , understanding and language of sexuality , ethics , and morality are the main cultural determinants of sexual norms in the iranian society . in her linguistic analysis of sexuality expression of iranian women , merghati - khoei has revealed the ways of developing terminology and cultural explanations , which are juxtaposed with the exploration of the development of women 's sexuality . iranian women expressed their sexuality differently from western women even though they understood and talked about the same issues . there is , however , a hesitation when it comes to discussing or reporting the emotional aspects of sexual encounters . the power of culture in dictating daily language for females has been highlighted in morocco as an arab - islamic context . similarly , culturally meaningful ambiguity in language guides iranian people 's behavior . even though persian vocabulary , concepts and expressions about sexual matters exist in art , poetry and the beauty of nature , these linguistic resources are not applied in daily life to express sexuality . for example , the sexual attitude scale ( sas ) , certainly a well - designed tool for use within many cultures measures a person 's awareness toward her / his sexual interests and needs as well as sexual - self judgment . communication of sexual needs or interests with others is another feature of this tool . as a neglected subject matter in the iranian culture , questioning people about their sexual needs and interests or sexual - self concept seems impractical . sexuality is an unspoken issue , and individuals might not be linguistically skilled to communicate their sexuality with an interviewer . in sum , the art of using a rich vocabulary of metaphors and euphemisms is a characteristic of iranian speech , used to communicate and encapsulate matters not normally spoken explicitly . for example , for fluent english speakers phrase marital life may indicate all kinds of relationships within a marital framework , while in farsi zendegi - e - zanashoyi ( literally marital life ) has sexual connotations and is usually understood to mean the sexual relationship between husband and wife . another difficulty in asking about sexuality during a study is the struggle between conscious embarrassment and sexual talk . by conscious embarrassment , we mean the shame , prohibition and modesty about sex . the majority of the women who participated in merghati - khoei 's qualitative study pointed out that they were not culturally expected to be straightforward or frank in expressing sexual matters . for example , the sexual desire inventory tends to measure sexual desire as a biologic factor . out of 14 items , 4 focus on masturbation and 2 items ask about having interest in casual sex . in iranian contexts , none of these 6 items would be posed by researchers or responded to by the participants . why human have sex ? ( ysex ) is another example , which measures number of variables . for instance , some of the items focus on motivations leading people to out - of - wedlock or casual sex . although casual or extra marital sex happens in every society , questioning iranians about these behaviors is not ethically and religiously possible or feasible . this assertion is based on the common assumptions . in iran , people strongly hold onto the traditional culture of sexuality based on purity ( paki ) , chastity ( nejabat ) , honour ( aberoo ) and honesty ( sedagat ) underlying the family structure . there is also the belief commonly permeating iranian society that people are fairly innocent in terms of sexuality compared with non - muslim or western societies . however , with these assumptions we can not minimize the impact of factors such as modernization , worldwide communication and cultural transformations in younger generations and the way they learn about sex , practice and develop their sexual understandings . undoubtedly , these factors change behaviors and attitudes . social conduct and religiosity define the ethical aspect of sexuality in the iranian culture . in iran the teachings of islamic principles tie strongly to shia interpretations , which form the basis for iranians understandings of sexuality . the expression of sexuality is considered legitimate only within the framework of islamic marriage ( nikah ) . moreover , as shown in merghati et al . study , sexual obedience was seen as the primary goals of the committed muslim woman . the concept of nejabat ( modesty ) is the most important ethical code applied to an iranian woman who is not sexually expressive . in contrast , islamic scholar morteza motahari pointed out the islamic clear guidelines toward sexuality , leading neither to any sense of sexual deprivation and frustration , nor to any repressed or inhibited sexual desire . however , in the islamic doctrine freeing sexual desire and lifting of traditional moral restraints is not accepted . as a criterion , religiosity has significant effects on iranian women 's sexual understandings ; and that experts working in the fields of gender and sexuality need to be sensitive to the notion that some muslim women may not speak out their sexuality as an indicator of submission to religious codes , of modesty and of being an idealized muslim wife . in her linguistic analysis of sexuality expression of iranian women , merghati - khoei has revealed the ways of developing terminology and cultural explanations , which are juxtaposed with the exploration of the development of women 's sexuality . iranian women expressed their sexuality differently from western women even though they understood and talked about the same issues . . there is , however , a hesitation when it comes to discussing or reporting the emotional aspects of sexual encounters . the power of culture in dictating daily language for females has been highlighted in morocco as an arab - islamic context . even though persian vocabulary , concepts and expressions about sexual matters exist in art , poetry and the beauty of nature , these linguistic resources are not applied in daily life to express sexuality . for example , the sexual attitude scale ( sas ) , certainly a well - designed tool for use within many cultures measures a person 's awareness toward her / his sexual interests and needs as well as sexual - self judgment . communication of sexual needs or interests with others is another feature of this tool . as a neglected subject matter in the iranian culture , questioning people about their sexual needs and interests or sexual - self concept seems impractical . sexuality is an unspoken issue , and individuals might not be linguistically skilled to communicate their sexuality with an interviewer . in sum , the art of using a rich vocabulary of metaphors and euphemisms is a characteristic of iranian speech , used to communicate and encapsulate matters not normally spoken explicitly . for example , for fluent english speakers phrase marital life may indicate all kinds of relationships within a marital framework , while in farsi zendegi - e - zanashoyi ( literally marital life ) has sexual connotations and is usually understood to mean the sexual relationship between husband and wife . another difficulty in asking about sexuality during a study is the struggle between conscious embarrassment and sexual talk . by conscious embarrassment , we mean the shame , prohibition and modesty about sex . the majority of the women who participated in merghati - khoei 's qualitative study pointed out that they were not culturally expected to be straightforward or frank in expressing sexual matters . for example , the sexual desire inventory tends to measure sexual desire as a biologic factor . out of 14 items , 4 focus on masturbation and 2 items ask about having interest in casual sex . in iranian contexts , none of these 6 items would be posed by researchers or responded to by the participants . for instance , some of the items focus on motivations leading people to out - of - wedlock or casual sex . although casual or extra marital sex happens in every society , questioning iranians about these behaviors is not ethically and religiously possible or feasible . this assertion is based on the common assumptions . in iran , people strongly hold onto the traditional culture of sexuality based on purity ( paki ) , chastity ( nejabat ) , honour ( aberoo ) and honesty ( sedagat ) underlying the family structure . there is also the belief commonly permeating iranian society that people are fairly innocent in terms of sexuality compared with non - muslim or western societies . however , with these assumptions we can not minimize the impact of factors such as modernization , worldwide communication and cultural transformations in younger generations and the way they learn about sex , practice and develop their sexual understandings . undoubtedly , these factors change behaviors and attitudes . social conduct and religiosity define the ethical aspect of sexuality in the iranian culture . in iran the teachings of islamic principles tie strongly to shia interpretations , which form the basis for iranians understandings of sexuality . the expression of sexuality is considered legitimate only within the framework of islamic marriage ( nikah ) . moreover , as shown in merghati et al . study , sexual obedience was seen as the primary goals of the committed muslim woman . the concept of nejabat ( modesty ) is the most important ethical code applied to an iranian woman who is not sexually expressive . in contrast , islamic scholar morteza motahari pointed out the islamic clear guidelines toward sexuality , leading neither to any sense of sexual deprivation and frustration , nor to any repressed or inhibited sexual desire . however , in the islamic doctrine freeing sexual desire and lifting of traditional moral restraints is not accepted . as a criterion , religiosity has significant effects on iranian women 's sexual understandings ; and that experts working in the fields of gender and sexuality need to be sensitive to the notion that some muslim women may not speak out their sexuality as an indicator of submission to religious codes , of modesty and of being an idealized muslim wife . to investigate sexual behaviors in an iranian context , we recognize the importance of identifying or developing an instrument to assess sexual behavior domains among women in the particular context of iranian culture . we thought that such an instrument would be essential tool for achieving a more systematic and in - depth understanding of iranian women 's sexuality , may be useful in applied settings , and would advance sexuality research as a whole . no matter the context or use , however , measuring a construct such as sexual behavior is subjective and therefore entirely dependent on self - report . it has been argued that iranian women may not report properly if they believe sexuality has nothing to do with health . for example , a woman 's inability to gain sexual pleasure due to painful intercourse might not be defined as a sexual health problem to be reported , whereas other people would consider it as a sexual health problem for the woman . this suggests the idea that the culture of sexuality affects people 's interpretations of sexually related problems . developing a contextualized instrument to measure the domains of sexual behavior would allow sexuality and gender researchers to better answer questions related to the influence of culture in those domains , sexual scripts across diverse cultures , and other factors influencing sexual health outcomes . in the 1970s , gagnon and simon 's sexual conduct represented the first truly sociological analysis of sexual behavior . gagnon and simon defined sexual behavior within a new theoretical framework of social scripts. they produced a critique that moved us beyond the objective definition of sexual behavior : our concern here is to understand sexual activities of all kinds as the outcome of a complex psychological process of development , and it is only because they are embedded in social scripts that the physical acts themselves become possible it is neither fixed by nature or by the organs themselves . the very experience of sexual excitement that seems to originate from hidden internal sources is in fact a learned process and it is only our insistence on the myth of naturalness that hides these social components from us . the character through a cultural scenario in which they take up , internalize and enact culturally prescribed normative roles ; interpersonal scripts in which they make a suitable identity based on desired expectations and intrapsychic scripting in which they make the self in relation to social life . thus , sexual practice is separated from the biology of the body and one 's sexuality is strongly formed by the very complex social world . having investigated the history of human sexuality , we believe sexuality is influenced by the society , culture and era in which people live . within an iranian context , we therefore recognized existing instruments targeting non - risky sexual behaviors among heterosexual women are insufficient to measure sexual behaviors . we categorized instruments as culturally compatible or incompatible based on the sexuality domains they tend to measure . the instruments , by which the biological aspects of sexual behaviors are measured were found applicable for any given community or population , the iranian context included . alternatively , those measuring outcomes related to subjects attitudes , understanding or sexual scripts were identified as culturally incompatible . most of the tools seem reasonable candidates for use in the iranian culture and society with minor revisions [ table 1 ] . review of these instruments shows that most of them are functional based , such as the most used scale in literature , ( fsfi ) . other well known tools , such as the arizona sexual experience scale ( asex ) and the golombok - rust inventory of sexual satisfaction ( griss ) , are also functional based . some other tools are problem - focused [ table 3 ] and specific to measure only disorder - based outcomes . we argue that the medically oriented instruments employed in the field are drawn from concepts and meanings based on investigations conducted in western societies . yet as researchers , we sometimes found ourselves disappointed by the level of difficulty , which we professionals encountered in fitting our participants into those biomedical frameworks . the culture - bound nature of sexuality limits the research - based information in iran . in our society , the lack of information in the sexual domain will be most productively addressed first through research attention to subjective concepts . lack of sufficient knowledge in the field of sexuality in iranian contexts , makes it important to identify normative sexual behaviors qualitatively before applying problem - oriented tools in research . there are social and cultural challenges arising from the recognition that iranians use culturally specific sexual expressions . these expressions may construct different ways of perceiving sexuality that are not easily translatable or even understandable by outsiders . this means that sexuality is a complex phenomenon embedded in various meanings and understandings , not merely objective and measurable behavior . explaining those meanings and perceptions makes sexuality a dynamic phenomenon through one 's life time . how we know what we know changes periodically and therefore , creating an epistemological crisis in knowledge as well as the research process . however , scholars in science and human behavior such as skinner have powerfully questioned the reliability of subjective measures of private events such as sexual behaviors . we concluded that the published instruments are well - designed and used worldwide ; however , we must also acknowledge that the sexual scripts of iranian women define sexual behaviors differently , limiting their communication in the research setting and compromising the compatibility of these instruments . therefore , exploring and analyzing the lexicon and expressions used by the iranian women creates a ground for developing a culturally comprehensive measure , which can adequately examine how these women explain their sexual behaviors .

the cultural compatibility of sexually related instruments is problematic because the contexts from which the concepts and meanings were extracted may be significantly different from related contexts in a different society . this paper describes the instruments that have been used to assess sexual behaviors , primarily in western contexts . then , based on the instruments working definition of sexual behavior and their theoretical frameworks , we will ( 1 ) discuss the applicability or cultural compatibility of existing instruments targeting women 's sexual behaviors within an iranian context , and ( 2 ) suggest criteria for sexually related tools applicable in iranian settings . iranian women 's sexual scripts may compromise the existing instruments compatibility . suggested criteria are as follows : understanding , language of sexuality , ethics and morality . therefore , developing a culturally comprehensive measure that can adequately examine iranian women 's sexual behaviors is needed .

a level of pain that completely incapacitates one individual may cause only a minor annoyance in another . characterizing the genetic factors affecting susceptibility to chronic pain could lead to novel treatment or prevention strategies , which are desperately needed . a recent paper by costigan et al . the authors performed a multi - dimensional analysis using rat gene expression and human genetic association that identified a new genetic factor affecting susceptibility to chronic pain . this study was initiated by analysis of gene expression changes within rat dorsal root ganglia obtained after nerve damage was induced in three different models of chronic pain . the mrna for a potassium channel alpha subunit ( kcns1 ) was constitutively expressed in sensory neurons , but was downregulated after three types of nerve injury in these models . other studies have successfully identified a human genetic susceptibility factor on the basis of a candidate gene emerging from analysis of a rodent model . costigan and colleagues therefore examined snp alleles within the human homolog ( kcns1 ) in five independent cohorts ( two with chronic low back pain , two after limb amputation and one after mastectomy ) that have a high incidence of chronic pain . in four of the five cohorts studied , there was a significant association of the val allele at a nonsynonymous snp ( rs734784 , val / ile ) with increased risk for chronic pain . for example , homozygous val / val individuals had a 2.4-fold increased relative risk of failing to achieve pain improvement 1 year after back surgery . the proportion of individuals without phantom limb pain after leg amputation was 45% in those without the val allele , but fell to 22% in val / val homozygotes . among healthy volunteers that were subjected to multiple experimental pain stimuli , although experimental studies demonstrating an allelic effect were not presented , which is a substantial limitation of this study , these results make it likely that kcns1 alleles contribute to susceptibility to chronic pain . although kcns1 by itself does not have potassium channel function when tested in heterologous expression systems , its expression has been shown to modulate the currents formed by other channels [ 3 - 5 ] . voltage - gated potassium channels have important effects on neuronal function , including altering the resting membrane potential and the shape and frequency of the action potential . similarly , gain - of - function mutations in the nav1.7 sodium channel cause syndromes associated with increased pain sensitivity . chronic pain has a huge impact on our society , and its treatment is a major driver for the increasing cost of health care . an estimated 76 million people in the us have chronic pain , which costs the us public over $ 100 billion per year . approximately 25% of chronic pain is neuropathic ( caused by nerve damage ) , which is characterized by spontaneous pain that is burning or stabbing in nature . this can develop after limb amputation , mastectomy or lower back injury , or in individuals with a long history of diabetes . although multiple kinds of treatment can be used , they often have limited efficacy , and chronic pain is often managed by administration of opioids ( morphine and related synthetic compounds , including hydrocodone ) . the increased focus on pain management resulted in a six - fold increase in the per capita sales of prescription opioid medications in the us between 1997 and 2006 . hydrocodone - acetaminophen is prescribed over 100 million times per year in the us , which is far more than any other medication , including lipid - lowering and blood - pressure - lowering agents . this has led to a dramatic increase in the incidence of opioid misuse , emergency room visits due to opioid analgesic poisoning , and fatal opioid overdose . prescription opioids have now surpassed marijuana as the drug that is most commonly abused among the newly initiated . because of the enormity of this public health problem , we desperately need new approaches for the prevention or treatment of chronic pain conditions . it is likely that genetic discoveries can lead to new approaches for chronic pain , because genetic discoveries have catalyzed new approaches for related clinical conditions . for example , haplotype - based computational genetic analysis has identified causative genetic factors affecting analgesic medication and inflammatory pain responses , and it has identified four genes affecting narcotic drug responses [ 11,14 - 16 ] in mice . the latter genetic discovery generated a new treatment strategy for preventing narcotic drug withdrawal symptoms , which was shown to be effective in humans . moreover , the variable response to multiple classes of analgesic medications among inbred mouse strains was shown to be due to genetic variation within a genetic locus ( kcnj9 ) that also encodes a potassium channel ( girk3 ) . hopefully , characterizing the functional role of kcns1 in neuronal responses and pain perception , and the impact of the allelic differences , could lead to new approaches for treatment of chronic pain . although the tractability of kcns1 ( or other potassium channels ) as a therapeutic target remains to be determined , small molecules targeting potassium channels have been produced ( reviewed in ) , and one has attenuated neuropathic pain in animal models . although the kcns1 val allele explains only a small percentage of the total variance ( 4.6 to 7.8% ) in the chronic pain endpoints in the cohorts studied , the identification of an initial genetic factor for chronic pain is an important achievement for two reasons . first , it shows that genetic factors can be identified for this condition , and this study provides a template for subsequent studies . second , it is likely that a greater percentage of the genetic susceptibility to chronic pain can be explained by combining this genetic factor with other subsequently identified factors . moreover , pain is a subjective sensory symptom ; it is difficult to measure ; and there are substantial psychological and emotional components that contribute to the perception of pain . it has recently been found that there are different subtypes of chronic pain , which are distinct from the causative disease ( diabetic neuropathy , radicular back pain and so on ) . it is likely that genetic factors determine whether a chronic pain syndrome will develop and the specific subtype that emerges after exposure to a triggering cause . thus , the impact of a single genetic factor could be much larger if a specific pain subtype is examined relative to that measured in a large cohort of individuals with different types of chronic pain . beyond finding new drug targets , identification of genetic factors affecting susceptibility to chronic pain syndromes could be the lever that drives advances in this difficult clinical area . a genetic risk factor could have a substantial impact on clinical practice because individuals at increased risk for developing a chronic pain syndrome could be identified before a surgical procedure or immediately after a traumatic incident . the genetic information could be used to develop proactive methods for prevention of chronic pain syndromes , enabling interventions to be targeted to the high - risk subset . because of the great inter - individual variation in the response to painful stimuli and to analgesic drugs , knowledge of genetic risk factors could enable better stratification of chronic pain patients or aid in the selection of the appropriate therapy . rather than focusing on the disease that is etiologically associated with the chronic pain , study is hopefully one of the first of many subsequent genetic studies that could lead to entirely new ways to approach chronic pain syndromes . clearly , the clinical and research communities have a long way to go before genetically targeted therapies for chronic pain become a reality . however , kcns1 ( or other channels ) could perhaps become the next target for a new class of analgesics that are selectively used in the 20% of the population that are homozygous for the val alleles of kcns1 . the day might soon come when we no longer think of ' chronic pain ' as a single clinical entity , but have the tools to characterize the specific ' channelopathy ' that underlies the clinical presentation and targeted treatments for each subgroup . gp is a professor and mz is the director of statistical research in the department of anesthesia at stanford university medical school .

a recent publication that combined rat gene expression data and a human genetic association study has identified the first genetic risk factor for chronic pain in humans . in four of the five cohorts studied , there was a significant association of an allele within a gene ( kcns1 ) encoding a potassium channel ( kv9.1 ) with an increased risk for chronic pain . identification of genetic risk factors for chronic pain could catalyze new advances in this difficult clinical area that has become a major public health problem . genomic - medicine - based advances for chronic pain could include the development of a mechanism - based classification system for chronic pain , new treatment options , improved methods for treatment selection and targeted prevention strategies for high - risk individuals .

optical coherence tomography ( oct ) is a noninvasive method that evaluates biological tissues by in vivo imaging.1 since its introduction , oct has undergone several improvements and revolutionized the diagnostic , monitoring , and therapeutic approaches to many retinal diseases and glaucoma . computerized algorithms can be used on the high - resolution images obtained by modern oct devices in order to identify and measure the thicknesses of discrete retinal layers , including the retinal nerve fiber layer ( rnfl ) , macular ganglion cell complex ( gcc ) , and choroid.2,3 the choroid is the most vascular tissue in the eye and is known to have an important role in ocular nutrition , volume regulation , and temperature control.4 choroidal abnormalities are critical to the onset and progression of many chorioretinal disorders such as age - related macular degeneration ( amd ) , polypoidal choroidal vasculopathy , vogt - kayanagi - harada disease , and central serous chorioretinopathy.5 with the recent development of enhanced depth imaging ( edi ) , in vivo assessment of the choroid has become an area of interest . edi allows better visualization and quantitative evaluation of the choroid , which was not possible before . information about the choroidal thickness ( ct ) is useful in many clinical situations for decision making regarding the management and monitoring of disease progression.6 neurodegenerative diseases such as glaucoma preferentially affect the gcc , which is the sum of the three innermost layers : the rnfl , the retinal ganglion cell layer , and the inner plexiform layer . these contain the axons , cell bodies , and the dendrites of the retinal ganglion cells , respectively.7,8 recent studies have shown that measurement of macular gcc thickness has the same diagnostic potential for glaucoma as rnfl thickness.911 moreover , it has been reported that measurements of average gcc thickness had better sensitivity in detecting changes in retinal thickness in multiple sclerosis neurodegeneration than rnfl thickness measured by oct.12 both ct and gcc thickness measurements can be affected by several factors such as pupil size , scan quality , and cataract . in this study , we aimed to evaluate the effect of uneventful cataract surgery on subfoveal ct and gcc thickness measured by edi - oct . this prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . the present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus , hypertension , glaucoma , amd , diabetic retinopathy , retinal vein occlusion , uveitis , or other inflammatory and vascular pathologies . patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure ( iop ) or cystoid macular edema were also excluded . all patients had a full ophthalmic examination , including best - corrected visual acuity ( bcva ) assessment , as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation , and iop measurement by goldmann applanation tonometry . axial length ( al ) was measured by immersion echography ( axis ii ; quantel medical inc . , cedex , france ) and central corneal thickness ( cct ) was measured by ultrasonographic pachymetry ( pacline ; optikon 2000 spa , rome , italy ) . prior to the operation , tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . phacoemulsification was performed by one experienced surgeon ( ec ) using infiniti vision system ( alcon laboratories , inc . ) . a clear corneal incision of 2.8 mm , 600 mmhg vacuum , an aspiration flow rate of 30 ml / min , bottle height of 75 cm , and quick - chop technique were used in all surgeries . two different dispersive viscoelastics ( viscoat and bivisc ) and two different cohesive viscoelastics ( healon and bio - hyalur ) were used during cataract surgery . one - piece hydrophobic acrylic intraocular lens ( sensar model : aab00 ; abbott medical optics inc . , santa ana , ca , usa ) was used in all surgeries . viscoelastics were cleaned out with marked attention , even behind the intraocular lenses . the procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . the operative times ( ots ) and effective phaco times ( epts ) were recorded in each case . postoperatively , all patients were treated with topical antibiotics ( ofloxacin 0.3% ) for 1 week and topical steroid drops ( prednisolone acetate 1% ) for 4 weeks . cirrus edi - oct ( carl zeiss meditec ag , jena , germany ; software version 6.5.0.772 ) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis ( macular cube 512128 ) scan protocols , respectively . scans with misalignment , segmentation failure , decentration of the measurement circle , and poor illumination , or those out of focus , were also excluded . the one - line raster is a 6 mm line consisting of 4,096 a - scans . scan 3 of the five scans taken , which passes through the fovea , was used for all measurements . ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . to avoid the effects of mydriatic agents or diurnal changes , the ct was measured from the subfoveal region by using the cirrus linear measurement tool . when there was a discrepancy of > 10% of the mean of the two values between the observers in two eyes in the study , observers discussed and repeated the measurements , and the average results from the two observers were determined as the final ct readings . the cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers ( gcipl ) in six circular sectors centered in the fovea . it also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans , each composed of 512 a - scans . a built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . the annulus has an inner vertical diameter of 1 mm , which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately , and an outer vertical diameter of 4 mm , which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . the ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . the difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . similar to other cirrus printouts , it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . those that are abnormal at the < 5% and at the < 1% level are represented by yellow and red backgrounds , respectively . the primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes ; however , central macular thickness ( cmt ) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols , respectively , using the cirrus oct device . statistical analysis was performed using spss statistics for windows , version 20.0 ( ibm corporation 2011 , armonk , ny , usa ) . paired - samples t - test was used for equality of means , and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . this prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . the present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus , hypertension , glaucoma , amd , diabetic retinopathy , retinal vein occlusion , uveitis , or other inflammatory and vascular pathologies . patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure ( iop ) or cystoid macular edema were also excluded . all patients had a full ophthalmic examination , including best - corrected visual acuity ( bcva ) assessment , as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation , and iop measurement by goldmann applanation tonometry . axial length ( al ) was measured by immersion echography ( axis ii ; quantel medical inc . , cedex , france ) and central corneal thickness ( cct ) was measured by ultrasonographic pachymetry ( pacline ; optikon 2000 spa , rome , italy ) . prior to the operation , tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . phacoemulsification was performed by one experienced surgeon ( ec ) using infiniti vision system ( alcon laboratories , inc . ) . a clear corneal incision of 2.8 mm , 600 mmhg vacuum , an aspiration flow rate of 30 ml / min , bottle height of 75 cm , and quick - chop technique were used in all surgeries . two different dispersive viscoelastics ( viscoat and bivisc ) and two different cohesive viscoelastics ( healon and bio - hyalur ) were used during cataract surgery . one - piece hydrophobic acrylic intraocular lens ( sensar model : aab00 ; abbott medical optics inc . , santa ana , ca , usa ) was used in all surgeries . viscoelastics were cleaned out with marked attention , even behind the intraocular lenses . the procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . the operative times ( ots ) and effective phaco times ( epts ) were recorded in each case . postoperatively , all patients were treated with topical antibiotics ( ofloxacin 0.3% ) for 1 week and topical steroid drops ( prednisolone acetate 1% ) for 4 weeks . cirrus edi - oct ( carl zeiss meditec ag , jena , germany ; software version 6.5.0.772 ) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis ( macular cube 512128 ) scan protocols , respectively . scans with misalignment , segmentation failure , decentration of the measurement circle , and poor illumination , or those out of focus , were also excluded . the one - line raster is a 6 mm line consisting of 4,096 a - scans . scan 3 of the five scans taken , which passes through the fovea , was used for all measurements . ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . to avoid the effects of mydriatic agents or diurnal changes , the ct was measured from the subfoveal region by using the cirrus linear measurement tool . when there was a discrepancy of > 10% of the mean of the two values between the observers in two eyes in the study , observers discussed and repeated the measurements , and the average results from the two observers the cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers ( gcipl ) in six circular sectors centered in the fovea . it also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans , each composed of 512 a - scans . a built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . the annulus has an inner vertical diameter of 1 mm , which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately , and an outer vertical diameter of 4 mm , which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . the ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . the difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . similar to other cirrus printouts , it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . those that are abnormal at the < 5% and at the < 1% level are represented by yellow and red backgrounds , respectively . the primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes ; however , central macular thickness ( cmt ) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols , respectively , using the cirrus oct device . statistical analysis was performed using spss statistics for windows , version 20.0 ( ibm corporation 2011 , armonk , ny , usa ) . paired - samples t - test was used for equality of means , and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . a total of 30 eyes of 30 patients ( 16 male and 14 female ) , with a mean age of 60.24.2 years were included in this study . mean bcva improved in all eyes , and this improvement was statistically significant ( 0.460.15 logarithm of minimum angle of resolution [ logmar ] units preoperatively and 0.060.49 logmar units postoperatively ; p<0.001 ) . mean preoperative al was 22.80.6 mm ( range : 21.524.1 mm ) and mean cct was 551.522.1 m . mean ot was 8.62.7 minutes and mean ept was 6.92.5 seconds ( table 1 ) . the mean subfoveal ct was 294.439.2 m ( range : 201356 m ) preoperatively and 301.439.9 m ( range : 231367 m ) postoperatively . the mean gcc thickness was 85.04.4 m ( range : 7894 m ) preoperatively and 89.25.3 m ( range : 8199 m ) postoperatively . the mean cmt was 247.917.6 m preoperatively ( range : 211280 m ) and 249.017.8 m ( range : 213278 m ) postoperatively . the mean rnfl thickness was 97.45.4 m preoperatively ( range : 87110 m ) and 101.75.6 m ( range : 91113 m ) postoperatively . regression analysis showed that age , sex , al , cct , ot , and ept were not associated with ct changes ( p=0.834 , p=0.129 , p=0.203 , p=0.343 , p=0.547 , and p=0.147 , respectively ) and gcc thickness changes ( p=0.645 , p=0.542 , p=0.152 , p=0.664 , p=0.448 , p=0.268 , respectively ) after cataract surgery . in this study , we investigated the effect of cataract surgery on subfoveal ct and gcc thickness , as measured by edi - oct , and we found that both subfoveal ct and gcc thickness increased slightly after cataract surgery . cmt and peripapillary rnfl thicknesses were also evaluated as a secondary objective , and we observed statistically significant increases in cmt and rnfl thickness . our results indicate that cmt , subfoveal ct , as well as rnfl and gcc thicknesses are slightly affected after phacoemulsification surgery . although our study included a small group ( only 30 eyes ) , we assume that our data reliability is high because of the homogeneous nature of the patients ( similarities of age , cataract type and severity , cct , al , ot , ept and so on ) and the careful / detailed oct examinations performed by two observers , avoiding the effects of diurnal changes and mydriatic agents . zhao et al13 revealed significant diurnal variations of the ct at the macular region in young healthy female individuals . cataract surgery by phacoemulsification is one of the most common intraocular surgeries that improve the quality of vision . however , it is an invasive procedure leading to an inflammatory response mostly induced by the release of prostaglandins in the retina and choroid and , in many patients , can lead to worsening of preexisting retinal diseases such as diabetic macular edema.15,16 recent studies have shown subclinical increase of cmt and peripapillary rnfl thickness after uneventful cataract surgery.1719 however , the morphologic changes that this inflammatory insult produces in the choroid have not been studied well because of the lack of detailed imaging techniques for the choroid . after the introduction of edi , changes in ct in several pathologic ( central serous chorioretinopathy , choroidal neovascularization , and high myopia ) and physiologic ( age , sex , and al ) conditions can be easily assessed.2022 therefore , ct is being measured increasingly often and is becoming an accepted procedure for clinical and research applications . falco et al23 evaluated the effect of uneventful phacoemulsification on the subfoveal ct and cmt , and they reported that phacoemulsification induced nonpathologic increases in cmt ; however , these changes were not accompanied by significant changes in ct . they claimed that the inflammatory insult due to phacoemulsification mainly affects at the retinal level and seems to be independent of ct changes.23 by contrast ; our results support the recent studies by ohsugi et al24 and noda et al,25 which report significant increases in ct after uneventful phacoemulsification . ohsugi et al24 evaluated 100 eyes and emphasized that the al and changes in iop were critical for evaluating the changes in ct after cataract surgery . noda et al25 evaluated 29 eyes and observed that the increase in subfoveal ct did not subside to baseline even at 6 months postoperatively . they also found that male sex and thicker baseline ct predicted a larger magnitude of increase in ct after cataract surgery.25 in our study , interestingly , the parameters of age , sex , al , baseline ct , and cct did not influence the postoperative ct statistically in regression analysis . we also analyzed the effect of ot and ept on postoperative ct and we did not find any relationship . this may be explained by the short / similar results in terms of ots and epts , as well as the experience of the surgeon conducting a quick surgery with less anterior segment manipulation , thus reducing the inflammatory response after cataract surgery . a speculation is that it may be related to postoperative inflammation because proinflammatory prostaglandins and cytokines are considered to explain macular edema after cataract surgery , and inflammatory disorders are also known to increase the ct.26,27 another speculation is that cataract surgery introduces more light into the eyes , leading to greater metabolic activation in the retinal pigment epithelium , causing angiogenesis and inflammation.28,29 in the beaver dam and blue mountains eye studies,3032 it has been suggested that cataract surgery is associated with the onset of amd , resulting in visual impairment due to neovascularization originating from the choroid . noda et al25 report that cataract surgery influences the ct and this influence persists for as long as 6 months postoperatively and may affect the process of amd . choroidal abnormalities are central to chorioretinal disorders such as amd , polypoidal choroidal vasculopathy , and central serous chorioretinopathy . furthermore , it has been claimed that pathologic choroidal blood supply is an important factor in open - angle glaucoma leading to optic nerve damage.33,34 cts of glaucomatous eyes have been reported to be thinner in postmortem histologic studies . in addition , significant increases in choroidal extravascular volume have been confirmed in many primary angle - closure glaucoma eyes and abnormal ct has been hypothesized to be a contributing feature in these eyes.35,36 however , it is unclear whether these findings represent a risk factor or a consequence of these diseases . the hallmark of glaucoma is the loss of the retinal ganglion cell axons , which leads to a typical optic neuropathy . qualitative and quantitative evaluations of the optic nerve head and rnfl have been used to detect evidence of glaucomatous damage.911 in a recent study,8 it has been suggested that the gcc thickness may be the most relevant parameter to measure in glaucoma . nouri - mahdavi et al37 also reported that regional gcc measurements derived from cirrus hd - oct performed as well as regional rnfl outcomes for the detection of early glaucoma . moreover , macular gcc thickness has been found to have better structure function correlations than rnfl thickness with both visual function and central nervous system findings in multiple sclerosis neurodegeneration.12 as oct uses near - infrared light and is based on interferometry , its image quality is influenced by opacities in the optical path . loss of oct image quality is caused by attenuation of the light in the oct scanning spot on the retina.17,18 several studies have shown that lens opacity decreased image quality and postoperative rnfl thickness measurements increased significantly.3840 the more advanced the cataract , the less is the signal quality and the thinner are the recorded rnfl and gcc thicknesses . the coexistence of neurodegenerative diseases such as glaucoma and cataract among elderly patients is not unusual , and cataract may decrease the quality of fundus images , leading to an incorrect evaluation in the diagnosis and follow - up of these patients . nakatani et al7 examined the effect of cataract on the measurements of gcc and rnfl thicknesses , and they reported that all thickness parameters in gcc and rnfl increased slightly after phacoemulsification surgery . therefore , we decided that the presence of cataract may lead to an underestimation of the thicknesses of gcc and rnfl , and this should be taken into account when analyzing progression in glaucoma and/or neurodegenerative diseases such as multiple sclerosis . after cataract surgery , the examiners should consider obtaining new baseline measurements , as suggested in previous studies . this study has many limitations such as short follow - up time , relatively small sample size , and lack of eyes with glaucoma and neurodegenerative diseases . in addition , we analyzed just mean subfoveal ct and mean thickness of gcc and rnfl , leading to lack of segmentation of these parameters . scleral interface and measured ct with cirrus linear measurement tool . although we used scans with signal strength 6 for analysis and excluded poor images , the effect of signal strength index on measurements was not investigated . furthermore , we used postoperative topical steroids after cataract surgery ; thus , the true effect of phacoemulsification on oct parameters could not be evaluated and we could not determine whether and when the increased ct can return to baseline measurements . phacoemulsification surgery increased ct and it should be kept in mind that the small changes in ct may affect the onset of amd , which is a serious disease that results in central visual impairment . although the mechanism is unclear , it has been postulated that ct changes may also play a role in glaucoma . we suggest establishment of new baseline measurements after cataract surgery for monitoring patients with glaucoma and neurodegenerative diseases . further studies with a greater number of patients and longer duration of follow - up and conducted using new computerized oct algorithms are needed .

purposewe aimed to evaluate the effect of cataract surgery on subfoveal choroidal thickness ( ct ) and ganglion cell complex ( gcc ) thickness , as measured by enhanced depth imaging - optical coherence tomography ( oct).methodsthis prospective study included 30 eyes of 30 patients who had undergone uneventful phacoemulsification surgery for senile cataract but had no previous ocular surgery or other ocular abnormality . best - corrected visual acuity , slit - lamp biomicroscopy , intraocular pressure , axial length , and central corneal thickness were measured preoperatively . the operative times ( ots ) and effective phaco times were also recorded in each case . oct measurements were performed at the preoperative visit and 1 month after cataract surgery . study of ct and gcc thickness changes was the primary objective , but central macular thickness ( cmt ) and peripapillary retinal nerve fiber layer ( rnfl ) thicknesses were also obtained by oct.resultsthe mean subfoveal ct was 294.439.2 m preoperatively and 301.439.9 m postoperatively ( p<0.001 ) . the mean gcc thickness was 85.04.4 m preoperatively and 89.25.3 m postoperatively ( p<0.001 ) . the mean cmt was 247.917.6 m preoperatively and 249.017.8 m postoperatively ( p=0.029 ) . the mean rnfl thickness was 97.45.4 m preoperatively and 101.75.6 m postoperatively ( p<0.001 ) . regression analysis showed that age , sex , axial length , central corneal thickness , operative time , and effective phaco time were not associated with ct changes ( p=0.834 , p=0.129 , p=0.203 , p=0.343 , p=0.547 , and p=0.147 , respectively ) and gcc thickness changes ( p=0.645 , p=0.542 , p=0.152 , p=0.664 , p=0.448 , and p=0.268 , respectively ) after cataract surgery.conclusionour results indicate that all subfoveal ct , cmt , as well as rnfl and gcc thicknesses are slightly affected after uneventful phacoemulsification surgery . after cataract surgery , the examiners should consider obtaining new baseline measurements .

only 2030% of patients with group a beta hemolytic streptococcus ( gabhs ) pharyngitis presents with classical symptoms of the disease . reliance on clinical judgment alone has a poor predictive value and results in 80% to 95% overestimation of disease [ 2 , 3 ] . diagnostic strategies for acute gabhs pharyngitis are thus based on epidemiological factors , signs , and symptoms , as well as the result of throat cultures ( tcs ) . several studies have shown that the use of throat culture leads to more judicious use of antibiotics [ 57 ] . physicians prescribe antibiotics for acute pharyngitis as they are concerned that patients with this complaint may be suffering from gabhs infection that if left untreated might develop suppurative complications , such as , tonsillar abscess or nonsuppurative complications , such as , rheumatic fever [ 6 , 8 ] . antibiotics , however ; confer only minor symptomatic benefits for gabhs sore throat . they shorten the duration of symptoms by merely half a day on average [ 8 , 9 ] . more recent studies have shown that antibiotic use only reduced the incidence of rheumatic fever by a mere 0.5 cases per 100,000 . the importance of preventing rheumatic fever has lessened as the incidence of rheumatic fever and rheumatic heart disease has declined significantly in the last 20 years , from a mean annual incidence of 13.4 per 100,000 to 5 per 100,000 . prevalence has decreased as well from 5.7 per 1,000 in the eighties to 0.5 per 1,000 in 2000 [ 8 , 10 , 11 ] . this failure probably stems from the fact that about 20% of children with gabhs is infected with bacteria which contain m protein , a virulence factor located on the surface of the bacterial wall that confers resistance to commonly used antibiotics . newer beta - lactamase - resistant antibiotics did not prevent this treatment failure [ 13 , 14 ] . review of the literature from 1945 to 1999 , which includes 10,484 cases of gabhs sore throat , found that antibiotic treatment reduced the occurrence of acute otitis media , a common complication of this disease , by a mere 25% , compared to the placebo group and sinusitis by only 50% . rheumatic fever , a nonsuppurative complication , was reduced by less than 33% , compared to placebo [ 8 , 10 , 11 , 15 ] . in addition to the uncertainly in the scientific literature , parents seem to be uncertain regarding the benefits of antibiotic treatment for acute gabhs pharyngitis and tend to stop treatment earlier than prescribed . in a pilot study , we randomly followed 75 children with gabhs pharyngitis for 6 months and have found that more than 75% of them did not complete ten days of antibiotics . this finding led us to conduct a multisite , prospective cohort observational study , the results of which are reported here . the goal of this study was to determine whether noncompliance with antibiotic treatment affects short - term or long - term complications . two central , primarily rural , and agricultural regions of the largest health maintenance organization ( hmo ) in israel , comprising approximately one million patients . using a standard protocol , we located from our computerized data base 107,840 patients , aged 6 months to 18 years , who were examined by their primary care physician for upper respiratory tract infection , tonsillitis , pharyngitis , sore throat , tonsillopharyngitis , neck pain , cervical lymphadenopathy , pta , rpa , from january 1 , 1999 until december 31 , 2000 . we then accessed the charts of 78,473 of these children who were diagnosed with infected throat or one of the differential variants , excluding all children diagnosed as having viral upper respiratory infections . 47,000 of these patients were formally diagnosed with acute pharyngitis or acute tonsillitis and received a prescription for antibiotics , indicating that their physician suspected bacterial disease . in the index visit , 35,000 of these children had at least four out of five symptoms in the modified centor criteria used for this study and nadir modified breese epidemiological and clinical score card ( ecsc ) that has 91% sensitivity and 98% specificity when the score was above 15 ( score between 4 and 36 ) for the diagnosis of gabhs [ 1719 ] . colonies yielding beta - hemolysis were grouped for surface carbohydrate assessment by using a latex bead agglutination test ( figure 1 ) . of the 6336 children ( with positive cultures with 4 or more centor criteria and 15 or higher ecsc ) , 4,775 parents consented to enroll their children to the study ( figure 1 ) . excluded from the study were children who were diagnosed as gabhs chronic carriers or who had suffered from post - gabhs complications ; had any chronic illness , such as , renal or hepatic impairment ; had bleeding disorder ; had congenital or acquired immunodeficiency or suffered from malignancy . initial patient / parent contact was made by one of the authors ( m. sarrell ) within 3 to 5 days of the initial positive throat culture . at that time , initial information regarding the illness and whether a prescription for antibiotics was given by the primary care physician . the attending physicians of the two thousand study patients were contacted by email within 48 hours of the enrollment by one author ( m. sarrell ) . the physicians were requested to inform the authors of any additional cultures taken during therapy , and to request that they obtain two additional throat cultures and engage in improve adherence strategies by providing information , counseling , reminders , reinforcement , and if needed personal attention or supervision . while repeated cultures are not routinely recommended for asymptomatic patients who have completed a course of antimicrobial therapy , in light of the poor compliance with treatment in the pilot study , performance of such follow - up cultures was considered important for the purposes of the study . the first follow - up culture was performed within 10 to 14 days of the initial positive culture regardless of treatment status . the purpose of this culture was verification of antibiotics treatment failure or persistence of gabhs in the oropharynx of the untreated patients . the second additional throat culture was taken between days 21 and 30 after the initial positive culture , regardless of treatment status , to ascertain the presence of residual gabhs or recurrences . treating physicians were also requested to obtain blood for liver enzymes , renal function tests , and urine analysis from all the participants and to perform annual follow - up evaluation thereafter . a second patient / parent contact was made by our study coordinator within 10 to 14 days of initiation of antibiotic treatment . she collected information about demographic characteristics , past medical history , febrile status , need for repeat throat culture during the treatment period ( that was not part of the study protocol ) , and type of medication prescribed . during this contact she obtained information about the number of days of actual treatment , omission compliance and complications , the patients / parents perceived as deriving from the treatment ( or lack thereof ) . the computerized charts of the participants were searched within 2 to 4 weeks of the second patient / parent contact for additional information , including demographic characteristics , medical and environmental history , initial clinical data , such as , in - office fever evaluation , results of the physical examination , additional culture taken , type of antibiotics used , and disposition of prescription received . a second search of the computerized medical charts was performed by our research assistant between 30 and 90 days of initiation of medical treatment , to ascertain that the 2 requested throat cultures were obtained . relapse or recurrence of clinical or bacterial pharyngitis , suppurative or nonsuppurative complication , or even whether the participants complained of any sore throat within 30 days of completion of treatment were also evaluated . in order to assure that all possible short - term complications that occurred within 90 days of the index case were obtained , an additional comprehensive search of the hmo database was done within 120 days of the second computer search . we ascertained that findings that were either not available on the original computerized chart or were seen by other than their primary care physician , ( e.g. , emergency departments , patients that relocated ) , were not overlooked . the charts of the participants were then reviewed by one of the authors on a yearly basis , from january 2000 to january 2010 , noting possible late nonsuppurative complications of gabhs infection . no patients were lost to followup , even if they had changed physicians , due to our ability to track them through the centralized database to their new physician or another hmo . the children that were enlisted to the army ( and thus not members of any hmo during their military service ) were contacted either through their former attending physician or the military physician . minor treatment failure was defined as any clinical or bacterial recurrence of pharyngitis during the short - term follow - up period and its correlation to compliance with treatment . major treatment failure was defined as retropharyngeal or peritonsillar abscess or long - term complications , such as rheumatic fever . suppurative complications were chosen as a model , because the nonsuppurative complications ( rheumatic heart disease , arthritis , carditis ) have been practically eradicated in our region . this cohort study was designed to analyze rare events ( according to the cioms classification 110 events per 10,000 children years ) . the annual incidence of peritonsillar abscess ( pta ) in our region is 24 cases per 100,000 and the incidence of retropharyngeal abscess ( rpa ) is 57 cases per 100,000 . power calculations suggested 6,500 to 7,000 person - years of intervention would be needed to detect a 22% difference in pta and rpa between the fully treated ( ft ) and partially treated ( pt ) arms of the study population . furthermore , one - sided alpha of 0.025 , a statistical power of 95% , and the pta / rpa incidence given above showed that approximately 19,000 children - years would be needed to show the noninferiority of ft versus pt . since the primary outcome of interest is the pta / rpa hazard ratio between ft and pt . the null hypothesis to be tested is hr pta / rpa > 2 ( i.e. , the pta / rpa hazard ratio for ft versus pt is higher or equal to 2 ) . the pta / rpa hazard ratio was calculated for ft versus pt . for purposes of analysis , participants were divided into four subgroups based on length of treatment : 1st subgroup ( untreated ) , those who did not receive any treatment , 2nd subgroup ( partially treated ) , children that received antibiotics for 1 to 3 days , 3rd subgroup ( mostly - treated ) , children treated for 4 to 6 days , and 4th subgroup ( fully - treated ) children treated between 7 to 10 days . data are presented as proportions ( with 99% confidence intervals [ cis ] ) , means ( with sds ) , or medians ( with interquartile ranges ) , using pearson tests , student 's tests , or fisher exact test . comparisons of length of treatment according to time and treatment were assessed using the repeated measures and analysis of variance and the paired t test . a 2-tailed p value of.05 was used to determine the statistical significance of differences observed between groups and to calculate confidence intervals around differences in sample means and odds ratios . we used the mcnamara test to measure the changes between the groups and their subgroups regard to the length of antibiotic treatment . over half of their children ( 1023 , 51% ) were between the ages of 6 months and 6.9 years , and over half 1,039 ( 52% ) were female . the majority of children ( 1,821 , 91% ) were prescribed penicillin or amoxicillin , allergic or intolerant to penicillin were treated with cephalosporin 25 ( 1.5% ) , erythromycin 109 ( 5.5% ) , and azithromycin 45 ( 2.5% ) , all medication were prescribed twice daily for 10 days , except azithromycin once daily for 5 days . no statistical correlation was found between the type of antibiotics , the children received , or the demographic characteristics and the complications found in the later medical examinations . only 196 children ( 9.8% ) actually completed 10 days of antibiotic treatment . despite having received a prescription from their physician , two hundred and thirteen participants ( 11% ) did not start taking any treatment whatsoever , including those who did not even purchase antibiotics . as shown in table 1 a no statistical correlation was found concerning length frequency and duration , palatability , number of daily dose of treatment , but a statistically significant difference was found between all the subgroups concerning the length of antibiotics treatment ( p < the majority of children ( 1192 , 59.6% ) had 3 days or less of fever , defined as any rectal temperature less than 38.5c or oral temperature less than 37.8c . the majority of children ( 1591 , 80% ) received medication for four to six days at the most ( partially treated subgroup ) . as illustrated in table 1 , the association between the duration of fever and the number of days of treatment was statistically significant ( p < .0001 ) . of the 306 ( 15.3% ) children with clinically diagnosed recurrent tonsillopharyngitis , only 236 ( 12.3% ) had positive gabhs findings on the throat culture taken within 10 to 14 days after conclusion of the primary infection . an additional thirty - four ( 1.7% ) had a positive second study culture ( taken 2130 days after the index positive gabhs culture ) . of note is the fact that the majority ( 156 , 66% ) of the positive study culture at 1014 days were found among the subgroup treated for 7 to 10 days . no such positive results were found in the subgroup treated for 1 up to 3 days . furthermore , no positive gabhs throat cultures were found on the second study culture in the untreated group . the majority ( 26 , 76% ) of positive gabhs cultures were in the mostly treated subgroups . as illustrated in table 2 , these findings were both statistically significant ( p < .0001 ) ( table 2 ) . cervical lymphadenitis , acute otitis media , and impetigo were the only suppurative complications noted . 110 ( 5.5% ) children developed cervical lymphadenitis , most ( 52 , 47% ) among the 6 to 7 days treatment subgroup and 33 ( 33% ) among the 4 to 5 days treatment subgroup , a significant difference among the treatment subgroups ( p < .0001 ) . additionally , no children developed nonsuppurative complications during 10 years of followup nor did any of the children develop iga nephropathy during the follow - up period . furthermore , they were no association between the five modified centor criteria and development of complication , even when stratified by type of antibiotics or the season of the year . altogether , 304 ( 15% ) new onset cases of acute otitis media ( aom ) were diagnosed within 30 days of the initial diagnosis . however , only 31 ( 10% ) of those were in the untreated subgroup , as compared to 141 ( 46% ) in the 7 to 10 days treatment subgroup , 98 ( 38% ) in the 4 to 6 days treatment subgroup and 98 ( 33% ) in the 1 to 3 days subgroup , a statistically significant difference among the all treatment subgroups ( p < .0001 ) . attempting to elucidate the possible causes for the differences between the recurrence of gabhs and the length of antibiotic treatment or clinical score on enrolment or illness severity , a multivariate stepwise logistic analysis was performed . the duration of fever was the most significant predictor for such recurrence , age under 6 years being less significant , while treatment for 7 to 10 days had no significant influence on the recurrence of gabhs . of note is that the contribution of the duration of fever was apparent even after controlling for the concomitant influence of age , gender , medical history , single- or two - parent home , type of antibiotics or the season of the year , and concomitant illnesses , such as , conjunctivitis , otitis media , upper respiratory infection , gastroenteritis , or lymphadenitis , which may have otherwise explained the influence of the length of treatment in relation to those illnesses ( table 3 ) . this study found a very poor parent / child compliance to antibiotic treatment prescribed for symptomatic , culture - positive gabhs tonsillopharyngitis . 11% of children did not start taking any treatment at all , and only 10% completed a full course of treatment . the reason for this low rate of compliance is unclear , but it coincides with other reported studies [ 14 , 16 ] . we speculate that a large proportion of lack of compliance is the parent 's sensation that antibiotics are potentially dangerous and overprescribed [ 18 , 19 ] . despite this poor compliance , in our study as in others , there was a very low rate of suppurative complications . furthermore , despite the poor compliance , we found no increase in the incidence of acute rheumatic fever , the most dreaded complication of gabhs , in our patients . in fact , since the year 2000 , the incidence of rf in our region has declined from 2.2 per 100,000 to 0.2 per 100,000 in 2008 , according to the epidemiological department of the israeli ministry of health . this is in concordance with other developed countries , including , the united states , where the original recommendation for 10 days of antibiotic treatment of gabhs originated . in that country , currently there are only 10 cases of rf per 100,000 patients with gabhs pharyngitis , and only 1 case per 10,000 patients with acute rheumatic fever develop rheumatic heart disease [ 2022 ] . in fact , concomitant with the increased use of antibiotics , recurrence of gabhs in the usa rose from 9% and 10.7% in the years 1975 to 1979 , respectively , to 25.9% and 37.5% in the years 1995 to 1996 , despite the decline in acute rheumatic fever . 14% of patients in our study had a recurrent infection , proven by a gabhs - positive throat cultures . this percentage is similar to other studies which found that penicillin failed to eradicate gabhs from the throat in approximately 13% to 26% of the patients evaluated [ 23 , 24 ] . the majority of recurrences in our study were in the younger group ( mean age of 10.2 years , and 60% of them younger than 9.9 years ) . this is consistent with other published studies where such recurrences were more frequent among children aged 1 to 8 years than among children aged between 13 and 19 years [ 24 , 25 ] . historically , prescribing 10 days of oral penicillin began in the 1950s , substituting the intramuscular injections of long - acting parenteral penicillin , based on surrogate markers of eradication of gabhs from the tonsillopharynx . however , no study has conclusively proven that this practice unequivocally prevents acute rheumatic fever [ 26 , 27 ] . even though orally prescribed penicillin appeared to be equally effective for clinical and laboratory resolution of signs and symptoms , it is difficult to administer and expensive , considering the staggering financial burden of approximately 140 office visits per annum per 1,000 children younger than 15 years [ 28 , 29 ] . substituting azithromycin or cephalosporins for penicillin was found to produce better bacteriological and clinical results and also required a shorter course of treatment [ 30 , 31 ] . our results may not apply to adults , sick people , or chronic gabhs carriers . it does not address the optimal length of treatment required to achieve appropriate eradication of the microbe or whether complete eradication is required at all . the method of pill / doses counts is not a good measure of adherence , but due to it simplicity and empiric nature it was found adequate for this study . we were unable to assess the true variation of the incidence of acute rheumatic fever , due to the fact that this disease has been practically eradicated from our population . our data suggest that the large majority of parents / patients stop administering antibiotics to their children who suffer from gabhs prior to the completion of the recommended course . a more judicious use of antibiotics would promote and improve compliance , cut costs , and prove more convenient to parents and children alike .

background . uncertainty exists concerning the necessity of 10-day antibiotic treatment of group a beta hemolytic streptococcus ( gabhs ) pharyngitis . objective . to assess the incidence of gabhs recurrence and suppurative and nonsuppurative complications in relation to compliance . methods . ( design ) . prospective cohort observational study . ( subjects ) . 2,000 children aged 6 months to 18 years with sore throat and positive gabhs culture . ( main outcome measures ) . recurrence of symptomatic culture positive gabhs pharyngitis , incidence of suppurative , and long - term , regional , nonsuppurative complications of gabhs pharyngitis , over a ten year period . results . 213 ( 11% ) of the children received no treatment . most children received antibiotics for only 46 days ( in correlation with the duration of fever , which in most cases lasted up to 3 days ) . three hundred and six ( 15.3% ) children had clinically diagnosed recurrent tonsillopharyngitis ; 236 ( 12.3% ) had positive gabhs findings within 10 to 14 days and thirty - four ( 1.7% ) within 2130 days after the index positive gabhs culture . the remaining 1.3% had no positive culture despite the clinical findings . almost all recurrences [ 236 ( 11.6% ) ] occurred within 14 days and 156 ( 7.6% ) in the fully treated group . the presence of fever during the first 3 days of the disease was the most significant predictor for recurrence . other predictors were the age younger than 6 years and the presence of cervical lymphadenitis . no increase in the incidence of nonsuppurative or suppurative complications was noted during the 10-year follow - up period , compared to the past incidence of those complications in israel . conclusions . our data suggests that the majority of children discontinue antibiotics for gabhs tonsillopharyngitis a day or two after the fever subsides . the incidence of complications in our study was not affected by this poor compliance .

neurofibromatosis affects 1:3,000 individuals , and characterized by largely benign but often debilitating tumors that grow in the nervous system . its course is unpredictable : it can cause a variety of benign nerve tumors including plexiform , dermal , and optic glioma tumors ; in some cases malignant peripheral nerve sheath tumors can develop in the plexiform tumours . down 's syndrome is one of the most common and easily recognized genetic conditions in humans . the estimated prevalence in the united states is approximately 15 per 10,000 live births ( ie , 1 out of every 700 ) most often , it is the result of nondisjunction on chromosome 21 during maternal meiotic division . we herein present the case of a 17-year - old boy with complaints of skin lesions over the back associated with mild itching since 3 months . he was a known case of down 's syndrome with a history of seizures in childhood [ figure 1 ] . the lesions gradually increased in size and number , and similar lesions started developing over his forehead since 23 weeks . on examination , multiple skin colored papules of varying size were present over the entire back and the forehead . velvety thickening of the skin and hyperpigmentation of the axillae suggestive of acanthosis nigricans was present . the characteristic features of down 's syndrome , including simian crease , mongoloid facies , and mental retardation were present . canities and a solitary keloid over the chest were also seen apart from the clinical features of down 's syndrome . on oral examination , scrotal tongue , abnormal dentition , and other investigations such as ct scan brain , 2d echo , and ecg were normal . histopathology of the nodule from the back revealed focally thinned out epidermis with intact basal layer ; the papillary dermis showed a mild perivascular infiltrate . deeper dermis showed a benign spindle cell proliferation suggestive of neurofibromatosis [ figure 3 ] . physical appearance of down 's syndrome ( a ) neurofibromas ( b ) sebaceous cyst ( c ) acanthosis nigricans , and axillary freckling ( d ) scrotal tongue focally thinned out epidermis with intact basal layer with the papillary dermis showing a mild perivascular infiltrate . the presentation of a patient with two unrelated genetic disorders is uncommon , although not statistically impossible . however , in two of these reports , a third medical condition was also present . in one report , breast cancer was reported . in the other , our patient had down 's syndrome , neurofibromatosis , dental anomalies , and ocular defects and keloids . in the large majority of cases , trisomy 21 there is no current evidence to support the idea that this is anything other than a chance occurrence . the two conditions are not related , and the likelihood of a person being born with these two conditions is approximately 1 in 2,700,000 births . they however overlap in their manifestations . both are associated with intellectual impairment to differing degrees . macroglossia occurs in both conditions , as may facial , dental , and occlusal abnormalities . hearing and speech are affected in both conditions , as may the ability to maintain an acceptable level of oral hygiene . mutations in the gene results in abnormal control of cell growth , differentiation , and aberrant myelination . our patient with of neurofibrmatosis type 1 with down 's syndrome is the first such to have a keloid and sebaceous cyst apart from myopia and dental anomalies . the unpredictable nature and course of the two genetic disorders along with multiple acquired conditions in this patient make it difficult for patients , teachers , care givers , and medical / dental providers to create and maintain long - term care plans . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

we report a patient with down 's syndrome and neurofibromatosis who presented with a keloid , sebaceous cyst and acanthosis nigricans , along with dental and ophthalmological defects . the coexistence of neurofibromatosis type 1 and down 's syndrome which are two unrelated genetic conditions is itself a rarity .

a 48-year - old man presented with acute lower extremity weakness and back pain developed while he was working in a crawl space hyper extending his back . muscle strength was 4/5 in right iliopsoas and hamstrings , and 0/5 in the remaining muscles of right and all muscles of the left lower limb . bilateral knee and ankle reflexes pinprick sensation was decreased over left leg up to t12 level , and right l3 through s2 dermatomes . vibration and proprioception sensation were normal . at a peripheral hospital , spine mri was unremarkable on day 1 . spine mri on day 2 showed a non - enhancing intra - axial bright t2-weighted signal preferentially involving the anterior part of the cord extending from the conus to t11 ( figure 1a&b ) . on day 4 , ncs showed normal sensory and motor studies except an unrecordable right peroneal motor response ( chronic , post ankle fracture ) . f - waves were not obtainable over the peroneal and tibial nerves ( figure 2a - c ) . cerebrospinal fluid protein , glucose , cell count , cytology and flow cytometry were normal . extensive metabolic , infectious , vasculitis , cardiac , malignancy and thrombophilia workup were unremarkable . he initially received intravenous methylprednisolone and plasma exchange for a presumed transverse myelitis . on day 10 , mri showed smooth enhancement of the ventral nerve roots and conus ( figure 1c&d ) . the diagnosis was revised to conus medullaris infarct based on the distribution and evolution of mri findings . . left peroneal and bilateral tibial compound muscle action potential ( cmap ) amplitudes decreased . left peroneal f waves reappeared with poor persistence ( 6.7% ) and prolonged latency ( 64 ms ) . although cmap amplitudes remained decreased , all f - waves were obtainable with a slightly prolonged latency and normal persistence ( figure 2g - i ) . spine mri axial ( a ) and sagittal ( b ) t2-weighted mri obtained 2 days after symptoms onset demonstrating t2 hyperintense signal within the conus extending to t11 . axial t2-weighted ( c ) and contrast enhanced ( d ) mri obtained 10 days after symptom onset demonstrating contrast enhancement of the anterior part of the conus and nerve roots . f - waves ( a - c ) f responses recorded by stimulating the left peroneal and bilateral tibial nerves at day 4 , ( d - f ) day 18 , and ( g - i ) day 56 after disease onset . m - direct motor response ; ml - minimal latency ; mv - millivolt , nr - no response ; ms - millisecond f - wave is not considered part of the workup in conus medullaris infarct . however , in the absence of upper motor neuron signs , especially with a normal early spine mri and absent f - wave , confusion about the diagnosis may arise . in a previous case report , f waves were absent in the hyperacute stage ( first 4 hours ) of anterior spinal cord infarct.2 in our patient , f waves were absent at the time of the first nerve conduction studies ( ncs ) ( day 4 ) and they may have been absent from the onset . hiersemenzel et al3 described reappearance of f waves after the stage of spinal shock in 12 patients with a traumatic paraplegia above t10 level , and attributed the early absence of f waves to reduced excitability of motor neurons at the stage of spinal shock . in our patient , the mechanism of injury was ischemia ( rather than trauma ) and the injury level was at the conus ( lumbosacral spinal cord segments ) with involvement of the motor nerve roots that showed enhancement on the follow - up mri . recovery of left tibial f waves on day 18 coincided with development of hyperreflexia in the same leg . in contrast , at the time of reappearance of right tibial f waves on day 56 , right ankle reflex was absent . thus , we hypothesize that the absent f - waves in the acute stages of conus medullaris infarct might have been due to temporarily unexcitable ahc as a result of spinal shock , spinal cord edema , and possibly oligemia ( figure 1 ) . we would not expect f waves to be recordable in the setting of a diffuse infarction of all ahc in the spinal cord segments subserving the tested nerve . we are not aware of any reports describing f wave as a prognostic indicator of conus medullaris infarct . we postulate that persistently absent f waves , beyond the stage of spinal shock ( 2 - 4 weeks ) , in conus medullaris infarct indicates severe damage to ahc in the lumbosacral region and poor chance for renervation through collateral sprouting . all f waves had reappeared by day 56 , at least 8 weeks before he regained walking . the area of infarct involved the ventral part of the spinal cord supplied by the anterior spinal artery . we suspect the mechanism of infarct was secondary to a hyperextension injury similar to the non - traumatic spinal cord infarct described in novice surfers ( surfers myelopathy ) . in such cases , the postulated mechanisms of ischemia include transient arterial compression during a prolonged back hyperextension in the setting of poor collaterals , vasospasm or thrombosis of the artery of adamkiewicz , and avulsion of perforating blood vessels.4,5 the prognosis of surfers myelopathy varies from complete recovery to persistent paraparesis.4,5 a normal mri in the first few days does not rule out spinal cord infarct . in reported case series , the earliest changes ( bright t2-weighted signals ) appeared after 1 - 2 days , followed by enhancement appeared within the first week and peaked at 14 -21 days.6,7 cauda equina enhancement post spinal cord infarct has been described in a few case reports and attributed to a possible disruption of blood - nerve barrier.2,8,9 in conclusion , further studies are required to test our hypothesis that reappearance of tibial and peroneal f waves has a prognostic value in predicting walking in patients with conus medullaris infarct .

absent f wave in the stage of spinal shock has been described in cases of traumatic spinal cord injury . the role of f wave in predicting prognosis after conus medullaris infarct has not been described . we describe herein a middle aged - man with a conus medullaris infarct . both tibial and peroneal f waves were absent on day 4 . the left tibial f wave reappeared in the following study on day 18 . all f waves reappeared on day 56 at which time the patient was still wheelchair bound . he regained walking on day 105 . we hypothesize that reappearance of initially absent f waves post conus medullaris infarct is a good prognostic sign for the return of ambulation . the applicability of this observation requires further research . we also discuss clinical and diagnostic caveats in this case .

west nile encephalitis is a mosquito - borne disease endemic to africa , asia , australia , and europe and the west nile virus is a single - stranded rna virus which belongs to the japanese encephalitis serogroup of flaviviruses ( 1 ) . it could be accompanied with various neurologic symptoms and signs including headache , mental confusion , optic neuritis , and muscle weakness ( 2 , 3 , 4 ) . after the first two cases of west nile encephalitis were diagnosed in 1999 in new york ( 5 ) , many neurological abnormalities were noted in patients of west nile encephalitis , and the flaccid muscle weakness is a distinctive feature of this disease ( 2 , 3 , 6 ) . until now , west nile encephalitis was never diagnosed in korea , and it is the first report of west nile encephalitis in korea . a 58-yr - old man was admitted with headache for 2 months in june 2012 . the patient had been on a business trip in guinea , west africa since 7 months ago . he denied other symptoms such as nausea , vomiting , neck stiffness , and dizziness . the headache and cognitive impairment had been worsening and accompanied general weakness and weight loss 10 kg for 2 months . he had no hypertension , cardiac diseases , chronic renal disease , and liver disease . he had a 20 pack - year history of smoking and no recent alcohol intake . the patient denied using any medications except for antimalarial drug ( sulfadoxine 500 mg , pyrimethamine 25 mg , artemether 80mg / ml ) and analgesics in africa . at admission to our hospital , he was afebrile ( 36.4 ) and his vital signs were blood pressure 121/78 mmhg , heart rate 97/min , and respiratory rate 20/min with normal findings of physical examinations . findings on initial neurologic examinations were normal except minimental state examination ( mmse ) with score 20 . admission complete blood count revealed hemoglobin of 12.7 g / dl , leukocyte count of 16.2 10/l with 79% neutrophils , and a platelet count of 388 10/l . the patient 's calcium level , chemistries , and liver function tests were normal and crp was slightly increased . analysis of cerebrospinal fluid ( csf ) demonstrated 0 rbc/l , 214 wbcs/l ( with 80% lymphocytes and 12% neutrophils ) , a protein level of 161.9 mg / dl , a glucose level of 93 mg / dl and ada level of 4.8 . a brain magnetic resonance imaging ( mri ) revealed multifocal patchy t2 high signal lesions in both temporal lobes , basal ganglia , internal capsule posterior limb , and corona radiata , which suggested encephalitis . electroencephalogram showed frequent semirrhythmic medium amplitude delta slowing on both hemispheres , with frontal dominancy . on hospital day 1 , his body temperature was 38.0 and malaria test was negative . follow up csf tapping was done on hospital day 3 and 6 rbc/l , 340 wbcs/l ( with 67% neutrophils and 28% lymphocytes ) , a protein level of 164.6 mg / dl , a glucose level of 70 mg / dl and ada level of 6.0 . the patient complained of both leg weakness and the motor strength of bilateral lower extremity was 4+/4 + . he also complained of both leg pain and paresthesia , deep tendon reflex on both lower extremities were areflexia . there was no remarkable finding on nerve conduction study ( ncs ) and needle electromyogram ( emg ) , but arachnoiditis and myelitis were observed on lumbar spine mri ( fig . polymerase chain reactions ( pcr ) for hsv , vzv , ebv , cmv , enterovirus , tuberculosis , and mycoplasma pneumonia were negative in csf . serological test for brucellosis , lyme disease , leptospirosis , hantann virus and tsutsugamuchi disease were negative at admission an additional serological study for specific antibody for west nile virus was done , the total antibody titer of west nile virus was 1:337 , and follow up titer after 2 weeks was 1:1,632 in serum by plaque reduction neutralization test . treatment with interferon- 3mu was started and maintained for 2 weeks . follow up csf tapping was done after one month later and 9 rbc/l , 9 wbcs/l ( with 16% neutrophils and 67% lymphocytes ) , a protein level of 99.9 mg / dl and a glucose level of 66 mg / dl . encephalitis with severe muscle weakness is a common symptom and may provide a diagnostic clue . to confirm a diagnosis , it needs identification of west nile virus in serum or csf . igm antibody is usually detectable only during a few days of illness ( 7 ) . another method is the identification of a change by a factor of four or more in the antibody titer or virus - neutralization tests ( 7 ) , the case was positive for the virus - neutralization tests . serological test and pcr for other infectious agents were all negative and autoimmune encephalitis such as nmda receptor encephalitis was excluded by clinical symptoms ( 8) . many studies indicated that the weakness was probably caused by infection and inflammation in the anterior horn cells of the spinal cord and brainstem like poliomyelitis ( 6 , 9 , 10 ) . electrophysiologic studies showed reduced compound muscle action potentials , and normal sensory nerve action potentials , which is distinguished from guillain - barre syndrome ( 3 , 6 , 9 , 10 ) . a few autopsy studies demonstrated that the inflammatory changes with severe leukocytic inflammation of the anterior horns with striking loss of motor neurons ( 4 , 11 ) . but this case did not present any abnormalities in the electrophysiologic study . he complained about severe sensory symptoms with leg pain and arachnoiditis was observed in lumbar mri . only a few reports noted a caudal equine enhancement ( 4 ) and weakness due to acute radiculitis ( 12 ) . it 's hard to say that his weakness was caused by only radiculopathy , because paralysis of the west nile encephalitis generally results from anterior horn cell damage . but in this case , radiculopathy also contributes the motor weakness of the west nile encephalitis . at present there is no established treatment for west nile encephalitis and basic treatment is a supportive care . but because it 's a fatal disease , sometimes it was treated with plasmapheresis or administration of iv immunoglobulin ( 5 ) . interferon- has been reported to inhibit west nile virus replication , and some successful cases were reported using of interferon- to treat west nile encephalitis ( 13 ) . korea is not an endemic area of west nile virus , but an increasing overseas trip poses a risk of the influx of overseas diseases and may contribute to the global spread of infectious diseases .

west nile encephalitis was first identified in 1937 , but until now , it was never diagnosed in korea . a 58-yr - old korean man was admitted with headache and cognitive dysfunction . the patient had been on a business trip in guinea . cerebrospinal fluid ( csf ) showed pleocytosis . the patient complained of both leg weakness , and arachnoiditis and myelitis were observed on lumbar magnetic resonance imaging ( mri ) . a specific neutralizing antibody for west nile virus was positive in serum . after a treatment with interferon- 3mu , follow up csf findings recovered completely after 3 months later . the first case of west nile encephalitis in korea was imported from guinea , and was cured successfully.graphical abstract

pathogens have evolved ingenious strategies to circumvent the host immune response as part of the constant evolutionary process - taking place in all living organisms . chief among these strategies is the prevention of the inflammatory response or seizure of the anti - inflammatory mechanism in place to protect tissue integrity . the manipulation of macrophage ( m ) polarization is one of the main targets to accomplish this , since this antigen presenting cell represents the first line of an active defense system in the host , and if successfully done , it can then undermine adaptive immunity ( benoit et al . , 2008 ) . m polarization is a dynamic process governed by mechanisms dictating their tissue distribution and functional capacities in response to endogenous and exogenous signals ( martinez et al . , 2009 ) . polarized ms are broadly classified into two groups : classical ( m1 ) and alternative ( m2 ) activated . on one hand , m1 program is a direct response to type-1 inflammatory conditions ( e.g. , ifn- ) and pathogen challenge , and it has been associated to resistance to intracellular pathogens and to some form of tumors . on the other hand , the m2 program is driven by type-2 inflammatory signals such as il-4 and il-13 ( m2a ) ; immune complexes , toll - like receptors ( tlrs ) agonists , or il-1 receptors ( m2b ) ; and immunosuppressants including il-10 , transforming growth factor- ( tgf- ) or glucocorticoids ( m2c ; table 1 ) . m2 ms participate in diverse activities including the suppression of inflammation , enhancement of phagocytosis , promotion of tissue remodeling and repair , elimination of parasites , and unwanted tumor angiogenesis ( sica et al . , 2008 ; martinez et al . furthermore , it is becoming clear that m polarization supports different , and in some cases , opposing biological functions , that influences tissue homeostasis , and numerous pathological situations , including infectious diseases ( benoit et al . given the pivotal role ms play as sentinels of the immune system , they represent ideal cell targets for subversion by successful intracellular pathogens . priming stimulus for the classical ( m1 ) and alternative ( m2a - c ) activation of macrophages . the purpose of this short review is not to provide a comprehensive summary of m polarization ; others have recently reviewed this growing research area ( martinez et al . also , we will not address the multiple ways by which the pathogens in question circumvent the immune system , as there are excellent reviews covering this subject ( deretic et al . , 2004 ; carter and ehrlich , 2008 ; meena and rajni , 2010 ; hajishengallis and lambris , 2011 ) . instead , we will focus exclusively on the significance of m polarization in the context of pathophysiology caused by mycobacterium tuberculosis ( mtb ) and human immunodeficiency virus ( hiv ) . the world health organization reports tuberculosis ( tb ) is still one of the leading causes of death due to a single infectious agent ( mtb ) with 1.7 million deaths and 9.4 million new cases in 2009 , and estimates that about one - third of the human population may be latently infected ( who global tuberculosis control report 2010 , 2010 ) . active tb may occur directly after infection or through the reactivation of latent infection that is confined in granulomas . the elaboration and maintenance of granulomas depends on a dedicated immune response , which is not fully understood . recently , however , it was demonstrated mycobacteria exploits m activation to turn the granuloma into an effective tool for pathogenesis ( davis and ramakrishnan , 2009 ; volkman et al . , 2010 ) . therefore , a better understanding of m polarization during mtb infection might yield further clues about how mtb circumvents the immune system . as aforementioned , m polarization is mainly driven by type-1 and type-2 inflammatory signals ( table 1 ) . type-1 inflammatory cytokines are essential in the defense against mtb since their expression often correlates with efficient anti - mtb immune responses , and genetic deficiencies of these factors lead to increased tb susceptibility ( quintana - murci et al . , 2007 ) . ifn- drives the m1 program characterized by m capacity to kill most mycobacteria and restrict the replication of the remainder ( ehrt et al . , 2001 ) . the early phase of the anti - mtb immune response is marked by m1 m polarization in multiple animal models and reminiscent of the clinical data collected from patients with active tb ( figure 1 ; benoit et al . , 2008 ) . at the transcriptome level , the gene modulation induced by mtb in ms highly overlaps , and in some cases synergizes , with that induced by ifn- to establish the m1 phenotype ( ehrt et al . m1 m polarization is evident in mice between 7 and 30 days after mtb infection when high levels of ifn- and inos are also detected within this structure and around the alveolar compartment ( redente et al . , 2010 ) . all in all , polarization of m1 ms is part of the common host response against intracellular bacteria characterized by high expression of inos and consequent nitric oxide ( no ) production ( characteristic of murine models ) , secretion of pro - inflammatory cytokines and chemokines , release of proteolytic enzymes and anti - microbial peptides , enhanced phagocytosis , and development of a toxic intracellular environment reflected in the fusion of microbial phagosomes with acidic and hydrolase - rich lysosomes ( ehrt et al . , 2001 ; deretic et al . , 2004 ; martinez et al . , 2009 ; cairo et al . , 2011 ; murray and wynn , 2011 ) . it remains to be demonstrated whether transcription factors [ e.g. , p65 and interferon regulatory factor ( irf5 ) ] or regulators ( e.g. , ship1 ) that dictate the m1 program of macrophage polarization also play a role in tb infection ( martinez , 2011 ) . considering this hostile environment created by m1 ms , it is not surprising mtb has evolved strategies to interfere with m1 polarization . indeed , mtb inhibits ifn- activation of ms by secreting virulence factors such as lipoarabinomannan that halters phagosome maturation , or early secretory antigenic target-6 ( esat-6 ) that prevents the activation of nf-b and ifn- regulatory factors downstream of tlr-2 ( deretic et al . , 2004 ; benoit et al . , 2008 ) . indirectly , mtb blocks m1 polarization by the transcriptional inhibition of ifn--responsive genes through a bystander effect involving il-6 ( sibley et al . , 1990 ; benoit et al . , 2008 ) . a model illustrating the putative involvement of macrophage polarization during mtb or hiv-1 infection . perhaps the best strategy to avoid the challenges posed by m1 ms is to shift their program into m2 ms . tb susceptibility parallels with elevated levels of type-2 inflammatory signals ( e.g. , il-4 , il-13 ; kahnert et al . , 2006 ; raju et al . , 2008 ; almeida et al . , 2009 ; schreiber et al . , likewise , high levels of il-10 ( mostly derived from ms ) correlate with active tb patients ( barnes et al . , 1993 ; verbon et al . , 1999 interestingly , the predominant type-2 inflammatory environment shifts back to type-1 after successful treatment of pulmonary tb in infected patients ( verbon et al . , 1999 ; 2008 ) . these observations in humans parallel with those reported in mtb - infected mice ; that is , there is an early type-1 immune response characterized by ifn- during the first 3 weeks after infection , followed by a type-2 immune response that contains high levels of il-4 ( figure 1 ; orme et al . , 1993 ) . a type-2 inflammatory environment drives the m2 program that renders ms immunomodulatory and poorly microbicidal ( raju et al . , 2008 ; , this seems to be the case in mice since m2 ms displayed a diminished inflammatory response to mtb as reflected by a reduced no production and increased of iron availability , suggesting these phagocytes offer a permissible intracellular environment for bacterial replication ( kahnert et al . , 2006 ) . indeed , ifn--induced no production is essential for host survival with respect of experimental tb , while iron - starvation is key to bacteriostasis ( ehrt et al . , 2001 ; forbes and gros , 2001 ; cairo et al . , 2011 ) . it remains to be seen if mtb also influences the expression level of kruppel - like factor 4 ( klf4 ) or any other transcription factor / regulator recently shown to be critical for both the establishment of the m2 program and the inhibition of m1 polarization ( e.g. , stat6 , cot / tpl2 ; liao et al . , it has been demonstrated that both il-4 and il-13 inhibit autophagy in m1 ms resulting in enhanced survival of mtb , an impairment that might also extend to m2a ms ( harris et al . , 2007 ) . at the granuloma level in mice , inos continues to be expressed within this structure but a significant shift from m1 toward m2 ms [ inos arginase-1 ( arg1 ) ] occurs around the alveolar compartment starting at day 35 and continuing up to day 60 after mtb infection , accompanied by high levels of type-2 inflammatory signals ( ly et al . 2010 ) . given the development of fibrosis is a key characteristic of caseous granulomas during mtb dissemination , and that m2 ms have been implicated in the inhibition of fibrosis development , the shift from m1 into m2 program might represent an attempt by the host to halter the pathophysiology caused by mtb or a microbial strategy to shield from immune attack ( dorhoi et al . , 2011 ) . for instance , mtb might influence all tlr - dependent signaling by targeting dc - sign to induce il-10 and counteract the pro - inflammatory response , as shown in dendritic cells ( geijtenbeek et al . likewise , the mannosylated lipoarabinomannan from mtb enhances the production of il-10 and other immunosuppressants through recognition by the mannose receptor ( mr ) in immature dendritic cells ( chieppa et al . , 2003 ) . although alveolar ms express dc - sign and mr , their role in m2 ms has yet to be demonstrated ( chroneos and shepherd , 1995 ; tailleux et al . , 2005 ) ( 2009 ) reported mtb - induced il-10 in ms promotes the m2 polarization program displaying diminished anti - mycobacterial effector mechanisms . indeed , m - specific overexpressing il-10 transgenic mice were indeed susceptible to mtb infection , displayed a specifically suppressed il-12 in infected tissues , and were characterized by lung ms with a m2 phenotype permissive to mtb infection ( schreiber et al . , 2009 ) . these observations correlate well with another study in mice where mtb was shown to promote its survival and ability to cause disease through a myd88-dependent induction of arg1 . arg1 inhibits no production by ms by competing with inos for arginine ( the common substrate ) , thus rendering these cells permissive to mtb infection ( el kasmi et al . , 2008 ; hajishengallis and lambris , 2011 ) . taken together , these observations suggest the reprogramming toward m2 ms by il-10 , and other immunosuppressants such tgf- and glucocorticoids ( hernandez - pando et al . , 2006 ) , might be yet another adaptation by mtb to survive and thrive inside of ms ( figure 1 ) . however , it should be noticed that this phenomenon might also represent a control mechanism by the host to preserve the integrity of mucosal sites as uncontrolled type-1 inflammatory responses against mtb result into lung immunopathology ( hernandez - pando et al . , 2006 ; human immunodeficiency virus-1 is another successful intracellular pathogen responsible for a worldwide pandemic . according to 2009 estimates by the united nations , there were about 33.2 million people worldwide living with hiv-1 infection and 2.6 million individuals had been newly infected ( cohen et al . , 2011 ) . in the absence of antiviral therapy , hiv-1 infection progresses through acute and asymptomatic stages leading to the eventual failure of the host immunological functions and acquired immunodeficiency syndrome ( aids ) . a reason is that hiv-1 targets cells from the mononuclear phagocyte lineage that drive an effective antiviral response and simultaneously serve as reservoirs of latent or productive infection ( goodenow et al . , 2003 ) . among these cells , ms are critical to pathogenesis because they contribute to early transmission , systemic dissemination , and persistence of hiv-1 . indeed , hiv-1 evades immune surveillance by hiding and thriving inside ms despite anti - retroviral treatment , and when infected , they persist for months displaying insensitivity to viral cytopathic effects . in addition , ms continuously secrete high level of viral particles over prolonged time periods by storing assembled virus in specialized endosomal compartments ( orenstein et al . , 1988 ; benaroch et al . , thus , they represent powerful long - term viral reservoirs ( goodenow et al . , 2003 ; carter and ehrlich , 2008 ; herbein and varin , 2010 ; cohen et al . , 2011 ) . in light of recent evidence suggesting that m1 and m2 ms influence hiv-1 pathogenesis , there is a surging interest to study the viral effects in m polarization . in vitro , this m response includes production of type-1 pro - inflammatory cytokines ( ifn- , il-2 , il-12 , tnf , il-1 , il-6 , il-18 ) and chemokines ( ccl3 , ccl4 , mip- , mip- , rantes ) , increased no and respiratory burst , up - regulation of mhc - ii molecules , and down - regulation of hiv - entry receptors ( e.g. , cd4 , ccr5 , cxcr4 ) , and endocytic receptors ( e.g. , cd163 , cd206 ; swingler et al . , 1999 ; cassol et al . , 2009 , 2010 ; herbein and varin , 2010 ) . although few studies have examined thoroughly hiv - induced polarization of ms in vivo , there is a predominance of ms displaying a m1 phenotype during the acute stage ( figure 1 ; cassol et al . , 2010 ; herbein and varin , 2010 ; cohen et al . , 2011 ) . whether m1 ms are beneficial to the host during hiv-1 infection remains an open question since m functions vary according to the experimental context . for instance , in vitro infection of m1 ms in the presence of ifn- and tnf is associated with a suppression of hiv-1 replication , a sharp decrease in hiv-1 dna synthesis at 48 h , and a decrease in the accumulation of hiv-1 proteins ( cassol et al . , 2009 ) . in addition , other studies demonstrate that m1 ms inhibit viral entry , assembly , and budding , suggesting the m1 program can be beneficial to the host ( cassol et al . , 2010 ; herbein and varin , 2010 ) . however , it is also known that pro - inflammatory signals deriving from m1 ms favor the formation of viral reservoirs with increased transcription of hiv-1 ltr ( long terminal repeat ) , alluding m1 ms might benefit hiv pathogenesis ( cassol et al . , 2010 ; herbein and varin , 2010 ) . this is supported by multiple observations that immune activation driven by ms correlates with hiv-1 pathogenesis ( goodenow et al . , 2003 ; lamers et al . , 2009 ; cohen et al . , 2011 m1hiv since it displays a pro - inflammatory state with increased production of cytokines independently of tlr - pathway . the authors argue that while hiv-1 stimulates ms through a variety of signaling pathways to promote a tailored inflammation in its favor , the tlr recognition of viral replication is impaired and could serve as a viral evasion strategy . given that prolonged pro - inflammatory m activation during chronic hiv - infection contributes , not only to a permissive environment for the formation of viral reservoirs with strong transcriptional activity , but also to disease progression and hiv - induced tissue damage , the proposed m1hiv polarized state may render ms detrimental to the host ( goodenow et al . , 2003 ; brown et al . , 2008 ; lamers et al . , 2009 ) . as hiv - disease progresses from the acute to asymptomatic stage , there is a switch from a type-1 toward a type-2 inflammatory environment ( figure 1 ; vasilescu et al . , 2003 ; becker , 2004 ) . at the transcriptional level , lymphatic tissue microarray analyses from hiv-1-infected subjects at different clinical stages revealed that each stage has a unique gene profile ( li et al . , 2009 ) . the asymptotic phase , however , down - regulates the acute phase gene profile to baseline level while it displays an increased expression of immunosuppressive genes ( li et al . , 2009 ) . based on these immunological systemic changes , it is likely that a polarization switch occurs in ms from a m1 program during the acute phase to the m2 programs through later stages . although there is no overwhelming evidence confirming the abundance of m2 ms in either the asymptotic or aids phase in vivo , the fact cd163 ( a m2 m cell surface marker ) is considered as a potential biomarker for hiv-1 disease progression may allude to the presence of m2 ms in hiv-1-infected individuals ( burdo et al . , 2011 ; tippett et al . , similar to m1 ms , it is not known whether m2 ms benefits the host during hiv-1 infection . in vitro activation of m2a ( il-4-treated ) ms results in inhibition of virus replication ( cassol et al . , 2009 ) . other studies have demonstrated that both il-4 and il-13 down - regulate viral entry receptors and hiv-1 reverse transcription in ms ( cassol et al . , 2010 ) . furthermore , activation of m2c ( il-10-treated ) ms strongly inhibits reverse transcription , transcription of hiv-1 ltr and viral assembly ( herbein and varin , 2010 ) . based on these observations , it might be tempting to conclude that m2 ms are beneficial to host immunity against hiv . however , the progression of aids is characterized by the loss of il-2 and increase of il-10 correlating with hiv viremia ( brockman et al . . moreover , the haplotypes of both il-4 and il-10 genes have been associated recently with aids progression ( vasilescu et al . , therefore , the switch toward a m2 m program might simply be part of a defensive mechanism by the host to control hiv - induced tissue damage since they participate in suppression of inflammation and promotion of tissue repair ( figure 1 ; martinez et al . recently , a functional proteomic analysis of hiv - infected ms in the presence of regulatory t cells showed that a deviation of m1 to m2 m program is associated with neuroprotection in the case of hiv - associated neurocognitive disorders , suggesting m2 ms may curtail the m1hiv polarized activity resulting in tissue damage ( huang et al . , 2010 ) . conversely , the switch toward the m2 m program might also occur as an evasion strategy by hiv to promote its own survival . a recent study demonstrated that hiv up - regulates both programmed cell death ligand 1 ( pd - l1 ) and pd - l2 expression , members of the b7:cd28 family , and pd-1 ligands , in ms ( porichis et al . , 2011 ) . given the importance of these molecules in t cell exhaustion during hiv infection , the ability of il-10 to both activate the m2c m program and induce pd - l1 , and the fact that il-10 production and increased expression of pd - l1 correlate in hiv - infected patients , the authors propose the manipulation of pdl expression in ms as a strategy to evade immune responses ( trabattoni et al . whatever the true role of m2 ms in hiv infection , it is clear they influence the establishment of hiv pathogenesis , and more studies are needed to examine thoroughly hiv - induced polarization of ms in vivo . tuberculosis is the most common opportunistic infection in aids and often used as a clinical parameter for undiagnosed aids cases ( deretic et al . , 2004 ) . while the synergy between mtb and hiv is evident at the clinical level , the mechanisms accounting for it are poorly understood . 2004 ) proposed the interference with endosomal sorting machine as a molecular mechanism contributing to the synergy between these two pathogens . likewise , we envision the pathogenic modulation of m polarization as a cellular mechanism that might influence this synergism . as aforementioned , it is estimated that about one - third of the human population may be latently infected with mtb ( who global tuberculosis control report 2010 , 2010 ) , suggesting that one in three of the 2.6 million people newly infected with hiv-1 in 2009 ( cohen et al . , 2011 ) latent mtb is confined in solid granulomas composed of mainly by ms and t cells that maintain their stability . the coinfection with hiv-1 results in a dramatic increase in the odds of latently infected people progressing into overt tb to a staggering annual risk of 10% ( deretic et al . , 2004 ; swaminathan et al . , hiv - driven immune perturbation , reflected in the loss of cd4 t cells and abnormal low levels of tnf causes the loss of granuloma integrity and efficiency in anti - microbial containment leading to post - primary reactivation state ( paige and bishai , 2010 ) . these events may increase both m necrosis and release of intracellular bacilli accounting for the extrapulmonary tb manifestation diagnosed in patients with hiv - driven immunosuppression ( swaminathan et al . the awakened mtb then might induce m1 ms to drive an excessive tnf response ( together with other mechanisms such as mmp secretion ) to deliberately promote parasitic granuloma formation , resulting in the recruitment of additional nave ms and the tissue pathology ( davis and ramakrishnan , 2009 ; paige and bishai , 2010 ; volkman et al . , 2010 ) . excessive levels of tnf , may not only contribute to the classical symptoms of cachexia in tb , but also to the augmentation of hiv-1 transcription and accelerated formation of viral reservoirs ( deretic et al . in the absence of an efficient adaptive immune response due to hiv - driven impairment , uncontrolled inflammation can result in lung immunopathology , and consequently , the host may induce tissue repair responses . the shift from m1 to m2 m program may become pronounced and prolonged in the sterile attempt to restore tissue integrity , elevating the level of il-10 that is typical of disease progression by both pathogens , and thus contributing to the failure of all immunological functions and clinical collapse . while highly speculative , this scenario highlights the importance to understand m polarization in the context of immune activation and pathogen - driven disease , and its potential to be yet another convergence point targeted by mtb and hiv to circumvent the host immune system . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

in the arms race of host microbe co - evolution , macrophages ( ms ) have been endowed with strategies to neutralize pathogenic challenge while preserving host integrity . during steady - states conditions , ms perform multiple house - keeping functions governed by their differentiation state , tissue distribution , and signals from the microenvironment . in response to pathogenic challenge and host mediators , however , ms undergo different programs of activation rendering them either pro - inflammatory and microbicidal ( m1 ) , or immunosuppressants and tissue repairers ( m2 ) . an excessive or prolonged polarization of either program may be detrimental to the host due to potential tissue injury or contribution to pathogenesis . conversely , intracellular microbes that cause chronic diseases such as tuberculosis and acquired immunodeficiency syndrome exemplify strategies for survival in the host . indeed , both mycobacterium tuberculosis ( mtb ) and human immunodeficiency virus ( hiv-1 ) are successful intracellular microbes that thrive in ms . given these microbes not only co - circulate throughout the developing world but each has contributed to prevalence and mortality caused by the other , substantial insights into microbe physiology and host defenses then rest in the attempt to fully understand their influence on m polarization . this review addresses the role of m polarization in the immune response to , and pathogenesis of , mtb and hiv .

computational probe mapping is frequently applied in structure - based drug design ( sbdd ) to identify potential binding pockets along a protein surface ( i.e. , hot spots ) . however , mapping is usually limited by the implementation of gas - phase minimizations , wherein protein flexibility and solvent competition are ignored . this leads to a rugged potential surface and many local energy minima , where druggable sites are indistinguishable from irrelevant minima . furthermore , predictions based on a static receptor structure often neglect important binding - site features , such as the presence of transient cavities or bridging water molecules . as a result , appropriately modeling the binding potential continues to be a challenge for sbdd . several computational and experimental models have emerged that address the limitations of traditional solvent mapping . the multiple protein structure ( mps ) method addressed the issue of protein flexibility by creating pharmacophore models from consensus mapping of conformational ensembles with solvent probes . while this technique has demonstrated success in mapping the binding sites of pharmaceutically relevant targets , it can not account for desolvation penalties or water - bridging contacts , which may be essential to accurate prediction . the multiple solvent crystal structure ( mscs ) technique uses x - ray crystallography to determine receptor structures in the presence of competing water and organic solvent . this paved the way for fragment - based methods by providing the first experimental identification of preferred sites for probe binding . mscs results have demonstrated high potential for use in drug development and have inspired several new computational approaches . simultaneous protein flexibility and solvent mapping was first implemented in 2009 when molecular dynamics ( md ) simulations were used as a tool to map hot spots . seco et al . performed md simulations with seven proteins that were solvated by a 20% volume / volume ( v / v ) solution of water and isopropanol ( ipa ) . although their method sought to detect binding sites and predict druggability , it did not address aromatic hotspots and was unable to reproduce some of the experimental binding sites . yang and wang have conducted similar studies that included phenol with the alcohol / water mix to address aromatic interactions . another md - based approach , site identification by ligand competitive saturation ( silcs ) , was implemented with a ternary - solvent box composed of 1 m benzene ( bnz ) , 1 m propane , and water to locate hot spots and to reproduce experimental binding sites . the authors introduced a dummy atom with a virtual repulsion term for both bnz and propane in order to solubilize the hydrophobic solvents and prevent probe their fragment maps for bnz along the trypsin surface found abundant local minima that were mapped equally or better than the valid binding site . in addition , guvench and mackerell noted that the repulsive term may also prevent the mapping of secondary binding sites due to the unnatural physical interactions that are enforced between the hydrophobic probes . the most recent technique was developed by bakan et al . , which uses mixtures of water with multiple probes simultaneously ( ipa , acetamide , acetic acid , isopropylamine ) . although each of these md - based techniques have shown some success in identifying binding sites , they were limited in their ability to selectively map hot spots without the use of either a weighting or artificial repulsion term . many spurious minima were also identified . over the past few years , we have worked to develop mixed - solvent molecular dynamics ( mixmd ) using amber , with an aim of solvent mapping across a wide range of targets . instead of simply showing that probes could occupy regions of known binding sites , we sought to establish a stronger foundation that would allow for application of our method to target systems without requiring a priori knowledge of the binding sites . our initial study with mixmd used a 50% w / w solvent box composed of acetonitrile ( acn ) and water , and the results showed that mixmd reproduced binding sites from mscs studies with excellent convergence to the true hot spots and no spurious minima . we were interested in extending the probe set for mixmd to fully enable identification of hydrogen - bonding sites , hydrophobic contacts , protein protein interactions , and aromatic pockets . here , we show what happens when poor parameters are used in mapping a protein . we then use md simulations of water - probe boxes to examine which remain evenly mixed . it is important to identify additional functional sites , particularly aromatic sites , for mixmd to be most useful for sbdd . we also identify additional probe solvents that expand the ability of mixmd to locate important interaction profiles in protein ligand binding : acetone ( ace ) , n - methylacetamide ( nma ) , imidazole ( imi ) , and pyrimidine ( 1p3 ) . we highlight the importance of developing a solid foundation for mapping with md , particularly as it relates to the validation of parameters through detailed evaluation of metrics for probe probe dispersion . solvent probes were selected to represent specific interaction types for use in mixmd ( table 1 ) . parameters for ace , acn , and nma were obtained from studies of liquid solvent with amber . therefore , we explored the suitability of opls parameters , which were developed to work with tip3p water and to reproduce the empirical data for pure liquids . the adaptation of nonbonded parameters from opls for use in amber simply required the conversion of to rmin , where 2 /2 = rmin . this is necessary because boss and amber use different combining rules for van der waals ( vdw ) parameters . a comprehensive list of the parameters used in our mixmd simulations is given in table s1 and figure s1 of the supporting information . pure solvent boxes of no fewer than 200 probe molecules were built using the tleap module in ambertools . md simulations were performed in amber10 through the sander module with shake and a 1 fs time step . each solvent box was subjected to 1000 steps of steepest descent followed by 49,000 cycles of conjugate gradient minimization and then heated over 20 ps from 10 to 300 k at constant volume . two nanoseconds of equilibration were followed by a 5 ns simulation at constant pressure with the temperature held at 300 k by a weak - coupling algorithm . the berendsen coupling method was chosen because it was used in the original publication of amber parameters for the solvent molecules used in this study . the behavior of the pure - probe boxes was used to confirm proper behavior in our setup . the final boxes of organic solvent were also used in the setup of the mixed boxes . mixed - solvent boxes were created in tleap by solvating a single probe molecule with a layer of tip3p water and then a layer of the equilibrated box of pure , organic solvent . the size of the outer box of probes is adjusted to achieve the desired solvent ratio . at least 2500 water molecules and the equivalent mass of probe molecules necessary to achieve a 50% w / w solution were used in order to ensure that realistic solvent behavior would be observed ( table s3 , supporting information ) . each layered mixed - solvent box underwent the same procedure for minimization , heating , equilibration , and production simulation as described for the pure - solvent boxes above . it should be noted that berendsen s coupling method can sometimes be problematic for simulations of mixtures because differential heating can occur for different components of the system . our use of the method with the small boxes could be considered a worst - case scenario , where probes may ( or may not ) have a small bias to aggregate . any mixture of water and probe solvent that displays homogeneous behavior under these circumstances is likely to be robust to a wide range of applications . in our examination of thermolysin with ipa+water , we chose the andersen coupling method to be consistent with our other published applications of mixmd . there is no bias from the temperature coupling that can cause the differences seen in the two parameter choices for ipa when used in mixmd of thermolysin . we characterized the distribution of probe density by computing atom atom radial distribution functions ( rdfs ) with the radial command in ptraj . the final 1 ns of production time was used to calculate the rdf with a bin size of 0.1 . when the solvent probes are fully solubilized into water , they are well dispersed throughout the solvent box , and the rdf converges toward unity at the vdw cutoff . when phase separation occurs in simulations of mixed solvent , too many probes and too little water occupy the local microenvironment around each probe . this makes the local environment too dense with organic probes , and the rdf will not converge to unity within the vdw cutoff . accordingly , we used these rdf descriptions of mixed vs aggregated solvent systems to evaluate the structure of each mixed - solvent box . the carbon carbon ( rcc ) , carbon oxygen ( rco ) , carbon nitrogen ( rcn ) , nitrogen nitrogen ( rnn ) , nitrogen oxygen ( rno ) , and oxygen oxygen ( roo ) distributions were assessed between probe probe , water water , and probe water as was relevant for each probe molecule . for brevity , cumulative density distributions of roo for water water distances in mixed solvent were also examined to determine the number density of water within the volume sphere . for the case of a 50% w / w mixed - solvent box , equitable distribution of solvent probes and water within the system will give a cumulative plot for roo of water the distribution of water molecules relative to one another is less than in pure solvent because obviously the presence of a miscible probe reduces the number of water molecules that can occupy the local microenvironment around each water . earlier papers ours included have used the behavior at the edges of the simulation box to prove even mixing of probes and water . the reasoning was that the box edges are far from the protein , outside the vdw cutoff , and should have minimal bias in the ratio of probe to water . if the edges of the box had the same ratio as the setup solvent box , then there was even mixing . however , taking a step back to these basic simulations of solvent boxes provided a better means of measuring mixing : the use of a rdf . rdfs of the solvents relative to one another can be calculated for simulations of proteins , and we recommend they be used in all mixed - solvent simulations to judge whether the behavior of the probes and waters are reasonable . the rdf , or pair correlation function g(r ) , can be used to describe the structure of the solvent based on atom / molecule pairs in a system . the function g(r ) enables a precise description of how likely a probe molecule will have another probe present at a separation distance of r. this function may be simplified for a liquid system in a solvent box , where g(r ) is proportional to the observed number density ( o ) divided by the expected number density ( e ) . the value o is the total number of atoms at a given distance , and e is the number expected at that same distance if the solvent were uniformly distributed . if the probe solvent and water are evenly mixed , the rdf will converge to 1.0 at long ranges . if it converges to a larger number , this indicates phase separation ( further discussion below ) . previously , mixmd was validated with acn as a probe , which specifies hot spots for amphipathic nitrogen - containing ligands . we applied amber parameters for acn from grabuleda et al . , and they accurately reproduce experimental data from mscs . for completeness , we ran simulations of acn+h2o boxes to confirm that the rdf demonstrated appropriate convergence to unity ( figure s2 , supporting information ) . in trying to reproduce simulations of thermolysin in ipa+h2o published by seco et al . , we found that the ipa phase - separated from water ( figure 1a ) . it is unclear whether the authors observed this same behavior , and they do not note which temperature - coupling method was used . they noted partial phase separation in the paper , and they rescaled the reference values for probe density to account for the solvent behavior . however , the bulk phase separation we observed signifies the implementation of inadequate parameters for liquid ipa , which were derived from the amber parameter files for threonine with charges assigned after resp calculations ( ipaseco ) . snapshots of mixmd simulations of thermolysin in ipa+h2o executed using the protocol outlined in seco et al . ( a ) fully flexible simulations of thermolysin were performed with a mixed box of 50% w / w ipaseco+h2o . five independent runs were calculated , and every simulation resulted in the solvent layers separating between 4 and 5 ns and remaining separated when simulations were extended to 10 ns . ( b ) the same simulation using ipaopls resulted in solvent remaining well distributed for the entire equilibration and 10 ns of production in all five independent simulations . we turned to the work of jorgensen et al . for alcohol parameters because they were developed to specifically reproduce a variety of pure - solvent behaviors and interactions with tip3p water . for small boxes without protein , we compared our simulations of ipaopls+h2o to experiments by langdon and keyes that determined the density for ipa+h2o systems at different temperatures . we found that our mixed box of ipaopls+h2o reproduced the experimental density at 308 k ( 0.830g / ml ) to within 0.06% . our ability to replicate this fundamental data confirmed the use of good parameters for our ipa+h2o systems . indeed , mixmd simulations of thermolysin resulted in proper mixing using the ipa parameters from jorgensen et al . the rdfs for the ipa probes ( roo and rcc ) and water ( roo ) in mixed - solvent boxes further justified the use of opls parameters . figure 2 compares the o o rdf for both mixed - solvent and pure - solvent boxes of ipa . the roo for ( ipa - ipa)opls converged to unity with an appropriately shorter peak than that of pure liquid alcohol . however , the roo for ( ipa - ipa)seco remained well above 1.0 at 8 and featured a large peak for the first solvent shell , the same as seen in the simulation of a pure ipa box . all simulations with ipaseco feature rdfs that indicate aggregation of the solvent molecules . for this first example , the count data behind the rdfs will be discussed in detail to further explain our interpretation , which is supported by viewing the boxes . water and ( b , c ) probe probe in a mixed - solvent environment with ipa . the impact of different parameters for ipa is demonstrated by the poor convergence to unity for o o in simulations of 50% w / w ipaseco ( b ) when compared to the appropriate convergence obtained for the same mixed solvent with ipaopls parameters ( c ) . ipa and the great majority of organic solvents chosen for this study are miscible with water ( table 3 ) . the definition of miscibility is that the two solutions are homogeneously distributed at all ratios of the two solvents . it might be reasonable to find some bias or local structure in the first and second solvent shells , but bulk separation should not be seen at long ranges . this has critical correspondence with one of the basic underlying assumptions of rdfs . rdfs are founded on a uniform distribution of solvent to describe e ( solvent count / box volume ) ; any deviations from uniformity ( 1.0 ) reflects the local structure of the solvent . in all the rdfs in figure 2 , water has its maximum g(r ) at 2.75 , and the ipa maxima are at 2.85 . for pure water at 2.75 , the maximum g(r ) is 2.6 , and the cumulative count of water is 1.4 molecules ( table 4 ) . if we define the first solvent shell between 2.45 and 3.15 , it contains 3.9 neighboring water molecules . when simulated in a mixture with ipaopls , there are 1.1 waters within 2.75 and 2.9 waters within 3.15 ( table 4 ) . of course , both counts are reduced because some of the interactions are occasionally fulfilled by ipa . this is also incorporated into e , where the larger box volume reduces the expected density . it is that reduced expectation ( actually 0.244 at 2.75 ) that results in a g(r)of 4.6 . this is higher than the g(r ) maximum from the pure water simulation , but it does not reflect more interactions with water molecules . within a 7.95 radius sphere , the observed and expected counts are nearly equal and g(r ) approaches 1.0 , showing that the molecules have a uniform distribution over larger scales . both o and e depend on the volume of the simulation box ; this drops out and makes each g(r ) = no(r)/ne(r ) . the values in this table have been rounded ; the g(r ) in the figures are the exact values . values for ne(r ) are based on the volume of the count sphere , the number of solvent in the whole box , and size of the whole box during the simulation . for the mixture of water and ipaseco , the expected count of water was 34.0 , but the observed count was 51.6 water molecules ( table 4 ) . clearly , there are too many waters close to one another ! furthermore , phase separation ( as in figure 1a ) is best quantified by high g(r ) at long r , but even the local changes reflected the phenomenon . within 2.75 and 3.15 , there are 1.3 and 3.4 waters ( table 4 ) , respectively , which is much closer to the values shown in the pure - water boxes . the mixed box equilibrated to a slightly larger volume ( 1% change ) , which is reflected in the slight differences in the expected counts between ipaseco+water and ipaopls+water . however , the 1% change does not explain the maximum g(r ) of 5.1 , which is based on the increase in number of water molecules . the same patterns are seen in the rdfs of the ipa oxygens ( table 4 ) . pure ipa simulations show appropriate behavior for both sets of parameters ( black lines in figure 2bc , table s2 and text in supporting information ) . though the rdfs may imply ipaopls has slightly tighter solvent shells , they both sum to the same number of ipa molecules in the first solvent shell ( 2.1 for ipaopls and 2.0 for ipaseco ) . for mixed simulations , both ipaseco and ipaopls show lower counts than pure - solvent boxes at their g(r ) maxima ( 2.85 ) and within their solvent shells ( 3.95 ) . influenced by the discrepancies in solvent behavior observed for ipaseco and ipaopls , we carefully examined the behavior of existing parameters for additional solvent probes to determine their applicability in mixmd . the inclusion of various probe types , including complicated molecular fragments , would facilitate the development of accurate pharmacophores for druggable hot spots , ligand binding sites , and protein protein interactions . the selection of additional probes for expanding the functional groups represented in mixmd was based on existing mscs data and common interaction types found in protein ligand systems . ipa maps both hydrogen bond - accepting and -donating locations , but additional hydrogen - bonding probes are needed to develop robust pharmacophore maps of binding sites . ace and nma were selected because each molecule represents a different hydrogen - bonding profile . ace locates where the protein donates hydrogen bonds , and nma represents protein protein or peptide binding . specific parameters for ace and nma had been developed for use with amber and opls . simulations of ace+h2o and nma+h2o were performed using each parameter set to assess their suitability . the g(r ) was calculated for probe probe , probe water , and water analysis of the resulting rdf plots indicated that in each case all g(r ) converged to unity near 8 . we found that both the amber and opls parameters for liquid ace and nma demonstrated correct behavior according to rdf plots and visual inspection ( figure 3 ) . the o o rdf for water from the mixed - solvent simulations also illustrate that solvent mixing has occurred . analysis of the cumulative plots for the roo of water in solution with ace or nma showed that the number density of water within the local volume sphere was half the number density observed in pure water simulations , which is the expected result for a well - distributed system of 50% w / w probe and water . visual analysis of the trajectory snapshots served to corroborate the occurrence of proper solvent mixing . however , the rdf s deviation from unity at 8 was slightly higher for the opls descriptions of ace and nma ( table 2 ) . for this reason , we would recommend that the amber parameters be used for ace and nma in our mixmd method , especially when the amber parameters are also used to describe the protein . ( a , d ) o o and ( b , e ) n n radial distribution functions for probe probe and o o rdfs for water water ( c , f ) in a mixed - solvent environment with ace and nma . the impact of optimized parameters from amber versus general solvent parameters from opls is shown in the slightly better convergence to unity for simulations using amber parameters ( d f ) compared to opls ( a c ) parameters . in particular , we wanted to find parameters for soluble heterocycles that could map aromatic hot spots without requiring an artificial repulsive term . no other md approach for probe mapping has successfully integrated aromatic probes without an artificial interaction term . futhermore , we were particularly interested in developing probes that matched common heterocycles used in modern pharmaceuticals . as a result , a highly polar diazole , imi , is miscible with water and is present as a functional group in a wide range of biologically active molecules , including mercaptopurine ( anticancer ) , ketoconazole ( antifungal ) , and moxonidine ( antihypertensive ) . imi parameters were derived from jorgensen and mcdonald , and then simulations of pure imi and imi+h2o were conducted to assess the potential of imi as a probe for mixmd . the proper convergence of the rdf plots to 1.0 showed that imi was properly solubilized and well distributed within the binary - solvent system ( figure 4 ) . our pure - solvent rdfs were consistent with the results of jorgensen and co - workers and further validated our parameter choice and the accuracy of our protocol . also , visualization of simulation snapshots confirmed that the imi probe was well dispersed in the aqueous solution . ( c ) water water in a mixed - solvent environment with ( a ) imi and ( b ) 1p3 . opls parameters for pyr , 1p2 , 1p3 , and 1p4 were each simulated in a binary water probe solution . visualization of the simulations of pyr+h2o , 1p2+h2o , and 1p4+h2o clearly showed that they underwent phase separation . this was proved by rdfs that converged to values well above 1.0 at 8 ( figure 5 ) . however , 1p3+h2o yielded a well - dispersed system with well - behaved rdf plots ( figure 4 ) . probe probe radial distribution functions for mixed solvent environments composed of ( a ) bnz+h2o , ( b ) pyr+h2o , ( c ) iph+h2o , ( d ) 1p2+h2o , and ( e ) 1p4+h2o clearly showed phase separation , with g(r ) values well above 1.0 at 8 . we also examined two nonpolar aromatic probes , bnz and iph , simply to confirm that they are not appropriate probes when an artificial repulsive term is not used . comparison of the resultant rdf plots to the reference rdf plot of pure water showed that the g(r ) values for bnz+h2o and iph+h2o do not converge to unity within 8 ( figure 5 ) . in fact , the g(r ) for bnz+h2o indicated convergence to a value of 2 . the simulations with iph as a probe molecule showed a converged g(r ) of approximately 1.45 at 8 . visualization of snapshots from these simulations showed significant aggregation of the probe molecules , verifying that these probes are not be soluble enough for use with mixmd without inclusion of a repulsive term . because bnz is one of the solvent probes used in silcs , we were able to compare the rdf results for our mixmd simulations of bnz+h2o to the published results from silcs mapping studies . the bnz probe used in silcs was parametrized based on the charmm force field and contained a repulsive term to correct for probe probe aggregation . although a nonbonded cutoff of 8 with a 58 switching term was applied in silcs , the probe probe distance for bnz converged to 1.0 at 13 ( solvent box size was 72 58 43 ) . in comparison , the soluble aromatic probes applied in mixmd converged at approximately 67 . the first solvent shell of bnz in silcs was observed at 9 , while we observed the first peak at 45 . the presence of the second hydrophobic probe ( propane ) in silcs simulations may have influenced these differences in rdf . however , the use of a repulsive term to prevent probe probe aggregation does not correspond with the experimental properties of bnz , suggesting that silcs simulations with high concentrations of bnz may yield incorrect results . to determine whether bnz and iph would disperse more readily in the presence of an intermediary probe , ternary mixed - solvent boxes were constructed to contain 1 m aromatic probe and 1 m ipa in water . we hypothesized that the introduction of the ipa molecule would enhance available interactions and aid in the dispersion of the aromatic probes into water . however , this was not observed in simulations of the ternary - solvent boxes . at 5 ns of production time , the bnz molecules were predominantly clustered along one side of the solvent box , while the ipa molecules were dispersed throughout the water . an additional 20 ns of production time did not change the observed aggregation ( figure 6 ) . ternary - solvent simulations with iph indicated a similar result ; after 5 ns of production time , iph was separated with some dispersion toward the interior . elongating the run time out to 25 ns representative snapshots of md trajectory for solvent boxes of 1 m aromatic probe ( yellow ) and 1 m ipa ( purple ) in water ( cyan ) for ( a ) bnz+ipa+h2o at 25 ns and ( b ) iph+ipa+h2o at 25 ns . although all of the probes selected for use in our validation study had experimental data establishing their solubility in water , not all of these probes were soluble in simulation ( table 3 ) . the solvation conditions applied in a mixmd simulation included high probe concentrations , which may have affected probe solubility . to determine whether the outcomes we observed were justified based on experimental data , we considered their miscibility . the terms soluble and miscible are frequently used interchangeably ; however , they are two distinct properties . solubility is dependent upon temperature and pressure and refers to the ability of a solute ( solid , liquid , or gas ) to dissolve into a solvent ( solid , liquid , or gas ) , thereby forming a homogeneous mixture . miscibility refers to the ability of two liquids to form a homogeneous solution , independent of proportion . all probes that have been experimentally categorized as miscible were well dispersed in our computational simulations , with two exceptions ( table 3 ) . pyridine is a weak base with a nontrivial concentration of protonated pyridinium ions ( pka of 5.3 ) following solubilization into water . this effect was not simulated in our studies , and we suggest that the charged species may be responsible for solubilizing the uncharged pyridine molecules into water . a similar explanation may also be extended to pyridazine . one advantage of using silcs as a tool for solvent mapping has been that the ternary - solvent box can enable the development of a complete pharmacophore model from a single md run because the three probe types identify aromatic , hydrophobic , and hydrogen - bonding sites . using three of the probes with parameter sets we have validated for mixmd , we constructed a ternary system with imi ( aromatic probe ) , ipa ( hydrophobic and hydrogen - bonding probe ) , and water at a concentration of 33% w / w by probe . visualization of the trajectory data indicated appropriate solvent mixing , and our rdf results showed that both organic probes were fully dispersed into solution ( figure 7 ) . total run time required a comparable amount of simulation time to a binary mixmd simulation ; the total cost was an additional 4 or 8 h of production time on an 8-core cpu as compared to a binary ipa+h2o or imi+h2o simulation , respectively . ( a ) final snapshot from the production simulation of a mixed - solvent box containing imi+ipa+h2o illustrates that the probes are well mixed within the system . ( b d ) probe probe and water water radial distribution functions for imi and ipa establish convergence to unity within the trisolvent environment . we suggest that the use of this ternary mixmd model may offer several advantages over the silcs approach . we have shown that mixmd can be used to identify maximally occupied sites without recovering additional spurious minima , which is a feature that was not consistently seen in the silcs studies . in addition , instead of requiring water to act as a hydrogen - bonding probe , in mixmd , the druggable hydrogen - bonding sites can be identified in competition with water using ipa as the probe . this is important for ascertaining whether a drug - like molecule that contains a similar function group could displace water molecules at the proposed hydrogen - bonding site . a further advantage of using mixmd is the array of available probes , which can be tailored to an investigator s mapping needs . for example , either imi or 1p3 could be used in the ternary box to locate aromatic hotspots that are specific to the size of the ring . the ternary mixture of 33% w / w ace+acn+h2o was also simulated , and the results depicted a well - distributed solvent system for use in hot spot mapping with mixmd ( figure s3 , supporting information ) . we have examined solvent parameters for neat liquid from the literature for use in hot spot mapping through mixmd . our work highlights the importance of parameter validation and analysis of simulation data when performing computational solvent mapping in order to obtain appropriate behavior . influenced by the poor results obtained using a standard set of parameters used in the field for liquid isopropanol in water , we identified a set of functional group probes with specific parameters available for liquid simulation . we pursued validation of these parameters for neat and mixed - solvent simulations and identified six probes for use with tip3p in mixmd : acetonitrile , acetone , imidazole , isopropanol , n - methylacetamide , and pyrimidine . of course , these probes should be used with more proteins beyond thermolysin ( figure 1 ) to prove their applicability . we have previously applied opls ipa parameters to also examine elastase , hewl , hewl , p53 core , and rnase a. furthermore , we are currently using all of these probes to map 10 additional protein systems , but these are separate studies in their own right . the extent of ideal dispersion is most easily quantified by using the cumulative rdf of roo for water . although published results for pyridine and pyridazine indicated that they are miscible with water , these two probes were observed to separate from the aqueous phase during our simulations , rendering them unsuitable for solvent mapping in our context . we hypothesized that this disparity between experiment and computation was caused by the presence of the corresponding ionized species , which were not simulated . in order to avoid unphysical mapping or reduced accuracy , our simulations did not employ the use of an artificial repulsion or weighted density term . we have successfully expanded the range of probes that can be incorporated into mixmd studies to allow for the mapping of druggable sites , including hydrogen - bonding regions , aromatic pockets , and protein , a full pharmacophore model could be created from the simulation of a single protein+probes+water system , which would greatly reduce the computational costs of mixmd studies . this is a clear advantage of the silcs method that we would like to incorporate . ultimately , investigators could mix and match the probes used in mixmd to identify hotspots with a greater degree of specificity .

probe mapping is a common approach for identifying potential binding sites in structure - based drug design ; however , it typically relies on energy minimizations of probes in the gas phase and a static protein structure . the mixed - solvent molecular dynamics ( mixmd ) approach was recently developed to account for full protein flexibility and solvation effects in hot - spot mapping . our first study used only acetonitrile as a probe , and here , we have augmented the set of functional group probes through careful testing and parameter validation . a diverse range of probes are needed in order to map complex binding interactions . a small variation in probe parameters can adversely effect mixed - solvent behavior , which we highlight with isopropanol . we tested 11 solvents to identify six with appropriate behavior in tip3p water to use as organic probes in the mixmd method . in addition to acetonitrile and isopropanol , we have identified acetone , n - methylacetamide , imidazole , and pyrimidine . these probe solvents will enable mixmd studies to recover hydrogen - bonding sites , hydrophobic pockets , protein protein interactions , and aromatic hotspots . also , we show that ternary - solvent systems can be incorporated within a single simulation . importantly , these binary and ternary solvents do not require artificial repulsion terms like other methods . within merely 5 ns , layered solvent boxes become evenly mixed for soluble probes . we used radial distribution functions to evaluate solvent behavior , determine adequate mixing , and confirm the absence of phase separation . we recommend that radial distribution functions should be used to assess adequate sampling in all mixed - solvent techniques rather than the current practice of examining the solvent ratios at the edges of the solvent box .

the epstein barr virus ( ebv ) was described by michael anthony epstein and yvonne barr , who isolated ebv virus particles from endemic burkitt lymphoma specimens . ebv is a double stranded dna virus of the herpes virus family and is the most common virus affecting humans . the infection occurs through oral transmission via saliva . by the age of 40 , over 90 % of the world population has experienced an ebv infection . ebv also plays a pathogenic role , mainly in the development of several t cell and b cell lymphoproliferations ( see below ) but is also associated with different types of rare epithelial and mesenchymal neoplasms . ebv is mainly a b - lymphotropic and epitheliotropic virus and rarely infects t cells and nk cells . the life cycle of ebv is composed of a lytic state and a latency state , the latter allowing lifelong persistence of the virus in the host . the replication cycle of ebv is composed of three phases : ( i ) entry into either epithelial cells or naive b cells after oropharyngeal transmission , ( ii ) lytic replication , and ( iii ) latency . once a cell has become infected , the viral capsid dissolves and the dna is transported into the cell nucleus . in epithelial cells , the entry of ebv into the cell is usually directly followed by the lytic replication of the virus , which leads to production of infectious virions using the virus s own replication machinery . ebv persists in b cells throughout the differentiation from naive b cells to germinal center cells to long - lived memory b cells . in memory b cells , dependent on the expression of specific ebv - associated proteins and rnas , three different viral latency types have been described , which can be reproduced to a certain extent by immunohistochemical markers ( table 1).table 1ebv - latency types and expressed antigenslatency typeebv - associated antigens0/ieber , ebna1ii ( default program)eber , ebna1 , lmp1 , lmp2biii ( growth program)eber , ebna1 - 6 , lmp1 , lmp2a + blyticall lytic antigens ( e.g. , zebra ) ebv - latency types and expressed antigens most humans become infected with ebv during childhood , when the infection usually proceeds asymptomatically or with mild symptoms that are undistinguishable from other viral infections . if the infection occurs in young adults and adolescents , it can manifest as mononucleosis ( glandular fever ) . ebv tonsillitis differs from unspecific tonsillitis related to other causes in its histologically detectable polymorphic lymphoid infiltration with abundant plasmablasts and occasional hodgkin - like cells expanding in the interfollicular area of the tonsils . in affected lymph nodes , an extension of the interfollicular areas with a similarly polymorphic infiltration to that in tonsils can be observed . within the peripheral blood and bone marrow , cd8 positive lymphoblasts ( pfeiffer cells ) might be observed . the histomorphological picture of primary ebv infection shares histomorphological features with other ebv - driven lymphoproliferative diseases ( lpd , table 2 ) . therefore , the definite diagnosis of infectious mononucleosis is based on the following features : ( i ) young patient age , ( ii ) sole or main manifestation in the tonsils , ( iii ) absence of immunosuppression in the patient , and most importantly ( iv ) a serological test indicating ebv primary infection ( positive igm titer).table 2spectrum of polymorphic ebv driven lpd with variable neoplastic potential spectrum of polymorphic ebv driven lpd with variable neoplastic potential following the primary infection , patients become asymptomatic virus carriers with a very limited number of ebv - positive b cells . a tissue correlate of this phenomenon is the occasional presence of single small ebv - encoded rna ( eber)-positive b cells , so called bystander cells , within lymph nodes , mucosa - associated lymphatic tissue , or lungs . rare complications of primary ebv infection , such as the chronic active ebv ( caebv ) disease and/or ebv - associated hemophagocytic syndrome have recently been reviewed in this journal . since the vast majority of humans have been infected by ebv in childhood and adolescence , ebv - associated lpds in adults usually arise from reactivation of the virus from ebv - infected memory cells . such reactivation is generally considered to consist in an escape from the normal immunosurveillance and preferentially occurs at sites representing the physiological reservoir of ebv - infected b cells , such as the lymphatic tissues and mucosa . in these patients , age - related decline in immunocompetence is assumed to be the cause of ebv - lpd [ 4 , 5 ] . however , the diagnostic pathologist should stress that a clinical work - up of the immune status is warranted once an ebv reactivation is detected . ebv reactivation in adults occurs primarily in the form of ebv - lpd presenting with a polymorphic histological and clinical picture . dependent on the clinical scenario and the preferential site of involvement , different types of polymorphic ebv - lpd can be distinguished . they are outlined in table 2 and include polymorphic posttransplantation lymphoproliferations ( ptld ) , ebv - positive mucocutaneous ulceration ( ebvmcu ) , and lymphomatoid granulomatosis . depending on the clinical scenario and the extent of tissue involvement , these conditions cause variable clinical symptoms . polymorphic ebv - lpd regress in most patients if immunocompetence can be reestablished , e.g. , by withholding immunosuppressive therapy . nevertheless , a subgroup of polymorphic ebv - lpd progress to more aggressive diseases , usually accompanied by a transformation to the morphology of a fully developed lymphoma ( designated as monomorphic polymorphic ebv - associated lymphoproliferations share histomorphological and immunophenotypical features , which are certainly not specific but should alert the histopathologist to an ebv - driven disease when analyzing lymphatic proliferations . these features include the following : tissue necrosisepithelial ulcerationangiotropic vasculitis - like lymphoid infiltratespolymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cellsintermingled large t cells ( often cd8 positive)b blasts , with weak cd20 expression and expression of cd30 epithelial ulceration angiotropic vasculitis - like lymphoid infiltrates polymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cells intermingled large t cells ( often cd8 positive ) b blasts , with weak cd20 expression and expression of cd30 if the clinical scenario ( immunosuppression ) or the histological features are suggestive of an ebv - lpd , testing by eber in situ hybridization should be initiated ( see below ) . when the diagnosis of an ebv - positive polymorphic lpd has been confirmed by detection of ebv in the tissue , the nomenclature ( classification ) of the disease is dependent on ( i ) the clinical context and ( ii ) the involved site ( table 2 ) . ebv - lpd histologically resembling a lymphomatoid granulomatosis might occur after organ transplantation , in which case the disorder is named according to both the clinical scenario and the histopathological picture as ebv - positive polymorphic ptld under the histopathological picture of a lymphomatoid granulomatosis . the most important diagnostic message from the pathologist to the treating clinican should be ( i ) the ebv association of the lesion and ( ii ) the clear distinction of polymorphic ebv - lpd from fully developed lymphomas.fig . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) f 400 histomorphology of ebv - associated lymphoproliferative diseases . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) are indicated . ebv - positive lymphomas occur in immunocompetent patients , but are much more frequent in immunocompromised patients . in the latter patient group , the latency type of ebv detectable in the lymphoma cells frequently but not exclusively indicates higher viral activity with expression of more ebv encoded proteins ( latency type 3 ) and sometimes active viral replication . lymphomas occurring in immunocompetent patients , such as hodgkin lymphoma , usually display latency types 0/1 or 2 with a more restricted pattern of ebv - associated proteins and no viral replication ( table 3 ) . the diagnostic criteria for these lymphomas have been described elsewhere and do not necessarily require the detection of ebv because of the presence of ebv in the lymphoma cells . in rare cases , detection of ebv is a mandatory diagnostic feature ( e.g. , in ebv - positive lpd of childhood ) . nevertheless , in certain lymphoma entities , e.g. , nasal and extranasal nk t cell lymphoma , angioimmunoblastic t cell lymphoma , primary effusion lymphoma , and plasmablastic lymphoma , ebv association remains an important clue towards the correct diagnosis . of further relevance might be the identification of ebv - positive diffuse large b cell lymphoma ( dlbcl ) of the elderly . histological features that are useful for identifying dlbcl candidates for screening for ebv have been described as angiotropic growth , necrosis , and polymorphic , plasmablastic or hodgkin - like differentiation and expression of cd30 .table 3ebv - positive lymphomas and latency types and their association with immunocompromised statelineageimmunocompetence of patientsentitylatency typeb cellcompetentclassical hodgkin lymphoma2endemic burkitt lymphoma0/1sporadic burkitt lymphoma0/1ebv + dlbcl of the elderlyvariableebv + dlbcl associated with chronic inflammation ( pyothorax lymphoma)predominantly 3compromisedprimary effusion lymphoma0/1plasmablastic lymphoma0/1lymphomatoid granulomatosis grade 3 and dlbcl arising from the former3monomorphic ptld variablelymphomas associated with hiv infection variablelymphoproliferative disease associated with primary immune disorders variableother iatrogenic immunodeficiency - associated lymphoproliferative disorders 3 t cellcompetentangioimmunoblastic t cell lymphoma0/1 or 2extranodal nk / t cell lymphoma2compromisedebv - positive t cell lymphoproliferative disease of childhood and young adults variable diseases under this category are further specified according to histopathology the immunodeficiency in many of these young patients is postulated but not always objectifiable dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease ebv - positive lymphomas and latency types and their association with immunocompromised state diseases under this category are further specified according to histopathology the immunodeficiency in many of these young patients is postulated but not always objectifiable dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease the diagnostic criteria for ebv - positive lymphomas are described in the current who classification . however , distinguishing polymorphic ebv - lpd and a fully developed ebv - positive lymphoma can be challenging . the most important feature , large sheets of ebv - positive blasts and absence of a polymorphic cellular picture , should raise suspicion of an ebv - positive lymphoma , especially a dlbcl . a specifically difficult task is the differentiation of a polymorphic ebv - lpd from a fully developed classical hodgkin lymphoma , because the latter may be composed of a similarly polymorphic cellular infiltrate to that of the polymorphic ebv - lpd . pathologists should be aware of the fact that proteins expressed in hodgkin lymphoma , such as cd15 , can also be observed in polymorphic ebv - lpd , like ebvmcu , and therefore the final diagnosis will depend not only on the histopathological features but also on the clinical context , including the site of involvement . for example , hodgkin lymphomas rarely involve epithelial barrier organs , but ebvmcu by definition always do ( table 2 ) . in our experience , clonality testing is not helpful in differentiating ebv - positive lymphoma from polymorphic lymphoproliferations , as viral - driven lpd can also result in a clonal expansion of t and b cells . testing for ebna2 expression is useful in some cases , since expression of ebna2 strongly argues against hodgkin lymphoma ( table 2).table 4differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphomafeaturehodgkin lymphomapolymorphic ebv + lpdhistomorphologymixed cellularity infiltration with hodgkin ( like ) cells , necrosispredominant epitheloid cells and eosinophilspredominant plasmablasts , plasmacells , angiotropismimmunophenotype of large cellscd30 > cd20cd20 > cd30expression of ebv antigens eber + cells equal to lmp1 + cells eber + large cells only ebna2 negative eber + cells more than lmp1 + cells eber + small and large cells ebna2 + /clinical scenariopredominant immunocompetentpredominant immunocompromisedsite mediastinum nodal > > extranodal extranodal > > nodal mucosa differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphoma the question as to which specimen should be tested for ebv depends on the histopathological findings on the one hand and on the clinical context on the other hand . table 3 summarizes our criteria and favored method of testing for ebv , fig . if pathologists are confronted with lymphoid tissue histologically resembling inflammatory ( reactive ) tissue in immunocompetent patients , the decision whether or not to test for ebv is primarily based on histological features ( table 3 ) . in immunocompromised patients , the method of testing should be eber in situ hybridization , which is most sensitive for detecting ebv , especially in lesions that can not necessarily be expected to express other viral antigens such as lmp1 ( table 3 ) . since indolent ( low grade ) b cell lymphomas are virtually never associated with ebv , testing can not be recommended . for other entities , which are listed in tables 3 and 5 , testing should be part of the standard diagnostic work - up . immunohistochemical staining for lmp1 is sufficient for hodgkin lymphoma , since these lymphomas express the latency type 2 . however , other lymphomas listed in table 5 , such as diffuse large b cell lymphomas , require testing by eber because lmp1 is frequently not detectable.fig . 2an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) . eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . d 400table 5recommendations for selecting tissue specimen and methods for ebv testinglymphomanodal and extranodal lymphatic tissue not presenting overt lymphoma ( polymorphic lpd)histopathological featuresclinical contexttesting by ebertesting by ebertesting by eberb cell lymphomas endemic burkitt lymhoma hodgkin - like large cells and hodgkin or reed - sternberg cells inherited immunodeficiency plasmablastic lymphoma primary effusion lymphoma posttransplantation pyothorax associated lymphoma dlbcl with histological features suspicious for ebv association necrosis drug induced immunocompromised state angiotropism of blastic cells any monomorphic ptld lymphomatoid granulomatosis plasmablastic infiltrate hiv infectiont cell lymphomas ailt extranodal nk / t cell lymphoma ebv - positive t cell lymphoproliferative disease of childhood and young adultstesting by eber or lmp hodgkin lymphomatesting by eber , lmp1 and ebna2 differential diagnosis between hodgkin lymphoma and ptld ptld posttransplantation lymphoproliferative disease , aitl angioimmunoblastic t cell lymphoma an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) . eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . original magnification a d 400 recommendations for selecting tissue specimen and methods for ebv testing ptld posttransplantation lymphoproliferative disease , aitl angioimmunoblastic t cell lymphoma although epidemiologic data are lacking , ebv - lpd distinct from overt lymphoma will be increasingly recognized . this is due on the one hand to the aging of many populations , with an increase in age - associated ebv reactivation . on the other hand , widespread access to commercial assays and automatization leads to an increasing use of in situ hybridization for eber by diagnostic pathologists . additionally , newly recognized subgroups of lymphomas , such as the ebv - positive dlbcl of the elderly , require the detection of ebv in more lymphoma entities as part of the diagnostic process .

lymphoproliferations associated with epstein barr virus ( ebv ) in adult patients pose a diagnostic challenge for pathologists for several reasons . first , the ebv lymphoproliferations represent a clinically and histologically very broad spectrum ranging from self - limiting lymphoproliferations to manifest malignant lymphomas . second , the classification of these diseases is not solely based on histopathology but rather requires a synopsis of clinical as well as pathological features . and third , a resource - efficient diagnostic procedure demands a deliberate strategy for selecting the tissue specimens that are to be tested for ebv . we describe how the clinical context and histological features may indicate to histopathologists which lymphatic tissues should be tested for the presence of ebv and how these features guide the classification . we provide recommendations as to which biopsy specimens should be investigated for ebv and which methods for detecting viral association are appropriate .

hepatitis c virus ( hcv ) is a global healthcare problem , with a prevalence of around 3% worldwide ( 1 ) ; it has six major genotypes ( types 1 6 ) . most infected patients will establish chronicity with long - term complications , including liver fibrosis , cirrhosis , and hepatocellular carcinoma ( hcc ) ( 2 ) . the success rates of current treatments with interferon ( ifn)-based therapy are highly variable and depend on both host and viral factors ( 3 ) . compared to other developing countries , egypt has a high prevalence of hcv , where 20% of egyptian blood donors are seropositive for hcv antibodies ( 1 ) . although hcv-4 causes about 20% of chronic hepatitis c in the world , it has not been subjected to enough research , most probably due to its restricted localization to the middle east and africa , where the management strategies for infected patients are not as well developed as for genotype-1 , -2 , and -3 ( 4 , 5 ) . micrornas are endogenous small non - coding rnas ( 22 nucleotides ) involved in the regulation of many cellular processes ( 6 ) . several host mirnas have been suggested to be involved in hcv entry , the establishment of viral infection , and the multi - step process of chronic hcv infection . the ability of the virus to take over certain cellular mirnas and its persistence are not fully understood ( 7 ) . the liver - specific mirna , mir-122 , positively modulates hcv infection through direct interaction with the 5 utr of the hcv genome and stimulation of hcv translation ( 8) . remarkably , reagents that can downregulate mir-122 have entered clinical development for hcv treatment ( 10 ) . other mirnas have also been reported to physically interact with the hcv genome and attenuate viral replication , namely let-7b , mir-196 , mir-199 , and mir-448 ( 11 ) . in hcv - infected patients , lower expression levels of mir-29 have been observed in liver , while its overexpression inhibits viral rna replication in hcv - infected hepatocytes ( 12 ) . mir-130a expression has been up - regulated in liver biopsies from hcv - infected patients , as well as in hcv - infected hepatocytes ( 13 ) . on hcv infection , the virus can modulate the function of certain cellular mirnas involved in ifn production and the innate antiviral immune response , thereby exacerbating the infection ( 14 ) . in addition , a number of reports have highlighted the importance of host cellular mirnas in modulating responsiveness to exogenous ifn treatment ( 15 ) . associated inflammation , fibrosis , and cirrhosis , as well as the initiation and progression of liver cancer ( 16 , 17 ) . in this study , we attempted to identify special characteristics of the egyptian hcv patients of predominantly genotype 4 by performing a selected 94 mirna profile in 50 chronically hcv - infected patients . the correlation between individual mirna expression profiles and clinicopathological variables was assessed for the sake of identifying potential biomarkers related to hcv-4 infection . fifty patients diagnosed with chronic hcv infection between 2011 and 2012 were enrolled in this study . . the study was reviewed and approved by the cairo university hospital research ethics committee ( rec ) , school of medicine , cairo university . tissues were divided into two parts ; one was used for histopathological examination , and the other was stored in rnalater ( qiagen , hilden , germany ) at -80c until use . liver biopsies obtained from normal liver transplantation donors were used as controls . for each biopsy sample , liver fibrosis and activity were scored according to the metavir classification system ( 18 ) . patients were considered hcv positive if their serum tested positive in an enzyme - linked immunosorbent assay ( elisa ; detect - hcv 3 , adaltis , milano , italy ) for hcv antibodies . all enrolled patients were hcv - rna positive and the viral load was determined using quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) assay ( artus hcv rg rt - pcr , qiagen ) . patients who were co - infected with hbv or human immunodeficiency virus ( hiv ) and/or who had liver cirrhosis were excluded from this study . liver function tests were conducted to determine patients levels of aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , total bilirubin , albumin , alkaline phosphatase ( randox , randox laboratories ltd . , bilharzial antibody titer was determined to assess the bilharzial status of the patients ( novatec immundiagnostica gmbh , germany ) . hcv genotyping was assessed using the gen - c reverse hybridization strip assay kit ( nlm diagnostic , italy ) . samples were first homogenized in qiazol lysis reagent , then rna was purified using mirneasy mini kit ( qiagen ) following the manufacturer s instructions . one microgram of rna was used to prepare cdna using miscript reverse transcription kit ( qiagen ) according to the supplied protocol . rna concentrations were quantified using a nanodrop spectrophotometer ( nanodrop technologies , wilmington , de , usa ) . the selected mirnas for this study were previously shown to be associated with liver health and chronic liver diseases . pcr reaction containing 1x sybr green master mix ( qiagen ) , 200 nm of mirna specific forward primer , and 200 nm of universal primers were performed in 96-well plates using 3 ng in 2.5 l cdna / well . the total reaction volume was 10 l / well with initial denaturation at 95c followed by 40 cycles at 94c for 15 seconds , 55c for 30 seconds , and 70c for 30 seconds . the qrt - pcr experiments were performed on an abi 7500 ( applied biosystems , foster city , ca , usa ) . the applied biosystems 7500 software was used to export the ct values of different mirnas to the microsoft excel program . the relative expression of mirnas was normalized using the mean expression value of all examined mirnas in each sample as a normalization factor , following mestdagh et al . all ct values above or equal to 35 were removed before calculating the mean of the remaining ct values . the mean ct value was calculated for each sample according to the equation 1 : the ct and fold changes were calculated according to the equation 2 ( fold change = 2 ) : heat - map and two - way clustering analysis were performed with a log2 fold of change using gene - e software ( broad institute , inc . ) . the chi - square test was used to test the observed distribution of mirna as up- or down - regulated to an expected distribution . the non - parametric mann whitney and kruskal wallis tests were used to analyze differences in mirna values according to categorical clinicopathological features . fifty patients diagnosed with chronic hcv infection between 2011 and 2012 were enrolled in this study . . the study was reviewed and approved by the cairo university hospital research ethics committee ( rec ) , school of medicine , cairo university . tissues were divided into two parts ; one was used for histopathological examination , and the other was stored in rnalater ( qiagen , hilden , germany ) at -80c until use . liver biopsies obtained from normal liver transplantation donors were used as controls . for each biopsy sample , liver fibrosis and activity were scored according to the metavir classification system ( 18 ) . patients were considered hcv positive if their serum tested positive in an enzyme - linked immunosorbent assay ( elisa ; detect - hcv 3 , adaltis , milano , italy ) for hcv antibodies . all enrolled patients were hcv - rna positive and the viral load was determined using quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) assay ( artus hcv rg rt - pcr , qiagen ) . patients who were co - infected with hbv or human immunodeficiency virus ( hiv ) and/or who had liver cirrhosis were excluded from this study . liver function tests were conducted to determine patients levels of aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , total bilirubin , albumin , alkaline phosphatase ( randox , randox laboratories ltd . , bilharzial antibody titer was determined to assess the bilharzial status of the patients ( novatec immundiagnostica gmbh , germany ) . hcv genotyping was assessed using the gen - c reverse hybridization strip assay kit ( nlm diagnostic , italy ) . samples were first homogenized in qiazol lysis reagent , then rna was purified using mirneasy mini kit ( qiagen ) following the manufacturer s instructions . one microgram of rna was used to prepare cdna using miscript reverse transcription kit ( qiagen ) according to the supplied protocol . rna concentrations were quantified using a nanodrop spectrophotometer ( nanodrop technologies , wilmington , de , usa ) . the selected mirnas for this study were previously shown to be associated with liver health and chronic liver diseases . pcr reaction containing 1x sybr green master mix ( qiagen ) , 200 nm of mirna specific forward primer , and 200 nm of universal primers were performed in 96-well plates using 3 ng in 2.5 l cdna / well . the total reaction volume was 10 l / well with initial denaturation at 95c followed by 40 cycles at 94c for 15 seconds , 55c for 30 seconds , and 70c for 30 seconds . the qrt - pcr experiments were performed on an abi 7500 ( applied biosystems , foster city , ca , usa ) . the applied biosystems 7500 software was used to export the ct values of different mirnas to the microsoft excel program . the relative expression of mirnas was normalized using the mean expression value of all examined mirnas in each sample as a normalization factor , following mestdagh et al . all ct values above or equal to 35 were removed before calculating the mean of the remaining ct values . the mean ct value was calculated for each sample according to the equation 1 : the ct and fold changes were calculated according to the equation 2 ( fold change = 2 ) : heat - map and two - way clustering analysis were performed with a log2 fold of change using gene - e software ( broad institute , inc . ) . the chi - square test was used to test the observed distribution of mirna as up- or down - regulated to an expected distribution . the non - parametric mann whitney and kruskal wallis tests were used to analyze differences in mirna values according to categorical clinicopathological features . liver biopsy and blood samples were collected from 50 chronically infected hcv patients . as expected , 94% of the enrolled subjects in this study were genotype 4 , and only 6% were of genotype 1 . liver biopsy samples revealed that most patients ( 90% ) had mild to moderate fibrosis . steatosis was mild in 78% and moderate in 12% of the samples ( table 1 ) . the log2 fold of change was used to generate expression profile heatmap using gene - e software . two - way clustering analysis showed an association between the enrolled patients and the expression levels of the studied micrornas ( figure 1 ) . forty - four out of the 94 analyzed mirnas were significantly deregulated ; 27 mirnas exhibited at least a 4.0-fold increase , and 17 mirnas exhibited at least a 4.0-fold decrease . to understand the role of the deregulated mirnas in association with hcv infection , their published functions and proposed targets were summarized , as shown in tables 2 and 3 . a number of these mirnas have been shown to be associated with hcv replication , pathogenesis , and associated liver complications . each column represents a sample and each row shows the fold change of each mirna in comparison to the average of normal samples . the differential regulation is indicated by red for up - regulated genes and blue for the down - regulated ones . remarkably , our data showed that the liver - specific mirna-122 was strongly down - regulated in the majority of our patients , which contradicts several other previously published papers showing an increase in this mirna correlated with its involvement in hcv infection and replication ( 20 , 21 ) . in contrast , our results were in agreement with spaniel et al . who found that levels of mir-122 were reduced in japanese patients with chronic hepatitis c ( 22 ) . expression levels of seven mirnas ( mir-29c , mir-30c , mir-126 , mir-145 , mir-199a , mir-199a-3p , and mir-222 ) tended to increase specifically in patients with an increasing grade of inflammatory activity ( figure 2 ) . expression of three other mirnas ( mir-95 , mir-130a , and mir-142 - 5p ) expression was inversely correlated with the serum albumin levels of the enrolled patients . no significant correlations were shown between the expression levels of analyzed mirnas and basic clinical and biochemical characteristics of the studied patients , including age , viral load , serum liver enzymes , afp level , presence of bilharziasis , or steatosis ( data not shown ) . a , mir-23c , p = 0.003 ; b , mir-30c , p = 0.009 ; c , mir-145c , p = 0.005 ; d , mir-199a , p = 0.005 . five mirnas , namely mir-21 ( p = 0.001 ) , mir-23a ( p = 0.046 ) , mir-126 ( p = 0.045 ) , mir-194 ( p = 0.04 ) , and mir-199a-3p ( p = 0.02 ) , showed down - regulation in male patients , while only one mirna ( mir-638 ) was significantly down - regulated in female patients ( p = 0.009 ; figure 3a and b ) . it illustrates the relation between a , the level of expression of mir-199a-3p ; and b , mir-638 with gender in the liver tissue of chronic hcv patients . the log2 fold of change was used to generate expression profile heatmap using gene - e software . two - way clustering analysis showed an association between the enrolled patients and the expression levels of the studied micrornas ( figure 1 ) . forty - four out of the 94 analyzed mirnas were significantly deregulated ; 27 mirnas exhibited at least a 4.0-fold increase , and 17 mirnas exhibited at least a 4.0-fold decrease . to understand the role of the deregulated mirnas in association with hcv infection , their published functions and proposed targets were summarized , as shown in tables 2 and 3 . a number of these mirnas have been shown to be associated with hcv replication , pathogenesis , and associated liver complications . each column represents a sample and each row shows the fold change of each mirna in comparison to the average of normal samples . the differential regulation is indicated by red for up - regulated genes and blue for the down - regulated ones . remarkably , our data showed that the liver - specific mirna-122 was strongly down - regulated in the majority of our patients , which contradicts several other previously published papers showing an increase in this mirna correlated with its involvement in hcv infection and replication ( 20 , 21 ) . in contrast , our results were in agreement with spaniel et al . who found that levels of mir-122 were reduced in japanese patients with chronic hepatitis c ( 22 ) . expression levels of seven mirnas ( mir-29c , mir-30c , mir-126 , mir-145 , mir-199a , mir-199a-3p , and mir-222 ) tended to increase specifically in patients with an increasing grade of inflammatory activity ( figure 2 ) . expression of three other mirnas ( mir-95 , mir-130a , and mir-142 - 5p ) expression was inversely correlated with the serum albumin levels of the enrolled patients . no significant correlations were shown between the expression levels of analyzed mirnas and basic clinical and biochemical characteristics of the studied patients , including age , viral load , serum liver enzymes , afp level , presence of bilharziasis , or steatosis ( data not shown ) . a , mir-23c , p = 0.003 ; b , mir-30c , p = 0.009 ; c , mir-145c , p = 0.005 ; d , mir-199a , p = 0.005 . five mirnas , namely mir-21 ( p = 0.001 ) , mir-23a ( p = 0.046 ) , mir-126 ( p = 0.045 ) , mir-194 ( p = 0.04 ) , and mir-199a-3p ( p = 0.02 ) , showed down - regulation in male patients , while only one mirna ( mir-638 ) was significantly down - regulated in female patients ( p = 0.009 ; figure 3a and b ) . it illustrates the relation between a , the level of expression of mir-199a-3p ; and b , mir-638 with gender in the liver tissue of chronic hcv patients . over the last few years , several studies have examined the expression levels of certain mirnas in association with hepatitis c viral infection . some of the obtained data were provided in the context of hcc ( 23 , 24 ) . in this study , we aimed to identify changes in mirna characteristics of nave egyptian patients with chronic hcv infection . we succeeded in identifying 44 mirnas with at least 4.0-fold change compared to the normal controls . the identified host - altered mirnas may contribute to the chronicity of hcv and mediated pathogenesis . the combined samples clustering with the expression pattern of the studied 94 mirnas could help to cluster the enrolled patients according to the progression of liver chronicity ; further investigations are necessary . to develop a better understanding of the role of the deregulated mirnas in association of chronic hcv infection , their suggested functions were manually curated from relevant published literatures ( tables 2 and 3 ) . the three liver - specific mirnas analyzed in this study ( mir-122 , mir-148a , and mir-194 ) were significantly down - regulated in the majority of the patients . our results are in agreement with a recent report regarding the reduced level of mir-122 in the tissues of chronic hepatitis c infected patients and chimpanzees . the decrease of mir-122 in our study may have occurred after an initial rise at the beginning of the hcv infection , which was then followed by a decline , since all of our subjects were in the chronic phase of infection . this initial rise of hepatic mir-122 levels followed by a decline was shown to occur in chimpanzees ( 25 ) . in recent studies , the hepatic mir-122 expression level has been shown to be reduced significantly with the severity of liver fibrosis in patients with chronic hcv infection ( 26 , 27 ) ; this might fit with the clinical data of the enrolled patients in this study to some extent . the reduced level of mir-122 was found to be associated with a poor response to inf - based treatment ( 28 ) . thus , the selection of patients with chronic hcv infection for effective inf - based therapy may be achieved based on the hepatic expression level mir-122 ; further investigations on this topic are necessary . other differentially expressed mirnas in this study have been shown to be associated with liver inflammation to fibrosis and hcc ( let-7 , mir-21 , mir-223 ( down - regulated ) , mir-105 , mir147 , mir-155 , mir-187 ( up - regulated ) ( 29 ) . notably , the down - regulated mir-223 can act as a negative regulator of inflammation during viral infection . mir-130a , a potential drug target in hcv treatment , was down - regulated in the examined hepatic samples ( albeit not below the threshold level we set for this study ) . it has been reported that mir-130a overexpression can inhibit hcv replication by restoring host innate immune responses and/or down - regulating pro - hcv mir-122 ( 31 ) . restoring the expression level of mir-130a in infected patients may be a useful strategy to combat hcv infection . we identified seven mirnas ( mir-29c , mir-30c , mir-126 , mir-145 , mir-199a , mir-199a-3p , and mir-222 ) that were significantly increased with the inflammation grade of the liver ( figure 2 ) . consistent with our data , it has been reported that members of mir-29 and mir-199 families correlate with the stage of liver fibrosis in hcv patients ( 32 , 33 ) . identification of possible markers for the inflammation stage can be helpful in monitoring the disease progression and in avoiding the invasive biopsies used in these assessments . liver fibrosis is the most common final path of chronic liver diseases ; this is influenced by the nuclear factor - kappa b ( nf-b ) signaling pathway ( 34 ) . mir-155 , which was up - regulated in the present study , has been shown to have a negative effect on regulation of the nf-b pathway through targeting different proteins , including myeloid differentiation primary response gene 88 ( myd88 ) . up - regulation of mir-155 was also reported in hcv - infected cells , leading to repression of the nf-b signaling pathway ( 35 ) . meanwhile , mir-16 , mir-199a , and mir-223 , which were down - regulated in the present study , can regulate the nf-b pathway by targeting key signaling protein genes ( 36 , 37 ) . it was shown previously that c - rel ( a member of the nf-b family ) was able to negatively regulate mir-1228 by binding directly to its promoter site ( 38 ) . in the present study , extremely high expression of mir-1228 - 5p it would be interesting to study the effect of increased mir-1228 on the nf-b pathway in infected cells . our results showed that a distinctive subset of mirnas up - regulated simultaneously in some patients ( mir-31 , mir-105b , mir-147 , mir-149 - 3p , mir-198 , mir-302b-5p , and mir-1228 - 5p ) , in which they exhibited a greater than 200-fold change compared to normal subjects . further studies are needed to reveal the significance of their expression level and the cellular machinery(ies ) governing the mirna - targeted relationship in chronically infected hcv patients . it is interesting to note that a number of the deregulated mirnas investigated in this study and their target genes , which are associated with advanced fibrosis or cirrhosis , play a critical role in the initiation and progression of hcc ( tables 2 and 3 ) . in addition , three mirnas , namely mirna-18a , mir-18b , and mir-21 , can promote cell proliferation and invasion and contribute to evasion of the host immune system . the role of several up - regulated mirnas in association with chronic hcv infection is not yet fully understood ( table 3 ) . in conclusion , many of the dysregulated mirnas in this study are associated with chronic liver disease and subsequent complications . this may reflect the potential mechanism(s ) of persistent hcv infection to drive normal hepatocytes to malignancy via changing expression of several mirnas , as a layer of gene expression control , in the host cell . moreover , the identified expression profiles of some examined mirnas might offer important points to consider for the treatment of nave patients and management of chronically infected hcv patients in egypt and around the world .

backgroundmicrornas ( mirnas ) have been repeatedly shown to play important roles in liver pathologies , including hepatitis , liver cirrhosis , and liver cancer . egypt has the highest hepatitis c virus ( hcv ) infection rate worldwide , predominantly involving genotype-4.objectivesin this study , we attempted to characterize the mirna profile of the poorly studied genotype 4 of hcv in chronically infected egyptian patients to obtain a better understanding of the disease and its complications and help in the design of better management protocols.patients and methodswe analyzed the expression levels of a selected panel of 94 mirnas in fresh liver biopsies collected from 50 egyptian patients diagnosed with chronic hcv infection using quantitative real - time polymerase chain reaction ( pcr ) assay . non - parametric tests were used to analyze the expression level of each mirna and association with the clinicopathological features of enrolled patients in this study.resultsour results revealed differential expression levels of the analyzed mirnas compared to the normal controls . twenty - seven mirnas ( including mir-105 , mir-147 , mir-149 - 3p , and mir-196b ) showed up - regulation , while 17 mirnas ( including mir-21 , mir-122 , mir-199a-3p , and mir-223 ) showed down - regulation . an inverse correlation was observed between levels of mir-95 , mir-130a , and mir-142 - 5p with the blood albumin level . increased expression levels of seven mirnas ( mir-29c , mir-30c , mir-126 , mir-145 , mir-199a , mir-199a-3p , and mir-222 ) were observed with severe chronic hepatic inflammation . several deregulated mirnas found in this study have been previously linked to chronic liver inflammation and the risk of hepatocellular carcinoma ( hcc ) development.conclusionsthe identified expression profiles of some examined mirnas might offer important points to consider for the treatment of naive patients and the management of chronically infected hcv patients in egypt and around the world .

epidermoid cysts are benign developmental malformations arising from abnormal epithelial constituents of ectodermal tissue formed during the fetal period . these lesions can be seen anywhere in the body , with the occurrence of approximately 7% in the head and neck region and their incidence in the oral cavity makes up for 1.6% of the total occurrences and they constitute < 0.01% of all the cystic lesions of the oral cavity . epidermoid cysts of the oral cavity , in adults , are mostly seen on the floor of the mouth and in other locations including the labial , palatine tonsil and in the soft palate . in infants incidence is highest in the floor of the mouth and lowest in the soft palate . only four cases have been reported in literature of epidermoid cysts in infants involving the soft palate . we present here a report of a 9-month - old female patient who underwent surgery for cleft palate and growth on the uvula of soft palate clinically diagnosed as a benign fibrous tumor which was confirmed by histology as epidermoid inclusion cyst . a 9-month - old indian infant was brought to the cleft care center for a surgery of cleft palate and associated growth in the soft palate . family history was not significant and her mother had an uncomplicated delivery . on physical examination , the patient was alert and intraoral examination revealed a cleft palate involving the soft palate and a whitish , oval shaped , solitary solid mass with pale overlying mucosa located behind the uvula and right side of the soft palate measuring approximately about 1 cm 1 cm [ figure 1 ] . no other external facial or neck cysts , sinuses or lesions were noted and the rest of the intraoral space was unremarkable . a provisional diagnosis of benign fibrous tumor was considered and differentials included salivary gland neoplasms and benign tumors of muscular origin . the patient was admitted to the hospital for the surgery of cleft palate as the medical history was irrelevant . surgery was performed under general anesthesia , surgery was uneventful , and the patient recovery was good . histopathological examination revealed a parakeratinized stratified squamous epithelium with flattened rete ridges and a cystic space with keratin flecks . the patient is under follow - up and there is no evidence of recurrence even after 1.5 years . clinical image revealing cleft palate with a whitish , oval shaped , solitary solid mass with pale overlying mucosa located behind the uvula and right side of the soft palate ( a ) gross specimen creamish white in color and soft in consistency ; ( b ) cut section of the gross specimen showing cystic space ; ( c ) photomicrograph demonstrating the cystic cavity containing keratin ( h&e stain , 40 ) ( d ) photomicrograph demonstrating parakeratinized stratified squamous epithelium with flattened rete ridges and the cystic space with keratin flecks ( h&e stain , 100 histopathological image demonstrating epithelium and keratin ( h&e stain , x40 ) high power view of the epithelium and keratin in the lumen ( h&e stain , x100 ) photomicrograph showing keratinizing epithelium and keratin flecks in the lumen ( h&e stain , 100 ) histopathological figure at 10 photomicrgraph showing keratin flecks ( h&e stain , x200 ) a thorough search using the keywords such as soft palate , pediatric / congenital , and epidermoid cyst / dermoid cyst/ in various combinations was made in pubmed . a keyword search using pidermoid cyst , palate , cleft in the pubmed literature revealed 10 cases , but none of them were associated with a cleft soft palate table 1 . reported cases of epidermoid cysts of soft palate and uvula dermoid cysts were classified by new and erich in 1937 as acquired implantation , congenital teratoma and congenital inclusion dermoid cysts and by meyer in 1955 as true dermoid cysts , epidermoid cysts and teratoid cysts . a true dermoid cyst is lined with keratinized epithelium and has skin appendages like hair follicles or sebaceous glands . an epidermoid cyst is lined with simple squamous epithelium and does not contain skin appendages . a teratoid cyst in addition to skin appendages contains other tissues such as muscle , bone and cartilage . according to new and erich , who conducted a study on 1495 cases , the common location was anal region ( 44.5% ) followed by ovarian ( 42.1% ) region . the overall incidence of pediatric head and neck cysts was 7% , with the periorbital region being the most common site . this is equally consistent with the study by pryor et al . which concentrated on pediatric dermoid cysts of the head and neck , in which 61% of head and neck dermoid cysts were periorbital in location . in neither of these series , were cysts of the soft palate mentioned . dermoid cysts and epidermoid cysts can be acquired or congenital , but in infants , they are usually congenital . during the 6 week of gestation , the secondary palate begins to develop . it starts initially as two outgrowths from the maxillary prominences which are oriented in a vertical direction and layout on either side of the tongue . during 8 week , these palatine shelves orient themselves in a horizontal direction and the tongue descends downward as mandible elongates and the fetal head tilts upward . the palatal shelves continue to grow toward each other and contact at the medial edge epithelia . a transient midline epithelium or midline seam is formed at this time and as the growth of head increases the seam thins into a single layer of cells and undergoes disintegration completely resulting in the merging of the mesenchymal portion of two palatal shelves by the process of fusion . this process of shelf elevation and fusion takes place about a week later in girls than in boys which explain why girls are more prone than boys for cleft palate formation . in contrast , after the fusion of palatal shelves the soft palate uvula forms through migration and proliferation of two confluenced sub epithelial mesenchymal growth centers at the posterior edge of the newly formed palate with a groove between them filled by merging . failure of the merging process during soft palate and uvula development can result in complete or partial clefts of the soft palate and uvula . and hence , in distinction to the previous epidermoid cysts that were reported on the soft palate , in the present case cleft of secondary palate might have developed due to lack of fusion and merging ; and the epidermoid cyst would have developed due to aberrant ectodermal entrapment in the uvular area followed by reactivation of these cleaved cells . identification of epidermoid cyst is essential in neonates as they may cause difficulty and obstruction in breathing and swallowing which might turn out to be fatal .

epidermoid cysts are benign malformations that can be encountered anywhere in the body and are rarely observed in the oral cavity accounting for < 0.01% of all cysts of the oral cavity . they can be classified as either congenital or acquired without any clinical or histologic differences . our literature search did not find any report of a congenital epidermoid cyst located in the soft palate associated with a complete palatal cleft in an infant . this is a case report of a 9-month - old female patient who had a cleft palate with an associated soft tissue mass at the junction of soft palate and uvula .

thirty years have passed after discovering human immunodeficiency virus ( hiv ) , the etiological agent of the acquired immunodeficiency syndrome ( aids ) [ 14 ] . two types of hiv are known : the most common hiv-1 , which is responsible to the worldwide aids epidemic , and the immunologically distinct hiv-2 , which is much less common and less virulent [ 6 , 7 ] but produces clinical findings similar to hiv-1 . the hiv-1 type itself includes four groups m ( main ) , o ( outlier ) , n ( non - m , non - o ) , and p [ 912 ] , which have different geographic distributions but all produce similar clinical symptoms . the m group further splits into 9 subtypes ( a through j ) [ 1315 ] , as well as at least 58 circulating recombinant forms ( crfs , http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html , last accessed 06 may 2013 ) and multiple unique recombinant forms ( urfs ) . the vast majority of reports on drug resistance deal with hiv-1 subtype b infections in developed countries , and this is largely due to historical delays in access to antiretroviral therapy on a worldwide basis . advances in antiretroviral therapy have revolutionized hiv management and the control of the spread of regional epidemics [ 1618 ] . currently , a combination of several antiretroviral agents , termed highly active anti - retroviral therapy ( haart ) , has been highly effective in reducing the number of hiv particles in the blood stream ( as measured by a blood test called viral load ) and delaying disease progression . clinical trials and observational studies have shown profound reductions in morbidity and mortality in patients infected with hiv as a result of combination antiretroviral therapy [ 16 , 1927 ] . of relevance , advances in hiv treatment have had a positive impact on all the affected demographic and behavioral risk groups , with an expected longevity for hiv - infected patients that is now 73 years . moreover , it should be considered that , thanks to the recent expansion in the number of antiretrovirals and antiretroviral classes , virological suppression has become achievable in most patients for whom numerous prior antiretroviral regimens had failed . in addition , antiretroviral therapy results in efficacious treatment of hiv-1 , regardless of the viral subtype . however , despite advances in antiretroviral therapy , some treatments still fail . a major cause of treatment failure is the development of drug resistance both in hiv-1 b and non - b subtypes [ 2834 ] . the extreme variability and the high evolution rate of hiv-1 favour the development of antiretroviral resistance . indeed , hiv-1 infection is characterized by a high degree of genetic variability within infected persons . this is explained by the fact that the virus population present at a certain time point within an infected person consists of a complex mixture of heterogeneous strains , termed the subsequent overgrowth or dominance of a certain viral strain over another is largely determined by its relative adaptation to a given intrahost environment , a factor particularly relevant to the emergence of drug resistant variants . indeed , the intrapatient virus population is a highly dynamic system , characterized by a high turnover rate and a high mutation rate [ 37 , 38 ] . these evolutionary dynamics are the basis for a diversified population that can quickly generate drug - resistant variants in response to the therapy [ 3942 ] . escape mutants that have a selective advantage under therapy become dominant in the population and lead to an increasing virus production and eventually to therapy failure . the shifted population may be hit with a new drug combination , but finding such a potent regimen after treatment failure is challenging , since many accumulated mutations confer drug resistance not only to the administered drugs but also to structurally and functionally similar compounds [ 41 , 43 ] . identifying and understanding hiv-1 drug resistance therefore helps clinicians to avoid minimally active antiretrovirals in favor of newer drugs that are fully or nearly fully active [ 4446 ] . for this reason , resistance testing has become an important diagnostic tool in the management of hiv infections [ 4752 ] . with the aid of hiv resistance tests pharmacoeconomic studies have shown that these tests are also cost effective [ 53 , 54 ] . for several years , national and international hiv treatment guidelines have been recommending the use of resistance testing both in drug - naive and drug - treated patients [ 4852 ] . antiretroviral drug design , resistance research , and interpretation systems have been largely based on hiv-1 subtype b because of its predominance in the wealthy countries in which antiretroviral drugs were first introduced , as well as the availability of assays for drug resistance in such locations . however , it should be noted that hiv-1 b subtype represents only about 10% [ 5557 ] of the overall subtypes in the world . several studies showed that non - b subtype hiv-1 infections have been rapidly increasing over time in previously subtype b homogeneous areas such as europe and north america [ 5873 ] . for example , in france , switzerland , and italy , it is estimated that non - b infections constitute roughly 15%35% of hiv-1 infections , with an increasing prevalence and complexity of intersubtype recombinants over the last years [ 59 , 64 , 66 , 7377 ] . although non - b infections are infrequent in north america , a study in new york identified non - b infections in a few us citizens who never travelled abroad , suggesting that transmission of non - b subtype occurs in the united states independently of travel history . moreover , new hiv-1 strains are constantly emerging [ 78 , 79 ] . due to the spread of hiv-1 non - b subtypes and the introduction of antiretroviral drugs in resource - limited settings ( known for the largest assortment of non - b subtypes ) , further knowledge concerning the responsiveness to antiretroviral therapy and hiv-1 drug resistance in non - b subtypes is required . in this regard , the development of specific mutations varies in different subtypes , and this can be explained mainly by the intrinsic properties of the virus and not only by a different pressure of antiretroviral drugs , as suggested in a recent study . to date , the improvement of the genotypic resistance test in terms of sensitivity , cost effectiveness and detection of drug resistance in non - b subtypes is a major topic . in order to achieve this goal , the introduction of new affordable assays is crucial . group m subtype independent genotyping assays for detection of hiv-1 drug resistance were recently developed . these assays could represent an alternative to commercial assays for hiv-1 drug resistance genotyping in routine diagnostics and for surveillance and monitoring of drug resistance in resource limited settings [ 8082 ] . several discrepancies are still present in the interpretation of resistance patterns present in hiv-1 non - b subtypes by using different interpretation algorithms . these discrepancies illustrate how hard it may be to reach an unambiguous conclusion , despite the efforts made over the last decade to interpret drug resistance in hiv-1 non - b subtypes [ 8387 ] . the increasing prevalence of hiv-1 non - b subtypes could have implications not only for the development of resistance but also for diagnosis , vaccine design , and the clinical management of hiv infection [ 65 , 90 , 91 ] . moreover , patients infected by certain hiv-1 non - b subtypes present accelerated disease progression [ 9297 ] and higher cognitive impairment . in the light of the above mentioned , the aim of this review is to provide a complete picture about hiv-1 genetic diversity and its implications . mutational escape results in a remarkable degree of viral diversity within hiv-1 and in its adaptation to both immune activity and antiretroviral therapy . however , not all escape mutations are advantageous to the virus since some of them can severely affect viral fitness [ 99 , 100 ] . the extensive genetic diversity of hiv-1 is due to its high replication rate , the error - prone reverse transcriptase , and recombination events that may occur during virus replication [ 101 , 102 ] . the molecular basis of hiv-1 variability is a highly error - prone reverse transcriptase enzyme . the activity of this enzyme , essential for viral replication , is specifically required for the conversion of single - stranded genomic rna into double - stranded viral dna , which is later integrated into the host genomic dna . for this reason , hiv-1 reverse transcriptase inhibitors are powerful inhibitors of hiv-1 replication and represent an important class of antiretroviral agents . hiv-1 reverse transcriptase is a multifunctional enzyme that possesses rna - dependent and dna - dependent dna polymerase activities as well as an rnase h activity that specifically degrades the rna strand of rna / dna hybrids . as an intrinsic property , and in contrast to other dna polymerases , hiv-1 reverse transcriptase lacks a proofreading function . this error - prone nature of reverse transcriptase , together with the high rate of virus production sustained by hiv-1 infection in vivo , strongly contributes to the continuous generation of new viral variants [ 13 , 106 , 107 ] . the rate of nucleotide substitutions introduced by reverse transcriptase is approximately 10 per nucleotide per cycle of replication , which is equal to one nucleotide substitution per genome during a single replication cycle . hiv-1 has a rapid turnover , and it is estimated that approximately 10 virions per day are generated in an infected individual . the composite lifespan of plasma virus and virus - producing cells is very short with a half - life of approximately two days , and an almost complete replacement of wild - type strains by drug resistant virus occurs in plasma within 24 weeks . during antiretroviral treatment , rapid viral turnover in combination with a high mutation rate is a primary factor behind the emergence of hiv variants with antiretroviral drug resistance . each retroviral particle contains two copies of single - stranded rna , and template switches occur frequently during reverse transcription , thus generating mutations and recombination by intramolecular and intermolecular jumps . recombination may link drug resistant mutations in hiv-1 , leading to increased resistance to a particular drug , or the generation of multidrug resistant variants . in addition , recombination may lead to the acquisition of mutations that compensate for a loss in viral fitness or replicative capacity due to previous acquisition of resistance mutations . since recombination can create a multiple drug resistant virus out of two single drug resistant strains , it is generally believed that the capacity of the virus to recombine facilitates the evolution of drug resistance [ 9 , 110 , 113115 ] . this rapid evolution of drug resistance in hiv remains a major obstacle for hiv therapy . recombination is a strategy for viral rejuvenation , and it is likely that recombination between hiv strains may lead to the evolution of fitter forms and viral strains acquiring drug resistance to all major classes of hiv-1 inhibitors . a different scenario could be that a fitter virus can be generated by recombining parts of two parental genomes with lesser fitness , or alternatively a less fit virus can be generated by breaking up favourable combinations of mutations in the parental genomes . this interaction between recombination , mutations , and viral fitness is highly intricate , but nonetheless , recombination and its mechanisms , especially at the level of diverse subtypes , warrant further investigation . the potential for genetic differences among subtypes to yield different patterns of resistance - conferring mutations is supported by natural variation among hiv subtypes in genetic content ( 40% variation in the env gene , and 810% variation in the pol / gag genes ) . this issue acquires special relevance in view of the fact that the hiv pol gene is the major target for all major classes of anti - hiv drugs and most hiv strains show hotspots for recombination in gag - pol and env regions . on the basis of genetic homology , hiv-1 has been classified into four groups : m ( main ) , o ( outlier ) , n ( non - m , non - o ) , and the recently identified p [ 912 ] . the m group further splits into 9 subtypes ( a through j ) [ 1315 ] , as well as at least 58 circulating recombinant forms ( crfs , http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html , last accessed 06 may 2013 ) and multiple unique recombinant forms ( urfs ) . in general genetic variation is of 2535% and 1520% between and within subtypes , respectively . for example , subtypes a and f have been subdivided into five ( a1 , a2 , a3 , a4 , a5 ) and two subtypes ( f1 , f2 ) , respectively . crfs are intersubtype recombinant hiv-1 genomes that have infected three or more subjects who are not epidemiologically related , so they can be assumed to make an epidemiologically relevant contribution to the hiv-1 m group epidemic . the crfs are labelled with numbers rather than letters , and numbered in the order they were first adequately described as the first time . urf variants are widely distributed worldwide , with recombination breakpoints different from those found in crfs . very few isolates of hiv-1 n group have been identified and sequenced from humans , while the diversity of sequences within the o group is nearly as great as the diversity of sequences in the m group . today , the hiv-1 m group , the cause of the worldwide pandemic , has a near global distribution , whereas the n and o groups are restricted to individuals of west african origin . the m and n hiv-1 groups have a common ancestor , one that is most closely related to the siv strain found in chimpanzees ( sivcpz ; pan troglodytes troglodytes ) [ 117 , 118 ] that live mainly in gabon , cameroon , and the republic of the congo . this hiv-1 progenitor probably was passed from chimpanzees to human hunters through blood borne transmission [ 119 , 120 ] . recent evidence suggests that the hiv-1 o and p groups may have originated in wild gorillas , in which the closest relatives of these two groups have been identified [ 10 , 121 ] . phylogenetic analysis of hiv-1 and related viruses from nonhuman primates suggests that three independent transmission events , which happened in early 20th century , spawned the hiv-1 m , n , and o groups [ 119 , 120 ] . in particular , the earliest direct evidence of hiv infection in humans was found retrospectively in a serum sample and in a lymph node biopsy specimen stored in 1959 and 1960 , respectively , in kinshasa in the democratic republic of the congo [ 122 , 123 ] . moreover , they have been instrumental in the validation and extrapolations of molecular clock computer programs used to estimate the time to the most recent common ancestors ( tmrcas ) and evolutionary rates of various hiv lineages [ 123 , 124 ] . the hiv-1 m group appears to be the oldest hiv lineage in humans , with an estimated time to the tmrca in the first two or three decades of the 1900s [ 123 , 124 ] . in particular , the tmrca shared between the m group and sivcpz is estimated at 1853 ( 17991904 ) . cross - species transfer is therefore inferred to have taken place sometime between 1853 and the early 1900s , although there is some uncertainty , given the size of the confidence intervals . hiv-1 m group subtypes can be considered as a result of the high error rate of reverse transcriptase enzyme during virus replication and selective pressure exerted by the immune system . since their introduction into humans , hiv-1 m group subtypes have expanded rapidly in west and central africa and established multiple epidemics around the world . regional expansion of hiv-1 has come in waves with the rapid emergence of specific subtypes due in part to specific modes and routes of transmission . for example , intravenous drug use in southeast asia in the mid-1980s and in eastern europe and russia during the early 1990s led to the rapid spread of crf01_ae and of subtype a , respectively [ 126 , 127 ] . a similar expansion of subtype b hiv-1 transmission occurred among men who have sex with men in north america and europe in the early 1980s however , hiv-1 subtype c ( the most dominant subtype in the world responsible for more than 50% of overall infections ) appears to have slowly emerged throughout the world over the past 10 to 15 years as a consequence of multiple introductions [ 126 , 128 ] . in recent years a substantial increase of recombinant forms has been observed as a consequence of the increased genetic complexity of the global epidemic [ 57 , 59 , 64 , 66 , 7377 , 129131 ] . however , the prevalence of recombinant forms ( estimated to be around the 20% ) is still underestimated . indeed , genetic complexity is not always detected , and this is mainly due to the subtyping of only one genetic region and not of the full genome . consequently , specimens previously considered pure variants may be classified as recombinants when additional viral genes are analyzed . the drugs currently approved for clinical use by the us food and drug administration ( fda ) and used to treat hiv infection belong to 6 distinct classes ( http://www.fda.gov/forconsumers/byaudience/forpatientadvocates/hivandaidsactivities/ucm118915.htm , last accessed 06 may 2013 ) : ( 1 ) seven nucleoside and one nucleotide reverse transcriptase inhibitors ( nrtis ) ; ( 2 ) five nonnucleoside reverse transcriptase inhibitors ( nnrtis ) ; ( 3 ) nine protease inhibitors ( pis ) ; ( 4 ) one fusion inhibitor ( fi ) ; ( 5 ) one cc chemokine receptor 5 ( ccr5 ) antagonist ; ( 6 ) two integrase inhibitors ( inis ) . each of these drug classes act at different steps in the hiv replication cycle ( figure 1 ) . the nnrtis and nrtis block the reverse transcription of the viral rna genome in cdna , which is catalysed by the reverse transcriptase . the inis block the integration of viral genome into the dna of the host cell . although it seems that combination antiretroviral regimens are effective against all hiv-1 group m subtypes , there is emerging evidence of subtype differences in drug resistance relevant to antiretroviral strategies in different parts of the world . for this purpose , extensive sampling of hiv genetic diversity and ad hoc analyses are required to tailoring of antiretroviral therapies in different parts of the world . hiv-1 non - b subtypes present clade - specific substitutions in positions related to drug resistance that could accelerate the emergence of drug - resistant viruses , change or induce alternative pathways of resistance , influence viral replicative capacity in vitro , impair the interpretation of genotypic resistance algorithms [ 70 , 77 , 134137 ] , and affect drug - binding affinity [ 138 , 139 ] . specific mutation development varies with different subtypes because of the intrinsic properties of the virus as well as the different pressures of antiretroviral drugs . in particular , three factors are involved in this phenomenon.intersubtype differences in codon usage : subtype differences in nucleotide and mutational motifs , which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs , may affect the genetic barrier for resistance , as shown in the development of some mutations in different hiv-1 proteins , such as ( i ) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes ( see section 4.2.2 ) [ 140 , 141 ] ; ( ii ) mutations at the protease codons 74 in c subtypes , 82 in g subtypes , and 89 in several non - b subtypes such as c and crf02_ag ( see section 4.2.3 ) [ 87 , 142145 ] ; ( iii ) mutations in the integrase gene at codons 140 , 148 , 151 , 157 , and 160 ( see section 4.2.4 ) [ 146 , 147].intersubtype amino acid differences involved in minor structural changes in the targets of therapy : in this situation , different mutations emerge under the same drug pressure ( e.g. , the protease mutation d30n ; see section 4.2.3).intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance ( e.g. , the reverse transcriptase mutation k65r ; see section 4.2.1 ) . intersubtype differences in codon usage : subtype differences in nucleotide and mutational motifs , which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs , may affect the genetic barrier for resistance , as shown in the development of some mutations in different hiv-1 proteins , such as ( i ) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes ( see section 4.2.2 ) [ 140 , 141 ] ; ( ii ) mutations at the protease codons 74 in c subtypes , 82 in g subtypes , and 89 in several non - b subtypes such as c and crf02_ag ( see section 4.2.3 ) [ 87 , 142145 ] ; ( iii ) mutations in the integrase gene at codons 140 , 148 , 151 , 157 , and 160 ( see section 4.2.4 ) [ 146 , 147 ] . intersubtype amino acid differences involved in minor structural changes in the targets of therapy : in this situation , different mutations emerge under the same drug pressure ( e.g. , the protease mutation d30n ; see section 4.2.3 ) . intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance ( e.g. , the reverse transcriptase mutation k65r ; see section 4.2.1 ) . the following subsections describe the impact of hiv-1 subtypes on the different classes of antiretrovirals so far available . table 1 provides an overview of the resistance associated mutations which are related to subtype diversity . the impact of subtype in terms of emergence of resistance in the nrti antiretroviral class is mainly due to the more rapid selection of primary resistance mutations in hiv-1 subtype c infected patients than in those infected by a subtype b virus . some of the differences in rates of acquisition of the mutation k65r or thymidine analogue mutations ( tams ) are doubtless due to treatment regimens and disease stage , as well as access to viral load testing in many developing countries . however , one study suggests that increased rates of k65r acquisition in subtype c may be due to the nature of the subtype c rna template . the lysine to arginine mutation at position 65 ( k65r ) is a major mutation that confers broad high - level resistance to most nrtis , except zidovudine . the nucleotide sequence at codons 64 - 65 - 66 ( containing a homopolymeric stretch of adenine bases ) differs between subtypes b and subtype c viruses . this leads to reverse transcriptase pausing during the synthesis of double - stranded dna from the single - stranded dna intermediate template , a process that is template - specific but independent of the reverse transcriptase enzyme [ 14 , 15 , 148 ] . subsequent misalignment of the subtype c template and primer leads to the aag to agg change responsible for the k65r mutation ( figure 2 ) . on the contrary , subtype b templates are prone to frequent pausing at codon 67 , facilitating the generation of mutation d67n and tams instead of k65r ( figure 2 , table 1 ) [ 148 , 150 , 151 ] . a recent study showed a very high rate ( > 65% ) of k65r for patients infected by subtype c who failed tenofovir - based first - line regimens in south africa . furthermore , a rate of 7 to 15% of k65r and/or k70e mutations was observed in subtype c endemic area in patients failing regimens including stavudine , didanosine , or zidovudine . studies from israel reported a high frequency of k65r with treatment failure in subtype c viruses from ethiopian immigrants , while in indian patients a rate of k65r around 1012% at failure of first line combination therapy including stavudine , lamivudine , and nevirapine was observed . by using ultrasensitive pyrosequencing , variants carrying k65r mutation showed a higher frequency in subtype c infected patients than those infected by subtype b ( 1.04% versus 0.25% ) . in addition to the recruitment for monitoring k65r prevalence in patients with subtype c hiv infection , also the tam pathway ( 67n/70r/215y ) deserves further attention . indeed , in patients infected by subtype c from botswana , india , south africa , and malawi tams were also observed at treatment failure with zidovudine and didanosine [ 153 , 155 , 157 , 158 ] . larger numbers of patients and followups will be required to determine whether any consistent effect of the emergence of k65r in subtype c is clinically relevant . the emergence of mutations associated with resistance to nnrtis occurs after a single dose of nevirapine ( sdnvp ) [ 159161 ] , a procedure recommended for the prevention of mother - to - child hiv transmission ( pmtct ) by initial guidelines from the world health organization ( who ) [ 162 , 163 ] . replacement of sdnvp may be necessary to reduce infant hiv infection risk and drug resistance development . indeed , high frequency of drug resistance was observed in 69% of pregnant women infected with subtype c , 36% of those with subtype d , 19% of those with subtype a , and 21% of those with the crf02_ag , despite the absence of resistance before the administration of antiretroviral therapy [ 163167 ] . moreover , by using ultrasensitive sequencing techniques , several studies revealed that in patients infected by subtype c a higher prevalence of nevirapine - resistance mutations ( k103n and y181c ) was found , reaching 70% to 87% , as compared with 42% of patients with subtype a with resistant viruses [ 168 , 169 ] . these findings underscore the role of viral subtype in facilitating the development of drug resistance , which is exacerbated by the fact that resistance to nnrtis can also be transmitted through breast feeding . indeed , the v106 m mutation is commonly selected in subtype c viruses ( in approximately 30% of patients ) after exposure to nevirapine or efavirenz , whereas a v106a substitution is only rarely selected ( in approximately 5% of patients ) by these two nnrtis in other subtypes ( table 1 ) . this peculiarity of subtype c results from the fact that v106 is encoded by gta in subtype b viruses and gtg in subtype c viruses [ 141 , 171 ] . a single g - to - a transition at the first position of codon 106 in subtype c viruses results in v106 m , which confers high - level resistance to efavirenz and nevirapine . in contrast , in subtype b viruses , v106 m requires two nucleotide substitutions ( gta - atg ) and therefore occurs infrequently [ 140 , 141 ] . k103n and y181c mutations remain important in both subtypes b and c , with k103n occurring in 40% of subtype b and 29% of subtype c viruses and y181c occurring in 23% of subtype b and 12% of subtype c viruses . in a recent italian study , the prevalence of mutation k103n in nnrti - treated patients was lower in subtype c infected patients compared with patients infected by b , f , or crf02_ag subtypes ( table 1 ) . conversely , the mutations y181c and y188l were found with higher frequency in patients infected with subtype c compared to patients infected with subtype b . another substitution that is seen more frequently among patients with a subtype c virus is g190a , which is also a result of naturally occurring g190a reverse transcriptase polymorphisms at residue 98 are common in subtype g , but they are also associated with nnrti resistance in subtype b ( table 1 ) [ 15 , 173 ] . the second - generation nnrti etravirine has demonstrated good efficacy across subtypes . a recent study highlighted the fact that hiv-1 genetic variant does not significantly influence the genotypic prediction of etravirine susceptibility in infected individuals failing efavirenz containing regimens . however , among the etravirine resistance associated mutations , some polymorphic substitutions resulted in drug - naive individuals infected with non - b subtypes , especially crf02_ag . noteworthy , in a recent study it was found that the mutation e138k was the first mutation to emerge in either subtype b , c , or crf02_ag clinical isolates under etravirine pressure ( table 1 ) . another study confirmed this finding for subtype c , while a preferential selection of y181c for the subtype b virus was observed ( table 1 ) . a novel mutation in the c - terminal domain of reverse transcriptase ( n348i ) has recently been reported to reduce susceptibility to etravirine in subtypes a , b , and c ( table 1 ) . in particular , in a south - african clinical trial , the n348i mutation was observed in 24% of subtype c infected patients failing a first - line nnrti regimen ( most commonly with nevirapine ) . this mutation is not included in standard mutation lists or algorithms , but more data are urgently required to determine its clinical relevance . to date , the efficacy and the resistance patterns of the novel second - generation nnrti rilpivirine seem to be similar across the different subtypes [ 181 , 182 ] . a study focused on hiv-1 subtype crf01_ae showed that the presence of mutations associated with resistance to rilpivirine are uncommon in patients infected by this subtype and failing a first - line nnrti - containing regimen . however , cross - resistance between rilpivirine and etravirine was high . moreover , approximately 60% of patients had a high level of etravirine resistance , thus suggesting that the role of etravirine in hiv-1 subtype crf01_ae infected patients in second - line therapy is limited in late nnrti failure settings . nnrti resistance associated mutations such as y181i , y188l , g190a , k101a , v106i , and v179i are natural polymorphisms in hiv-2 , thus hiv-2 proves to be naturally resistant to all nnrtis . nonpolymorphic substitutions in the protease gene have a greater impact on baseline susceptibility to pis than polymorphic mutations . however , intersubtype amino acid differences can create subtle structural differences in the targets of therapy . for example , subtype b infected patients receiving nelfinavir are more likely to develop d30n than those with viruses belonging to subtypes c , f , g and crf01_ae , which are more likely to develop l90 m or n88s ( table 1 ) [ 186 , 187 ] . more specifically , by comparing b and g subtypes , even in cases where the first mutation is l90 m in both these subtypes , indeed , in subtype b , l63p is the second mutation and occurs in almost all cases , suggesting that the progression of resistance is dependent on the emergence of this mutation , followed by the selection of v77i and other mutations . differently , in subtype g , in almost 100% of cases , l89i follows the emergence of l90 m , suggesting a role in subtype g similar to that of l63p in subtype b. in this regard , findings indicate that the association of l89i with l90 m may further increase the pi resistance of subtype g viruses when compared with l90 m alone . although subtype c isolates from ethiopian immigrants to israel favoured the l90 m pathway , patients with subtype c viruses in botswana did have d30n , suggesting that subtype c viruses from ethiopia and southern africa might be different [ 186 , 189 ] . the basis for the higher preponderance of d30n in subtype b than in other subtypes may be related to the flexibility of the protease flap region and destabilization of the pi complex in subtype b , whereas an accessory n83 t mutation may be needed to rescue fitness and confer resistance in subtype c [ 142 , 190 ] . recent evidence has suggested that the polymorphism at codon 36 in the protease gene ( m36 in subtype b and i36 in most other non - b subtypes ) affects both the patterns of resistance that emerge under drug pressure and viral replication capacity ( table 1 ) . similarly , the m89 polymorphism in subtypes a , c , and crf01_ae ( l89 in subtype b ) preferentially leads to the emergence under drug pressure of the m89 t mutation , which confers high - level resistance to nelfinavir , atazanavir , and lopinavir . there is also in vitro evidence that crf2_ag viruses with the 17e/64 m polymorphisms demonstrate hypersusceptibility to certain pis ( nelfinavir , atazanavir , and indinavir ) . differences in polymorphisms in the protease gene have been reported among several non - b subtypes ; these include the following protease residues : 10 , 20 , and 63 in subtype a ; 20 , 53 , 63 , 74 , and 82 in subtype c ; 13 and 20 in subtype d ; 10 , 14 , 20 , and 77 in subtype f ; 20 , 67 , 73 , 82 , and 88 in subtype g ; 20 , 63 , 82 , and 89 in the crf01_ae ; 20 and 89 in the crf02_ag ( table 1 ) . in particular , in patients treated with pis , l89v was predominantly found in crf02_ag , while the tipranavir resistance mutation t74p was predominantly found in the c subtype in comparison to b subtype . the mutation v82 m was mainly found in subtype g while the mutations v82a / f / s were mainly present in other subtypes ; the mutation n88s was found in subtypes c and crf02_ag , while n88d was present in subtype b . in southern brazil , scientists reported a lower relative frequency of primary resistance to pis in subtype c rather than in subtype b . despite clear evidence for preferential emergence , diminished susceptibilities among wild - type isolates have been found for crf02_ag recombinant viruses in three different studies of nelfinavir and atazanavir [ 138 , 173 , 194 ] . a study suggested that distortions in the k26 pocket of the a / g protease may be responsible for a lower binding energy of nelfinavir and lower susceptibility of a / g viruses . the protease and gag genes coevolve as a functional unit when hiv is subjected to drug pressure from pis . mutations in gag can act as compensatory substitutions that can increase both rates and levels of resistance to pis , as well as viral replication capacity . no genotypic system for the determination of drug resistance to pis currently monitors gag , despite the fact that relevant mutations in gag can increase resistance by a factor of 2 to 2.5 , depending on the subtype . it is likely that different subtypes could develop compensatory gag mutations at different rates ; this might ultimately justify the genotyping of gag in resistance testing . the primary mutations in the integrase gene associated with ini resistance are e92q , y143r / c , q148k / r / h , and n155h . the residues associated with primary resistance seem to be highly conserved across subtypes , but polymorphisms at the sites of secondary resistance mutations are more common in non - b subtypes [ 147 , 198200 ] . signature subtype differences in integrase at codons 140 , 148 , 151 , 157 , and 160 among hiv subtypes may affect the genetic barrier for resistance . these variations predict higher genetic barriers to the development of g140s and g140c mutations in subtypes c , crf02_ag , and a / crf01_ae , as well as higher genetic barriers to v151i in subtypes crf02_ag and crf01_ae [ 146 , 147 ] . regarding the impact of subtype on primary resistance development , it was recently observed that the subtype b integrase enzyme with the n155h mutation ( e92q ) exhibited increased resistance to raltegravir compared to the subtype c enzyme ( table 1 ) . in addition a new potential resistance pathway for raltegravir might include the mutation g118r , a substitution associated with lower susceptibility to raltegravir in crf_02ag subtypes . notably , polymorphisms such as t97a , v151i , and g163r , already known as raltegravir secondary resistance mutations , showed a considerable prevalence ( 9% ) in several recent studies also analyzing non - b subtype infected patients [ 203 , 204 ] . the additional ini - resistance associated mutations k156n and s230n mutations were more frequent in b subtype while v72i , l74i , t125a , v201i , and t206s were more frequent in certain non - b subtypes [ 200 , 205 ] . no differences in phenotypic raltegravir resistance were found in several non - b isolates tested . dolutegravir is a second - generation ini which recently entered the battery of antiretrovirals against hiv-1 infection . it showed a higher genetic barrier to resistance and retains activity against viruses with resistance to raltegravir or elvitegravir . interestingly , among the mutations associated with in vitro resistance to dolutegravir , the mutations l101i and t124a were polymorphic and significantly more prevalent in patients with non - b subtypes . in addition , mutation r263k seems to be preferentially selected in subtype b under dolutegravir pressure ( table 1 ) . among the eis today available in clinics , both enfuvirtide ( targeting gp41 hiv-1 protein ) and maraviroc ( targeting cellular ccr5 receptors ) have shown antiviral activity against hiv-1 b and non - b subtypes [ 207 , 208 ] . however , the high level of diversity ( 20 to 40% ) in the env region predicts that this class of drugs will probably have higher potential for natural and emergent drug resistance . clinical data have shown that the fi enfuvirtide is active against non - b subtypes owing to a highly conserved binding domain , although hiv-2 and hiv-1 class o viruses have natural resistance against this drug [ 209 , 210 ] . so far , some studies suggest that there are significant differences in baseline susceptibility to hiv eis among the predominant hiv-1 subtypes [ 211 , 212 ] . for instance , it was found that in drug - naive patients many more mutations associated with resistance to eis occur in subtype c compared with subtype b strains ( table 1 ) . due to the worldwide spread of non - b viruses and the introduction of antiretroviral drugs in developing countries ( known for the largest assortment of non - b subtypes ) , further knowledge concerning responsiveness hiv-1 drug resistance in non - b subtypes is required . moreover , it should be taken into account that the increasing prevalence of hiv-1 non - b subtypes could have several implications not only for the development of resistance but also for other topics such as the response to antiretroviral therapy , disease progression , and transmission . there is a solid body of evidence indicating that the type and degree of hiv-1 resistance to nrtis , nnrtis , and pis vary between different subtypes [ 136 , 186 , 213 , 214 ] . the development of nelfinavir resistance in subtypes b and g represents a classic example of this phenomenon . a different level of resistance has been observed among different subtypes . indeed , the recombinant form crf02_ag is more susceptible to nelfinavir and ritonavir than subtypes c and f ; subtype g is more sensitive to tipranavir and lopinavir than other subtypes , and the subtype c has accelerated risk in developing resistance to tenofovir [ 189 , 215 , 216 ] . an explanation for the extreme variability of hiv-1 subtypes in the response to antiretrovirals can be given by the presence of some polymorphisms that can influence both the emergence of drug - resistance mutations and the response to drugs . for example , polymorphisms at residues 20 and 36 of hiv-1 protease decrease the genetic barrier to tipranavir resistance in subtypes a , c , f , and g , while nucleotide heterogeneity at 64 and 65 positions in the reverse transcriptase accelerates development of k65r in subtype c [ 150 , 189 ] . in some cases , drug exposure may lead to amplification of such polymorphisms as a98g / s in reverse transcriptase and m36i , k20i , and l89 m in protease , leading to a potential for resistance . it is commonly accepted that , as a result of a high degree of polymorphism in the protease gene , drug - naive patients infected with non - b subtypes present a certain degree of resistance to the pis . however , the data which support this assumption are largely controversial , as highlighted by two studies that showed conflicting results . indeed , in a study analysing the hiv-1 phenotype from 42 patients ( 19 , subtype g ; seven , subtype c ; six , subtype f ; 10 , crf02_ag ) by the antivirogram assay ( virco bvba ) , no differences in baseline susceptibility to any pi were found , with the exception of an unexpected hypersusceptibility to nelfinavir with crf02_ag . on the contrary , a second study , investigating drug susceptibility of 39 isolates from treatment - naive ghanaian patients , mostly infected with crf02_ag , showed reduced susceptibility to several pis , especially nelfinavir . this reduced in vitro susceptibility is not in agreement with the available clinical data , which show no difference in time to undetectable viral load among different subtypes [ 217219 ] . continuous research on the role of polymorphisms in the development of drug resistance is therefore necessary . studies are needed to assess genotypes both before and after therapy in the context of possible associations between polymorphisms and drug resistance . this area of research could include polymorphism variability in variants of the same subtype in different geographic regions . this information might improve the efficacy of certain drug combinations over others in the context of second- or third - line therapeutic strategies . polymorphisms should improve current algorithms for interpretation of genotyping results in a subtype - independent way . as access to antiretroviral therapy in resource limited settings increases , where non - b subtypes are predominant , it remains imperative to establish appropriate treatment strategies for long - term clinical benefit that limits the emergence of drug resistance . the use of nontoxic , effective antiretroviral drugs should yield excellent clinical responsiveness , regardless of the viral subtype . subtype differences , suboptimal therapies , and deficiencies in health care delivery systems can create conditions for accelerated development of resistance . urgent recruitment for low - cost viral - load monitoring is needed to prevent and detect drug resistance , as well as to avoid unnecessary treatment switches [ 220 , 221 ] . unfortunately , pooling of resistance data often masks the role of regional differences in viral subtypes and antiretroviral therapies in the development of drug resistance . in resource - poor settings , such studies have used different nrti backbones ( e.g. , combinations of zidovudine and didanosine , zidovudine and lamivudine , or stavudine and lamivudine ) . in parts of africa , treatment failure has been reported in as many as 40% of patients after 2 years . of note , resistance rates in india to two drug classes were observed in more than 80% of patients who failed their first - line regimen using combinations of nrtis and nnrtis . larger longitudinal studies are necessary to determine the response to first - line combinations of antiretroviral drugs . the availability of genotypic resistance testing needs to be expanded to include all countries in which antiretroviral drugs are used . cross - resistance among drugs is important , especially in settings where treatment options may be limited . relatively few in vitro comparative data are available for pis in non - b subtypes , even if such data may be crucial for an understanding of cross - resistance to this class of antiretrovirals [ 191 , 224 ] . such data are important , since pis are often the only available option for drug sequencing in resource - limited settings after the failure of first- or second - line treatment . this means that resistance to pis usually requires the presence of large numbers of resistance mutations . for this reason , there is an advantage of using pis to avoid early resistance development . therefore , differences among subtypes with regard to drug resistance are likely to be more important for nrtis , nnrtis , and inis than for pis . our knowledge of the impact of hiv-1 subtypes on responses to antiretroviral therapy is very limited . most studies have been small or poorly designed , and only a few have had an evaluation period longer than 48 weeks . an important limitation of these studies was to compare subtype b with non - b subtypes grouped together , a necessary oversimplification when dealing with small numbers of patients infected with non - b subtypes [ 217219 , 225227 ] . a study by the paediatric european network for treatment of aids ( penta 5 trial ) investigated the response to therapy of 113 children infected with 7 different subtypes ( a , 15% ; b , 41% ; c , 16% ; d , 9% ; f , 5% ; g , 2% ; h , 1% ) and the two crfs crf01_ae ( 5% ) and crf02_ag ( 7% ) . however , these results should be used with caution because the study did not have the statistical power to rule out minor differences between subtypes . moreover , only four drugs were used ( zidovudine , lamivudine , abacavir , and nelfinavir ) . although polymorphisms at resistance - associated positions were highly prevalent in non - b subtypes , no negative impact of the mutations in virological responses was found ; therefore , the authors concluded that polymorphisms at resistance - associated positions in subtype b could not necessarily be interpreted as conferring resistance in non - b subtypes . retrospectively analysed the virological response in 362 patients : 265 europeans infected with subtype b and 97 africans infected with non - b subtypes . subtype was presumed from ethnic and epidemiological data , with confirmation further extrapolated from genotyping the samples from 60% of the africans and 30% of the european patients . there was a significant imbalance between the two groups in several important parameters , including gender , transmission - risk groups , cd4 cell counts , and antiretroviral regimens used . results showed no difference in time to undetectable viral load or recovery of cd4 count ; however , a significant difference was observed in viral load over time , with a continuous increase in the african group after 9 months , suggesting a higher rate of therapy failure . based on indirect evidence ( i.e. , the number of resistance mutations at time of failure ) , the authors concluded that the difference in virological response was mainly due to poorer adherence in the african group rather than the infecting viral subtype . as the study was not controlled for adherence , its conclusions remain controversial . a study which analysed retrospectively b and non - b subtypes ( mainly focused on pure subtype a or recombinants with a subtype a protease ) showed no differences in the time to undetectable viral load or in the viral load measured over time . however , the gain in cd4 count was significantly lower in the non - b group , which showed an increase of 161 cells / mm , compared with 236 cells / mm in the subtype b group . these results were consistent with those of camacho , who compared retrospectively the pathways to nelfinavir resistance in 101 patients ( 46 subtype b , 55 subtype g ) failing a first - line regimen including this pi . at the time of failure , the only parameter significantly different between the two groups was the cd4 count ( 423 cells / mm in the subtype b group and 360 cells / mm in the subtype g group ; p < 0.001 ) . assessed virologic and immunologic responses to starting haart in a large cohort of 2116 patients , with the specific objective of comparing outcomes in patients with subtype b infection and those with subtype c , subtype a , crf02_ag , and subtype d infection , the predominant non - b subtypes circulating in the united kingdom . overall , 1906 ( 90% ) patients achieved viral load undetectability within 12 months after they started haart , of whom 335 ( 18% ) subsequently experienced virologic rebound . in adjusted analyses , viral load suppression occurred more rapidly in patients infected with subtype c ( hazard ratio ( 95% confidence interval , ci ) 1.16 [ 1.011.33 ] , p = 0.04 ) and subtype a ( 1.35 [ 1.041.74 ] , p = 0.02 ) relative to subtype b infection . the virologic rebound occurred marginally faster in patients with subtype c infection ( 1.40 [ 1.001.95 ] , p = 0.05 ) , but the hazard of virologic rebound was similar with other subtypes . although patients infected by subtype b viruses showed higher baseline cd4 cell counts and maintained the advantage throughout therapy , cd4 cell count recovery occurred at similar rates with all subtypes . the effect of pretreatment hiv-1 drug resistance on the response to first - line combination antiretroviral therapy in sub - saharan africa has recently been evaluated in a large prospective cohort . pretreatment drug resistance results were available for 2579 ( 94% ) of 2733 participants . among them , 123 ( 5% ) had pretreatment drug resistance to at least one prescribed drug , and 52 ( 2% ) had pretreatment drug resistance and received fully active antiretroviral therapy . more than 50% of participants for whom it was possible to determine the subtype were infected by hiv-1 subtype c ( 1405 patients , 54% ) , while 638 ( 25% ) harboured subtype a , 296 ( 11% ) d , 117 ( 5% ) a / g recombinant , 68 ( 3% ) g , 48 ( 2% ) other recombinants , seven ( < 1% ) other subtypes , and five ( < 1% ) b. compared with participants without pretreatment drug resistance , the odds ratio ( or ) for virological failure ( or ( 95% ci ) : 2.13 [ 1.443.14 ] , p < 0.0001 ) and acquired drug resistance ( 2.30 [ 1.553.40 ] ; p < 0.0001 ) was increased in participants with pretreatment drug resistance to at least one prescribed drug but not in those with pretreatment drug resistance and fully active antiretroviral therapy . cd4 cell count increased less in participants with pretreatment drug resistance than in those without ( 35 cells per l difference after 12 months ; 95% ci 1358 ; p = 0.002 ) . further ad hoc studies are needed to better evaluate the therapeutic implications of specific non - b subtype differences in terms of long - term efficacy of different antiviral regimens . several studies on disease progression showed that , among non - b subtypes , subtypes c and d were found to be more aggressive , followed by g , ae , ag , and a , the least aggressive of all hiv-1 subtypes [ 9297 ] . in this regard , it should be noted that because the different hiv-1 subtypes are not uniformly dispersed , comparisons of virulence and transmissibility are hampered by potential confounders , such as ethnic , socioeconomic , and other epidemiological factors . the report by kiwanuka et al . concerning this topic provided a good opportunity to compare rates of disease progression associated with these different subtypes within a similar population , thanks to the cocirculation of hiv-1 subtypes a and d , and several intersubtype recombinants in the rakai district of uganda . in particular , kiwanuka et al . compared rates of progression among hiv-1 seroconverters who were followed as part of the rakai health sciences program . subjects identified during 19972002 were followed through 2004 , when antiretroviral therapy became available in rakai district . the primary end point was the time to achievement of a cd4 cell count of 250 cells / mm or death due to aids . progression to a cd4 cell count of 250 cells / mm was significantly less common among subjects infected with hiv-1 subtype a ( 20% ) , compared with subjects infected with subtype d ( 40% ) , recombinant forms ( 40% ) , or multiple subtypes ( 53% ) ( p = 0.03 ) . death from aids was also less common among subjects infected with hiv-1 subtype a. these differences were reflected in the longer time to aids onset for subjects infected with hiv-1 subtype a ( 8.05 years ) compared with those infected with non - a subtypes ( d = 6.49 years ; recombinant forms = 5.57 years ; multiple subtypes = 5.80 years ) . by multivariable models ( adjusting for viral load , age , and sex ) , subjects infected with non - a subtypes more likely progressed to aids compared with those infected with hiv-1 subtype a. similarly , subjects infected with non - a subtypes had an approximately 68-fold greater risk of death from aids . viral factors that could explain some of the hiv-1 pathogenicity are higher ex vivo replicative capacity , higher genetic diversity , and cxcr4 coreceptor usage [ 231233 ] . in this regard , a less common emergence of cxcr4-using ( x4 ) variants was found in hiv-1 subtype a infection compared with hiv-1 subtype d infection , thus explaining the apparent lower virulence of hiv-1 a [ 234 , 235 ] . a european study showed how subtype d has a fourfold higher rate of cd4 count decline , in the absence of antiretroviral therapy , compared with other subtypes in the study ( a , b , c , and crf02_ag , which had similar rates of cd4 loss ) , even when adjusted for baseline cd4 count . subtype d was also associated with higher rates of dementia in individuals with advanced immunosuppression if compared with subtype a . a study by kiwanuka et al . showed how in uganda subtype a viruses have a significantly higher rate of heterosexual transmission than subtype d viruses . the faster disease progression and the lower rate of heterosexual transmission of subtype d compared with subtype a can explain the changes in the proportions of subtypes a and d observed over time in uganda and kenya [ 238 , 239 ] . in particular , a significant decrease in the prevalence of subtype d with a concurrent increase of subtype a was observed . despite the importance of viral characteristics in determining the rate of hiv-1 disease progression , recent findings from genomic studies show that host genetic factors also play a crucial role . the genetic determinants that influence susceptibility to hiv-1 and limit aids vary in different populations and among individuals . meta - analyses of large cohort studies have identified several genetic variants that regulate hiv cell entry ( particularly chemokine coreceptors and their ligands with copy number variations ) , acquired and innate immunity ( major histocompatibility complex ( mhc ) , killer immunoglobulin - like receptors ( kirs ) , and cytokines ) , and others ( trim5- and apobec3 g ) that influence the outcome of hiv infection [ 240242 ] . of the various genes that contribute toward host genetic propensity , mhc turns out to be the major contributor because it is responsible both for restriction of cytotoxic t lymphocyte ( ctl ) epitopes and for the emergence of ctl escape mutants . leukocyte antigen ( hla ) alleles have been shown to be associated with the rate of disease progression in africans and caucasians [ 243245 ] . the interplay of viral , immune , and host genetic factors in the control of hiv-1 replication has been recently evaluated in hiv controllers [ 246 , 247 ] . similar efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to understand fully the complex interplay of virus and host in aids pathogenesis . hiv diversity plays a central role in the hiv pandemic ; for this reason , it is today imperative that global molecular epidemiology surveillance is continued and improved using rigorous sampling strategies . the improved knowledge of the significance of non - b subtypes for resistance evolution and interpretation , in response to antiretroviral therapy , disease progression and vaccine design are becoming mandatory today not only because antiretroviral therapy is being introduced in countries where non - b subtypes are driving the epidemic but also because the number of infections by these variants is increasing sharply in previously subtype b homogeneous areas such as europe and north america [ 5769 , 7173 , 248 ] . it is reassuring that current antiretroviral strategies appear to be effective against a broad spectrum of hiv subtypes . however , further ad hoc studies in larger and homogenous populations infected by different specific non - b subtypes are required to better evaluate their therapeutic implications in terms of long - term efficacy of different antiviral regimens . further elucidation of viral polymorphisms and properties associated with transmission , viral load set point , and disease progression may lead to new approaches to disease prevention and treatment . efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to fully understand the complex interplay of hiv and host in aids pathogenesis .

despite advances in antiretroviral therapy that have revolutionized hiv disease management , effective control of the hiv infection pandemic remains elusive . beyond the classic non - b endemic areas , hiv-1 non - b subtype infections are sharply increasing in previous subtype b homogeneous areas such as europe and north america . as already known , several studies have shown that , among non - b subtypes , subtypes c and d were found to be more aggressive in terms of disease progression . luckily , the response to antiretrovirals against hiv-1 seems to be similar among different subtypes , but these results are mainly based on small or poorly designed studies . on the other hand , differences in rates of acquisition of resistance among non - b subtypes are already being observed . this different propensity , beyond the type of treatment regimens used , as well as access to viral load testing in non - b endemic areas seems to be due to hiv-1 clade specific peculiarities . indeed , some non - b subtypes are proved to be more prone to develop resistance compared to b subtype . this phenomenon can be related to the presence of subtype - specific polymorphisms , different codon usage , and/or subtype - specific rna templates . this review aims to provide a complete picture of hiv-1 genetic diversity and its implications for hiv-1 disease spread , effectiveness of therapies , and drug resistance development .

every year more than 14 million cases of obstetric hemorrhage occur , resulting in an estimated 127,000 deaths.1 postpartum hemorrhage ( pph ) , blood loss of 500 ml or more , accounts for the majority of these hemorrhage deaths.2 pph is the leading cause of maternal death in low income countries and is the primary cause of approximately one - quarter of global maternal deaths.3 among pph survivors , an estimated 12% will suffer from the consequences of severe anemia.2 several factors contribute to the high pph mortality estimates . in most developing countries , 50% or more of deliveries are attended by unskilled providers at home.4 in addition , health facilities are often not adequately staffed or lack medicines that can address pph.5 these structural barriers are further complicated by difficulties in predicting who will develop pph . many women who develop pph do not present with any of the risk factors typically associated with the complication.6 consequently , pph is an obstetric complication that requires effective preventive interventions , tailored to the diverse needs of women and providers in resource - poor settings . the purpose of this paper is to present current perspectives on the prevention of pph , particularly in resource poor - settings , where pph is the leading cause of maternal mortality.3 we reviewed historical events and the current evidence related to pph prevention and highlight the progress in policy and program implementation to reduce this disease . we reviewed the current strategies being implemented to prevent pph , ranging from active management of the third stage of labor ( amtsl ) in health facilities to the use of misoprostol in home births , which was given particular attention . in addition , we looked at challenges to the implementation and scale - up of these interventions , as well as examples of ongoing efforts that could be positions as opportunities to increase access to pph prevention interventions . using pubmed , we conducted a review of the literature on randomized controlled trials ( rcts ) of interventions to prevent pph . we then searched pubmed and google scholar for nonrandomized field trials of interventions to prevent pph . we limited our review to interventions that are discussed in the current world health organization ( who ) recommendations for pph prevention , which we consider to be key interventions , and present evidence on the use of these interventions from 2000 to 2013 . the following search terms were used , among others , in varying combinations : postpartum hemorrhage , pph , pph prevention interventions , active management of the third stage of labor , amtsl , controlled cord traction , cord clamping , uterine massage , oxytocin , ergometrine , misoprostol , systematic review , cochrane review , randomized controlled trial , rct , operations research , low - resource settings , and developing countries . we assessed the evidence from rcts of pph prevention interventions , as well as evidence from field trials and implementation programs . we focused our review on comparisons of nondrug pph prevention interventions versus no intervention and of uterotonics versus placebo ; this review was not intended to decipher the relative effectiveness of uterotonic drugs . the interventions and conventional uterotonic drugs reviewed are those presumed to , either alone or in combination with other drugs , prevent pph . the nondrug interventions included were amtsl and the specific components ( ie , controlled cord traction , cord clamping , and uterine massage ) . the conventional uterotonics included were oxytocin , ergot - based alkaloid ( ergometrine ) , and misoprostol . to avoid duplication , we started with systematic reviews ( often cochrane reviews ) conducted since 2000 and then added individual studies conducted after the review . we also searched for other studies not included in the most recent reviews that would meet our search criteria , including peer - reviewed articles , documents in the gray literature , manuals , reports , clinical guidelines , and relevant publications from organizations working to promote pph prevention , such as the who , the international federation of gynecology and obstetrics ( figo ) , the international confederation of midwives ( icm ) , as well as the work of many other nongovernmental organizations . we recognize the barriers to implementation that developing countries may face and have described the challenges to and opportunities for scaling up recommended interventions . we focused specifically on scalability in light of limited access to services and shortages in skilled health care workers and commodities . figure 1 presents a timeline of the important milestones related to pph prevention , including discoveries , research publications , policies , and programs . to provide a historical perspective , we started in 1953 with the elucidation of the amino acid sequence of oxytocin , followed by its biochemical synthesis.710 following these discoveries , a landmark in the history of pph prevention occurred when the three components of amtsl were first described in 1962 : the administration of a prophylactic uterotonic drug , early cord cutting and clamping , and controlled cord traction.11 however , it was not until the 1980s that data from an rct of amtsl revealed a significant reduction in the incidence of pph compared with expectant management of the third stage of labor.12 more than two decades later , figo and icm released their first statement on amtsl.13 then , recognizing that amtsl could only be provided by skilled attendants , thus excluding women who deliver at home and limiting the coverage of this intervention , researchers were encouraged with the discovery and potential of a prostaglandin analogue in tablet form ( ie , misoprostol).14 in 2005 , the first placebo - controlled trial of misoprostol use for pph prevention at home births was carried out , with promising results.15 the findings paved the way for women with limited or no access to health facilities to have a pph prevention intervention delivered at home , but the use of misoprostol for this purpose also introduced policy and program - related challenges . a considerable impediment was that at the time , misoprostol was an off - patent drug that was only on the market for the treatment of gastric ulcers , and in many developing countries , a product needs to be registered for the specific indication for which it is marketed . in 2006 , the who organized a technical consultation meeting to review the evidence for interventions to prevent and treat pph ; the following year , the who published recommendations based on this meeting , which included support of the use of misoprostol for pph prevention in the absence of oxytocin but not at home births.16 by that time , individual country efforts to take advantage of misoprostol for pph prevention had already started . in 2006 , nigeria was the first country in the world to register the use of misoprostol for pph prevention.17 five years later , another important landmark policy reform occurred , which was the inclusion of misoprostol in the who model list of essential medicines in 2011,18 followed by a revision of the recommendations for the prevention and treatment of pph , in 2012.3 around this time , the evidence regarding the impact of misoprostol in reducing pph became clear , and its inclusion in the united nations commission on life - saving commodities for women and children in 2012 was a testament to its contribution.19 the nonprofit organizations and professional associations with maternal health programs that attend largely to rural populations have recently issued a call for the scaling up of pph prevention programs that include the use of misoprostol.20,21 in addition , programs to strengthen labor and delivery practices continue to take place.22 table 1 presents a description of the key pph prevention interventions and the who recommendations with respect to each . amtsl is considered the gold standard strategy to reduce the incidence of pph . the amtsl preferred uterotonic is oxytocin,3 and the relative importance of other components has changed over time . even though the who strongly recommends amtsl , it also provides recommendations on the relative importance of each component . for example , the practice of controlled cord traction has a weak recommendation level , only to be practiced if small reductions in blood loss or durations of the third stage of labor are perceived to be beneficial ( table 1).3 cord clamping is now subdivided into late and early cord clamping ; the latter is no longer recommended . similarly , sustained uterine massage is no longer recommended in women who receive prophylactic oxytocin , although it was initially a common component of amtsl . instead , it is recommended that abdominal tonus assessment be conducted by a skilled provider , for all women ( table 1 ) . however , the figo guidelines ( 2012 ) for the prevention of pph in low - resource settings defines amtsl as : administration of oxytocin , controlled cord traction , and uterine massage after the delivery of the placenta.23 uterotonic drugs , such as oxytocin , ergometrine , and misoprostol , are strongly recommended ( table 1 ) . some researchers feel that oxytocin , which has minimal side effects and is safe to use among women with hypertension and preeclampsia , is all that is needed.24 ergometrine , which has proven to be a powerful drug in reducing pph , especially when combined with oxytocin , is known to be associated with serious side effects , such as severe vomiting and hypertension , and retained placenta when given intravenously.25 misoprostol is similarly associated with side effects , but none have been shown to threaten the life of the mother or newborn.26 its use is associated with significantly higher incidences of shivering and fever among mothers compared with placebo.26 misoprostol has had recent success because of its heat stability and multiple routes of administration,27 which is critical in resource - poor countries . it is important to note that all of these recommended interventions , with the exception of misoprostol administration , are to be provided by skilled providers ; as a result , most of them are available only to women who attend facility deliveries . large numbers of women living in countries with high maternal mortality deliver at home.4 thus , the recent recommendation supporting the inclusion of community health workers in the provision of misoprostol for the prevention of pph was welcomed by the safe motherhood community.3,28 the current recommendations with regards to pph prevention are based largely on rct evidence . the clinical evidence of the initial amtsl package from the 1980s showed that pph could be reduced by 70% compared with expectant management.29 the most recent cochrane review of active versus expectant management of the third stage of labor includes seven studies ( table 2 ) , and the results indicate a significant reduction in the risk of pph.29 controlled cord traction was an initial component of amtsl , but since 2000 , three rcts assessing amtsl with and without cord traction were published , all of which found a nonsignificant difference in the risk of pph ( table 2).3032 regarding cord clamping , it was initially thought that early cord clamping was best , but recent findings indicate that the timing is not critical.3 in 2013 , the cochrane collaboration researchers updated a previous review on the subject , and their findings confirmed that there are no significant differences in the risk of pph between early and late cord clamping.33 uterine massage , although not officially in the initial amtsl package , was often included.3 however , a cochrane review of two rcts comparing uterine massage after birth and before or after the delivery of the placenta for the prevention of pph found the differences between groups to be insignificant.34 in contrast , the efficacy of lower uterine segment compression versus nothing , in addition to oxytocin , cutting and clamping of the umbilical cord , and controlled cord traction , was found to be associated with a significantly lower incidence of pph.35 table 2 also presents the recent clinical evidence on the conventional uterotonics , demonstrating their importance in the prevention of pph . a recent review of seven rcts investigating the efficacy of oxytocin , three with intramuscular ( i m ) and four with intravenous ( iv ) preparations , showed that this uterotonic can significantly reduce the risk of pph.36 in addition to the i m and iv preparations , two rcts investigating the intraumbilical administration of oxytocin also found significant reductions in blood loss when compared with a control saline solution.37,38 collectively , the results from these studies make it clear why oxytocin is the preferred uterotonic for pph prevention . in terms of the other uterotonics , a 2011 review of ergot alkaloids containing six studies ( rcts and quasi - rcts ) found a significant reduction of pph when these were administered in the third stage of labor.39 regarding misoprostol , three recent reviews of rcts comparing it to a placebo were identified , two published in 2012 and one in 2013.4042 results of these reviews showed both significant reductions and nonsignificant reductions of pph . the inconsistencies in the results could be due to the fact that misoprostol was assessed in different doses and routes and also that the individual studies inclusion criteria varied . the cochrane review included 72 rcts that assessed the effects of prophylactic prostaglandin use versus a placebo , eight of which assessed 600 g oral or sublingual misoprostol ( table 2 ) . based on data from one trial , the authors of the review concluded that sublingual misoprostol was associated with significant reductions in the incidence of pph . the data from seven trials on oral misoprostol were not totaled due to heterogeneity , but the authors concluded that compared with a placebo , oral misoprostol shows promising results.42 another 2012 meta - analysis of three rcts investigating oral or sublingual misoprostol sound significant reductions in both acute and severe pph.40 an even more recent meta - analysis of three rcts showed that misoprostol does not provide significant reductions in the incidence of pph when compared with placebo.41 of the three rcts included in these two latest reviews from 2012 and 2013 , two of the rcts were the same,15,43 while one was different.44,45 two seminal studies that were included showed a reduction in pph of 24%45 and 47%15 in home births . thus , factoring in the findings from the abovementioned reviews , all of which showed reductions in the risk of pph , even if nonsignificant , it is understandable that the who ultimately supported the use of misoprostol in the absence of other uterotonics . assessment of the nonrandomized data on the interventions to prevent pph supports the clinical evidence from the rcts . a quasi - experimental study of amtsl in vietnam ( table 3 ) showed similar levels of reduction in pph to those found in the rcts.46 an analysis of the independent or combined effect of uterotonic agents , controlled cord traction , and uterine massage from hospital - based data in four countries also showed similar results to the rcts.47 no recent nonrandomized or field trials were found reporting on the individual components of amtsl , nor for oxytocin or ergometrine use in pph prevention . on the contrary , evidence was found on the use of misoprostol to prevent pph in facilities and home births . all of the nonrandomized studies with blood loss and/or acute or severe pph as outcomes showed that misoprostol use in the third stage of labor can significantly reduce pph ( table 3 ) . the evidence demonstrates that misoprostol can be effective in the prevention of pph in home births.4850 these findings are especially important for developing countries , which contribute the majority of pph morbidity and mortality , where skilled attendance at delivery is limited , and where the use of the other two uterotonics is more challenging.51 table 1 presents a description of the key pph prevention interventions and the who recommendations with respect to each . amtsl is considered the gold standard strategy to reduce the incidence of pph . the amtsl preferred uterotonic is oxytocin,3 and the relative importance of other components has changed over time . even though the who strongly recommends amtsl , it also provides recommendations on the relative importance of each component . for example , the practice of controlled cord traction has a weak recommendation level , only to be practiced if small reductions in blood loss or durations of the third stage of labor are perceived to be beneficial ( table 1).3 cord clamping is now subdivided into late and early cord clamping ; the latter is no longer recommended . similarly , sustained uterine massage is no longer recommended in women who receive prophylactic oxytocin , although it was initially a common component of amtsl . instead , it is recommended that abdominal tonus assessment be conducted by a skilled provider , for all women ( table 1 ) . however , the figo guidelines ( 2012 ) for the prevention of pph in low - resource settings defines amtsl as : administration of oxytocin , controlled cord traction , and uterine massage after the delivery of the placenta.23 uterotonic drugs , such as oxytocin , ergometrine , and misoprostol , are strongly recommended ( table 1 ) . some researchers feel that oxytocin , which has minimal side effects and is safe to use among women with hypertension and preeclampsia , is all that is needed.24 ergometrine , which has proven to be a powerful drug in reducing pph , especially when combined with oxytocin , is known to be associated with serious side effects , such as severe vomiting and hypertension , and retained placenta when given intravenously.25 misoprostol is similarly associated with side effects , but none have been shown to threaten the life of the mother or newborn.26 its use is associated with significantly higher incidences of shivering and fever among mothers compared with placebo.26 misoprostol has had recent success because of its heat stability and multiple routes of administration,27 which is critical in resource - poor countries . it is important to note that all of these recommended interventions , with the exception of misoprostol administration , are to be provided by skilled providers ; as a result , most of them are available only to women who attend facility deliveries . large numbers of women living in countries with high maternal mortality deliver at home.4 thus , the recent recommendation supporting the inclusion of community health workers in the provision of misoprostol for the prevention of pph was welcomed by the safe motherhood community.3,28 the clinical evidence of the initial amtsl package from the 1980s showed that pph could be reduced by 70% compared with expectant management.29 the most recent cochrane review of active versus expectant management of the third stage of labor includes seven studies ( table 2 ) , and the results indicate a significant reduction in the risk of pph.29 controlled cord traction was an initial component of amtsl , but since 2000 , three rcts assessing amtsl with and without cord traction were published , all of which found a nonsignificant difference in the risk of pph ( table 2).3032 regarding cord clamping , it was initially thought that early cord clamping was best , but recent findings indicate that the timing is not critical.3 in 2013 , the cochrane collaboration researchers updated a previous review on the subject , and their findings confirmed that there are no significant differences in the risk of pph between early and late cord clamping.33 uterine massage , although not officially in the initial amtsl package , was often included.3 however , a cochrane review of two rcts comparing uterine massage after birth and before or after the delivery of the placenta for the prevention of pph found the differences between groups to be insignificant.34 in contrast , the efficacy of lower uterine segment compression versus nothing , in addition to oxytocin , cutting and clamping of the umbilical cord , and controlled cord traction , was found to be associated with a significantly lower incidence of pph.35 table 2 also presents the recent clinical evidence on the conventional uterotonics , demonstrating their importance in the prevention of pph . a recent review of seven rcts investigating the efficacy of oxytocin , three with intramuscular ( i m ) and four with intravenous ( iv ) preparations , showed that this uterotonic can significantly reduce the risk of pph.36 in addition to the i m and iv preparations , two rcts investigating the intraumbilical administration of oxytocin also found significant reductions in blood loss when compared with a control saline solution.37,38 collectively , the results from these studies make it clear why oxytocin is the preferred uterotonic for pph prevention . in terms of the other uterotonics , a 2011 review of ergot alkaloids containing six studies ( rcts and quasi - rcts ) found a significant reduction of pph when these were administered in the third stage of labor.39 regarding misoprostol , three recent reviews of rcts comparing it to a placebo were identified , two published in 2012 and one in 2013.4042 results of these reviews showed both significant reductions and nonsignificant reductions of pph . the inconsistencies in the results could be due to the fact that misoprostol was assessed in different doses and routes and also that the individual studies inclusion criteria varied . the cochrane review included 72 rcts that assessed the effects of prophylactic prostaglandin use versus a placebo , eight of which assessed 600 g oral or sublingual misoprostol ( table 2 ) . based on data from one trial , the authors of the review concluded that sublingual misoprostol was associated with significant reductions in the incidence of pph . the data from seven trials on oral misoprostol were not totaled due to heterogeneity , but the authors concluded that compared with a placebo , oral misoprostol shows promising results.42 another 2012 meta - analysis of three rcts investigating oral or sublingual misoprostol sound significant reductions in both acute and severe pph.40 an even more recent meta - analysis of three rcts showed that misoprostol does not provide significant reductions in the incidence of pph when compared with placebo.41 of the three rcts included in these two latest reviews from 2012 and 2013 , two of the rcts were the same,15,43 while one was different.44,45 two seminal studies that were included showed a reduction in pph of 24%45 and 47%15 in home births . thus , factoring in the findings from the abovementioned reviews , all of which showed reductions in the risk of pph , even if nonsignificant , it is understandable that the who ultimately supported the use of misoprostol in the absence of other uterotonics . assessment of the nonrandomized data on the interventions to prevent pph supports the clinical evidence from the rcts . a quasi - experimental study of amtsl in vietnam ( table 3 ) showed similar levels of reduction in pph to those found in the rcts.46 an analysis of the independent or combined effect of uterotonic agents , controlled cord traction , and uterine massage from hospital - based data in four countries also showed similar results to the rcts.47 no recent nonrandomized or field trials were found reporting on the individual components of amtsl , nor for oxytocin or ergometrine use in pph prevention . on the contrary , evidence was found on the use of misoprostol to prevent pph in facilities and home births . all of the nonrandomized studies with blood loss and/or acute or severe pph as outcomes showed that misoprostol use in the third stage of labor can significantly reduce pph ( table 3 ) . the evidence demonstrates that misoprostol can be effective in the prevention of pph in home births.4850 these findings are especially important for developing countries , which contribute the majority of pph morbidity and mortality , where skilled attendance at delivery is limited , and where the use of the other two uterotonics is more challenging.51 when considering scaling up pph prevention interventions , it is important to consider where deliveries are occurring . approximately 46% of all births worldwide take place outside of an institutional setting , attended by a traditional birth attendant , a relative , or no one.52,53 as previously mentioned , all who - recommended interventions other than misoprostol administration require a skilled birth attendant , and many require a facility - based delivery.3 one strategy for increasing access to these life - saving interventions is to encourage facility - based delivery , especially during prenatal care . however , one must keep in mind the limited number of skilled providers in settings where the most at - risk women deliver.4 in fact , it is in these high maternal mortality settings where the shortage of trained health providers is most acute . for example , 57 countries , many of which are among the least developed countries , have a shortage of approximately 2.4 million physicians , nurses , and midwives . in sub - saharan africa specifically , their health care workforce shortage is so severe that they have only 1.3% of the world s skilled providers , but 25% of the global burden of disease.54 most mortality from pph would be eliminated if women had access to a skilled birth attendant , yet only 35% of births are attended by a skilled health worker in the least - developed countries.55 it is estimated that the coverage of skilled attendance at birth is improving at a rate of less than 0.5% per year,56 thus it will be many years until we see adequate coverage at delivery ; alternative solutions are needed . for many developing countries , poor storage conditions and deficient public sector supply chains also contribute to the limited access to and utilization of uterotonic drugs . the storage of oxytocin and ergometrine can be particularly challenging , due to their instability in high temperatures and sensitivity to light.51,57 additionally , because these two drugs are only available in injection form , their administration is limited to skilled providers . finally , in some settings where oxytocin and ergometrine are accessible , procurement of unregistered drugs may be prolific and the quality of the drugs may be compromised . for example , in ghana , researchers found 89% ( n=90 ) of all ampoules of oxytocin and ergometrine tested did not meet the specifications for the active ingredient , which was not a result of being expired ; this problem was present in both the public and private sector.58 on the other hand , misoprostol is reportedly more stable , available in tablet form , and can be provided through multiple routes of administration.59 however , all uterotonics can be exposed to health system failures , such as the case of oxytocin in tanzania and ethiopia , where the drugs were available but not properly distributed to health facilities.60,61 even when available , uterotonics are not always used consistently to prevent pph . a survey of 15 tertiary level facilities ( including 452 vaginal deliveries ) conducted by the global network for perinatal and reproductive health showed that prophylactic oxytocin was used in 44% of the cases and was the least used of the three components of amtsl assessed ( use of early cord clamping was 79% and use of controlled cord traction was 70%).62 for home births without a skilled provider , misoprostol has been distributed using a variety of approaches with or without a safe delivery kit , including distribution at antenatal care visits , at household visits by community health workers during pregnancy , at home births assisted by traditional birth attendants , and in some instances , through a combination of these methods.63 none of these approaches are without challenges related to access and utilization of the drug . for example , antenatal care distribution is only effective in increasing access to this drug in those settings where the majority of women who will deliver at home also attend antenatal care . furthermore , if advanced distribution of misoprostol requires a specific gestational age among eligible women ( eg , in mozambique , this is 28 weeks gestation ; in bangladesh , this is 32 weeks),64,65 only those attending antenatal care after the required gestational age would receive the drug and be able to use it in home births . separate from the interventions themselves is the health care system in which these interventions are being implemented . the continuum of care is an important consideration because these interventions in isolation will likely not be enough to prevent maternal morbidity and mortality . these interventions are not a replacement for a weak health care infrastructure and limited health care personnel , which must not be forgotten in this discussion of pph prevention interventions . the implementation of effective approaches is also dependent on the timely translation of research findings into policies and programs , which remains a considerable barrier in accelerating pph prevention efforts . in general , the translation of research into clinical practice is often conceptualized as proceeding from awareness through acceptance to adoption.66 however , decades may pass before research findings are integrated into guidelines and routine clinical practice ( see figure 1 ) . there is no agreement or set of rules among policy makers regarding the amount of evidence needed ( ie , number and type of studies ) to prompt changes in policies and programs . as mentioned before , by 2007 , there was only one rct published on the use of misoprostol in home births , which prompted mathai et al to express , after the 2007 release of the who guidelines , that there was insufficient evidence for the safe use of misoprostol by lay providers in nonfacility settings.67 nonetheless , based solely on the evidence of its efficacy in clinical settings and encouraged by the potential impact that misoprostol could have in the absence of oxytocin , many countries had already begun conducting operations research to test its safety , feasibility , and acceptability for home births , even before consensus was reached and the official guidelines were in place.68,69 eventually , a placebo - controlled rct with traditional birth attendants was conducted , from 2006 to 2008 , in pakistan.45 similar to the history of misoprostol , but with less debate , amtsl was first described in 1962,11 but the first rct comparing amtsl with expectant management was not done until 1988,12 and the official guidelines and policies were not adapted until later,13 after many countries had already begun to incorporate this practice . the evidence of the relative importance of its components has only become available in the last 3 years , prompting the who to review its recommendations ( see table 1 ) . finally , the challenges in the use of evidence to recommend improvements in the quality of care are further complicated by the delays in provider uptake of practices based on new knowledge . even when health providers recognize and accept guidelines , they may fail to adopt them,70 and pph prevention efforts are no exception.62 as noted , the translation of research into policy is often a long and inconsistent process , and the prioritization of a health intervention based solely on costs or cost - effectiveness is an equally challenging proposition . however , in resource - poor settings , the reduction of maternal mortality in the most cost - effective way possible should be a priority to policy makers.71 in a modeling exercise , the prevention of pph with advanced distribution of misoprostol was found to be very cost - effective in resource - poor settings , relative to other maternal - health interventions included in the who mother - baby package , only preceded by family planning and safe abortion.72 the cost of each intervention recommended by the who might differ due to the cost of each uterotonic.67 the costs of services also depend on the level of health facility and provider involved . however , given that pph is the main cause of maternal death , saving lives with any pph prevention intervention is an effective way to reduce overall maternal mortality , which is the goal of every developing country , in accordance with the united nations millennium development goal ( mdg ) 5 . an economic assessment of the reduction of pph in developing countries estimated that the consistent use of a conventional uterotonic in every birth could avert 41 million cases of pph , resulting in an estimated 1.4 million lives saved.73 specifically with regard to misoprostol , an article utilizing modeling techniques determined that misoprostol use for pph prevention is a cost - effective intervention that could reduce maternal deaths by approximately 38%.74 one of the greatest opportunities in pph prevention is the fact that the currently available evidence is sufficient to establish policies and programs that will increase access to effective interventions in low - resource settings . in order to leverage this opportunity , countries must establish a supportive national policy , which starts with the adoption of national guidelines for pph prevention that reflect the latest research and the most recent who recommendations.3,20 early on , it is important to identify local champions who will engage policy makers and clinicians.75 with respect to misoprostol , countries must register it for pph prevention and ensure its inclusion on the national essential medicines list.20 the next step for any of the interventions is to secure adequate funding in the national budget to ensure the consistent availability of the drugs and the training of health care providers.20 with regards to misoprostol , correct use by community health workers and women could be increased if misoprostol is procured in indication - specific packaging , with 600 g packets for pph prevention,69 and if pph prevention information is included as part of information , education , and communication ( iec ) mass media campaigns . another crucial step is the building of community awareness and demand for services and drugs , which will help to ensure the success of any of the pph prevention interventions , home- or facility - based . specifically related to the community - based distribution of misoprostol , several countries have conducted successful pilot projects , and some , including ghana , nepal , niger , and bangladesh , are currently working to scale - up misoprostol access.20,28,76,77 country - to - county regional exchanges involving countries that have already begun to change and implement national pph prevention policies would provide a great opportunity to share experiences and best practices.75 moving from evidence to action , particularly with regard to misoprostol , is an important next step in improving pph prevention that needs to be prioritized in low - resource countries.75 involving community - based and lay providers , to the extent the evidence allows , should be an integral part of this step.28 community health workers are being engaged more regularly in task - shifting strategies to provide basic health services , including the prevention of pph.63,78,79 in a recent integrative review of 18 programs using lay health care workers to provide misoprostol via advanced distribution or at - delivery distribution , smith et al found that high coverage and use of misoprostol can be achieved via multiple routes of distribution.63 in addition , very low rates of incorrect use were found.63 other opportunities are on the horizon with regard to new drug formulations / drugs and delivery mechanisms . oxytocin is the preferred drug for preventing and managing pph.3 however , in its current formulation , oxytocin is not heat stable and therefore an impractical intervention in many low - resource settings where extreme heat is coupled with limited access to refrigeration.51 yet it is in these low - resource settings that we see the majority of maternal deaths ; therefore , research on oxytocin formulations that are heat stable is paramount . two research teams are currently leading efforts in this field.80 a team at monash university , australia is working to develop oxytocin for aerosol delivery,81 and this formulation would allow women to inhale oxytocin immediately after childbirth , with no refrigeration of the product required . a nonprofit pharmaceutical development group in the netherlands is attempting to stabilize oxytocin under tropical conditions.82 another way to potentially increase access to oxytocin is to diversify its route of delivery . oxytocin in the uniject auto - disable injection system ( uniject ; bd biosciences , franklin lakes , nj , usa ) is comprised of a plastic , nonreusable , disposable syringe that is prefilled with a single dose of 10 international units ( iu ) of oxytocin in 1 ml . given its simple design and safety features , uniject can be used by lay health workers , which is the reason oxytocin in uniject is an important innovation for resource - poor settings.83 oxytocin in uniject is produced by an argentine pharmaceutical distributor that has regulatory approval in eight countries in latin america . though it is not yet broadly available , access to oxytocin in uniject would be particularly important in countries where human resources are limited and where task - shifting to lower cadres of health professionals is necessary.84 field studies in several countries have demonstrated the acceptability of oxytocin in uniject among health workers with less training , due to its ease of use.8587 the oxytocin in uniject is not heat stable , but the product packaging provides a straightforward time - temperature indicator to allow health workers to monitor heat exposure.84 given the early success of oxytocin in uniject , the who has amended its model list of essential medicines to include oxytocin in uniject . however , the path to the expanded availability of oxytocin in uniject will require a concurrent increase in demand and supply to counter the challenges of low - volume / high - price production.84 another recent innovation in the prevention of pph is carbetocin , a long - acting oxytocin agonist , which mimics the action of oxytocin and helps to reduce blood loss.88 carbetocin is currently indicated for the prevention of uterine atony after delivery by cesarean section in spinal or epidural anesthesia in 23 countries , but it is not approved for vaginal births . however , it has had proven success in the prevention of pph , due to its longer duration of action and demonstrated fewer side effects in several studies.88 further research is necessary to determine the cost effectiveness of carbetocin as a uterotonic agent.88 it is also important to assess the feasibility and acceptability of carbetocin in the prevention of pph in vaginal deliveries in low - resource settings . fortunately , in the 2012 annual technical report of the who special program of research , development and research training in human reproduction , plans were announced for a multicenter , controlled trial in 2014 that will compare a new heat - stable formulation of carbetocin with oxytocin , for use in low- and middle - income countries.89 another intervention that presents an opportunity for preventing pph in home births but that has limited evidence of effectiveness is the home - based life - saving skills ( hblss ) package . a community- and competency - based program that aims to reduce maternal and neonatal mortality by increasing access to basic life - saving measures within the home and community and by decreasing delays in reaching referral facilities where life threatening problems can be managed.90 hblss is implemented by hblss guides who are selected by the community and who are then trained using a modular design that focuses on the prevention , recognition , and initial home management of life - threatening maternal and newborn problems and referral to a facility , where possible.91 these guides then share their hblss knowledge and skills with women , family caregivers , and homebirth attendants ( ie , people involved in delivery care and decision making ) by way of group discussions , demonstrations , and use of pictorial learning cards.90,91 when implemented within an existing health care infrastructure , the instruction of family and community members in techniques such as uterine fundal massage and emergency preparedness has the potential to reduce maternal morbidity and mortality due to pph.23 the potential effectiveness of this approach in relation to pph relies on early identification of hemorrhage and quick initiation of treatment . the findings from an evaluation of a field test of hblss in ethiopia were promising.91 pre- and posttraining tests of hblss guides pph knowledge demonstrated a statistically significant increase , and although lower , the knowledge remained much higher than the pretraining levels at 1 year posttraining.91 in addition , the management of pph ( according to postpartum interviews ) was significantly better among women who delivered with an hblss guide compared with another unskilled attendant.91 other similar programs implemented in low - resource settings have shown success in increasing the coverage of uterotonics and/or reducing pph by actively engaging women , the community , and traditional birth attendants in community - based interventions and using locally produced materials to gauge blood loss.9294 public private partnerships need to be further developed to ensure better collaboration in the procurement , distribution , and marketing of uterotonics . for example , local pharmaceutical manufacturers , distributors , or mobile network providers can be partners in creating demand through their extensive network of product retailers , that could support the dissemination of information about misoprostol.95 in addition , new mobile technologies are being tested to support supply chain management , provide training and diagnostic assistance for health workers , and disseminate information in hard - to - reach communities . text messaging is being used to collect and transmit a wide range of information , from the documentation of stock levels of commodities to the circulation of information to women about where and how to access maternal health care.96 one of the greatest opportunities in pph prevention is the fact that the currently available evidence is sufficient to establish policies and programs that will increase access to effective interventions in low - resource settings . in order to leverage this opportunity , countries must establish a supportive national policy , which starts with the adoption of national guidelines for pph prevention that reflect the latest research and the most recent who recommendations.3,20 early on , it is important to identify local champions who will engage policy makers and clinicians.75 with respect to misoprostol , countries must register it for pph prevention and ensure its inclusion on the national essential medicines list.20 the next step for any of the interventions is to secure adequate funding in the national budget to ensure the consistent availability of the drugs and the training of health care providers.20 with regards to misoprostol , correct use by community health workers and women could be increased if misoprostol is procured in indication - specific packaging , with 600 g packets for pph prevention,69 and if pph prevention information is included as part of information , education , and communication ( iec ) mass media campaigns . another crucial step is the building of community awareness and demand for services and drugs , which will help to ensure the success of any of the pph prevention interventions , home- or facility - based . specifically related to the community - based distribution of misoprostol , several countries have conducted successful pilot projects , and some , including ghana , nepal , niger , and bangladesh , are currently working to scale - up misoprostol access.20,28,76,77 country - to - county regional exchanges involving countries that have already begun to change and implement national pph prevention policies would provide a great opportunity to share experiences and best practices.75 moving from evidence to action , particularly with regard to misoprostol , is an important next step in improving pph prevention that needs to be prioritized in low - resource countries.75 involving community - based and lay providers , to the extent the evidence allows , should be an integral part of this step.28 community health workers are being engaged more regularly in task - shifting strategies to provide basic health services , including the prevention of pph.63,78,79 in a recent integrative review of 18 programs using lay health care workers to provide misoprostol via advanced distribution or at - delivery distribution , smith et al found that high coverage and use of misoprostol can be achieved via multiple routes of distribution.63 in addition , very low rates of incorrect use were found.63 other opportunities are on the horizon with regard to new drug formulations / drugs and delivery mechanisms . oxytocin is the preferred drug for preventing and managing pph.3 however , in its current formulation , oxytocin is not heat stable and therefore an impractical intervention in many low - resource settings where extreme heat is coupled with limited access to refrigeration.51 yet it is in these low - resource settings that we see the majority of maternal deaths ; therefore , research on oxytocin formulations that are heat stable is paramount . two research teams are currently leading efforts in this field.80 a team at monash university , australia is working to develop oxytocin for aerosol delivery,81 and this formulation would allow women to inhale oxytocin immediately after childbirth , with no refrigeration of the product required . a nonprofit pharmaceutical development group in the netherlands is attempting to stabilize oxytocin under tropical conditions.82 another way to potentially increase access to oxytocin is to diversify its route of delivery . oxytocin in the uniject auto - disable injection system ( uniject ; bd biosciences , franklin lakes , nj , usa ) is comprised of a plastic , nonreusable , disposable syringe that is prefilled with a single dose of 10 international units ( iu ) of oxytocin in 1 ml . given its simple design and safety features , uniject can be used by lay health workers , which is the reason oxytocin in uniject is an important innovation for resource - poor settings.83 oxytocin in uniject is produced by an argentine pharmaceutical distributor that has regulatory approval in eight countries in latin america . though it is not yet broadly available , access to oxytocin in uniject would be particularly important in countries where human resources are limited and where task - shifting to lower cadres of health professionals is necessary.84 field studies in several countries have demonstrated the acceptability of oxytocin in uniject among health workers with less training , due to its ease of use.8587 the oxytocin in uniject is not heat stable , but the product packaging provides a straightforward time - temperature indicator to allow health workers to monitor heat exposure.84 given the early success of oxytocin in uniject , the who has amended its model list of essential medicines to include oxytocin in uniject . however , the path to the expanded availability of oxytocin in uniject will require a concurrent increase in demand and supply to counter the challenges of low - volume / high - price production.84 another recent innovation in the prevention of pph is carbetocin , a long - acting oxytocin agonist , which mimics the action of oxytocin and helps to reduce blood loss.88 carbetocin is currently indicated for the prevention of uterine atony after delivery by cesarean section in spinal or epidural anesthesia in 23 countries , but it is not approved for vaginal births . however , it has had proven success in the prevention of pph , due to its longer duration of action and demonstrated fewer side effects in several studies.88 further research is necessary to determine the cost effectiveness of carbetocin as a uterotonic agent.88 it is also important to assess the feasibility and acceptability of carbetocin in the prevention of pph in vaginal deliveries in low - resource settings . fortunately , in the 2012 annual technical report of the who special program of research , development and research training in human reproduction , plans were announced for a multicenter , controlled trial in 2014 that will compare a new heat - stable formulation of carbetocin with oxytocin , for use in low- and middle - income countries.89 another intervention that presents an opportunity for preventing pph in home births but that has limited evidence of effectiveness is the home - based life - saving skills ( hblss ) package . a community- and competency - based program that aims to reduce maternal and neonatal mortality by increasing access to basic life - saving measures within the home and community and by decreasing delays in reaching referral facilities where life threatening problems can be managed.90 hblss is implemented by hblss guides who are selected by the community and who are then trained using a modular design that focuses on the prevention , recognition , and initial home management of life - threatening maternal and newborn problems and referral to a facility , where possible.91 these guides then share their hblss knowledge and skills with women , family caregivers , and homebirth attendants ( ie , people involved in delivery care and decision making ) by way of group discussions , demonstrations , and use of pictorial learning cards.90,91 when implemented within an existing health care infrastructure , the instruction of family and community members in techniques such as uterine fundal massage and emergency preparedness has the potential to reduce maternal morbidity and mortality due to pph.23 the potential effectiveness of this approach in relation to pph relies on early identification of hemorrhage and quick initiation of treatment . the findings from an evaluation of a field test of hblss in ethiopia were promising.91 pre- and posttraining tests of hblss guides pph knowledge demonstrated a statistically significant increase , and although lower , the knowledge remained much higher than the pretraining levels at 1 year posttraining.91 in addition , the management of pph ( according to postpartum interviews ) was significantly better among women who delivered with an hblss guide compared with another unskilled attendant.91 other similar programs implemented in low - resource settings have shown success in increasing the coverage of uterotonics and/or reducing pph by actively engaging women , the community , and traditional birth attendants in community - based interventions and using locally produced materials to gauge blood loss.9294 public private partnerships need to be further developed to ensure better collaboration in the procurement , distribution , and marketing of uterotonics . for example , local pharmaceutical manufacturers , distributors , or mobile network providers can be partners in creating demand through their extensive network of product retailers , that could support the dissemination of information about misoprostol.95 in addition , new mobile technologies are being tested to support supply chain management , provide training and diagnostic assistance for health workers , and disseminate information in hard - to - reach communities . text messaging is being used to collect and transmit a wide range of information , from the documentation of stock levels of commodities to the circulation of information to women about where and how to access maternal health care.96 as pph is the main cause of maternal mortality worldwide , pph prevention interventions need to be prioritized as an essential way to improve maternal health . each country must develop its own context - dependent policies and programs , incorporating myriad approaches that combine the most recent recommendations and reflect the experiences of other countries . though oxytocin is the recommended uterotonic , it is not readily available in settings with the highest risk for mortality and morbidity from pph , due to its sensitivity to heat and need for provision by a skilled provider . yet increasing access to prophylactic uterotonics , regardless of where deliveries occur , should be the primary means of reducing the burden of this complication . there is still some debate as to whether misoprostol is effective in pph prevention,97 and some have called for additional high - quality studies that demonstrate significant reductions in pph.48 but at the present time , based on the evidence available , the best way to reduce pph deaths in low - resource settings where women continue to deliver without access to a skilled birth attendant is to make misoprostol widely available . therefore , efforts need to be directed at increasing misoprostol supplies and supporting correct and consistent utilization by providers and by women themselves , in the case of home births .

backgroundpostpartum hemorrhage ( pph ) is the leading cause of maternal death in low - income countries and is the primary cause of approximately one - quarter of global maternal deaths . the purpose of this paper is to provide a review of pph prevention interventions , with a particular focus on misoprostol , and the challenges and opportunities that preventing pph in low - resource settings presents.methodsusing pubmed , we conducted a review of the literature on the randomized controlled trials of interventions to prevent pph . we then searched pubmed and google scholar for nonrandomized field trials of interventions to prevent pph . we limited our review to interventions that are discussed in the current world health organization ( who ) recommendations for pph prevention and present evidence regarding the use of these interventions . we focused our review on nondrug pph prevention interventions compared with no intervention and uterotonics versus placebo ; this review does not decipher the relative effectiveness of uterotonic drugs . we describe challenges to and opportunities for scaling up pph prevention interventions.resultsactive management of the third stage of labor is considered the gold standard strategy for reducing the incidence of pph . it combines nondrug interventions ( controlled cord traction and cord clamping ) with the administration of an uterotonic drug , the preferred uterotonic being oxytocin . unfortunately , oxytocin has limited application in resource - poor countries , due to its heat instability and required administration by a skilled provider . new heat - stable drugs and drug formulations are currently in development that may improve the prevention of pph ; however , misoprostol is a viable option for provision at home by a lay health care worker or the woman herself , in the interim.conclusionas the main cause of maternal mortality worldwide , pph prevention interventions need to be prioritized . increased access to prophylactic uterotonics , regardless of where deliveries occur , should be the primary means of reducing the burden of this complication .

the first n - terminal acetylation is catalyzed a variety of n - terminal acetyltransferases ( nats ) , which co - translationally transfer acetyl moieties from acetyl - coenzyme a ( acetyl - coa ) to the -amino although n - terminal acetylation is rare in prokaryotes , it was estimated that 85% of eukaryotic proteins are n - terminally modified ( 1,2 ) . the second type is n - lysine acetylation , which specifically modifies -amino group of protein lysine residues ( 38 ) . although n - lysine acetylation is less common , it s one of the most important and ubiquitous post - translational modifications ( ptms ) conserved in prokaryotes and eukaryotes ( 1,2 ) . moreover , the acetylation and deacetylation are dynamically and temporally regulated by histone acetyltransferases ( hats ) and histone deacetylases ( hdacs ) , respectively ( 48 ) . in 1964 , allfrey et al . ( 9 ) first observed that lysine acetylation of histones plays an essential role in regulation of gene expression . later and recent studies in epigenetics solidified this seminal discovery , and proposed acetylation as a key component of the histone code ( 6 ) . beyond histones , a wide - range of non - histone proteins can also be lysine acetylated , and involved in a variety of biological processes , such as transcription regulation ( 10 ) , dna replication ( 11 ) , cellular signaling ( 12 ) , stress response ( 13 ) and so on . aberrance of lysine acetylation and deacetylation is associated with various diseases and cancers ( 5,7,14 ) . in particular , acetylation was demonstrated to be implicated in cellular metabolism and aging ( 1517 ) , while one class of nad dependent hdacs of sirtuins might be potent drug target for promoting longevity ( 13,17 ) . although a great number of efforts have been carried out during the past four decades , the functional contents of lysine acetylation are still far from fully understood . in this regard , identification of acetylated substrates with their sites is fundamental for understanding the molecular mechanisms and regulatory roles of acetylation . in contrast with labor - intensive and time - consuming conventional experimental approaches , recent progresses in acetylome with high - throughput mass spectrometry ( ms ) have detected thousands of acetylation sites . in 2006 , kim et al . ( 14 ) performed a large - scale identification of acetylome with an anti - acetyllysine antibody . there were 195 acetylated proteins with 388 sites detected in hela cells and mouse liver mitochondria ( 14 ) . with a similar strategy , choudhary et al . ( 11 ) experimentally identified 3600 acetylation sites in human . in 2010 , zhao et al . ( 16 ) discovered 1047 acetylated substrates in human liver , and demonstrated acetylation playing a major role in metabolic regulation . furthermore , two acetylomic studies revealed that the functions of lysine acetylation are conserved in escherichia coli ( 18 ) and salmonella enterica ( 15 ) . since the number of known acetylation sites has rapidly increased , it is an urgent topic to collect the experimental data and provide an integrated resource for the community . recently , several public databases , such as phosphositeplus ( 19 ) , hprd ( 20 ) , sysptm ( 21 ) and dbptm ( 22 ) , have already contained protein acetylation information . in these databases , both of n - terminal and n - lysine acetylation data were curated , while lysine acetylation sites are usually only a limited part of total sites . for example , sysptm 1.1 contains 3001 acetylation sites in 2000 proteins , with only 345 lysine sites ( 11.5% ) in 397 substrates ( 21 ) . in dbptm 2.0 , 2071 experimentally verified acetylation sites were collected in 1525 proteins , with only 792 lysine sites ( 38.2% ) in 299 targets ( 22 ) . interestingly , hprd release 9 contains 4691 total sites in 1987 proteins , with 4420 lysine sites ( 94.2% ) in 1821 substrates ( 20 ) . however , hprd database only focuses on human protein information ( 20 ) , while thousands of lysine acetylation sites in other species still remain to be collected . with the motivation to meet the desire for complete acetylomes , here we developed a novel database of compendium of protein lysine acetylation ( cpla ) . from the scientific literature in pubmed , we manually curated 3311 acetylated proteins with 7151 lysine sites ( table 1 ) . in cpla database , the primary references and other annotations of these substrates were provided , while the protein protein interaction ( ppi ) information was also integrated . based on the gene ontology ( go ) and interpro annotations , we carried out an analysis of functional diversities and regulatory roles of lysine acetylation . as 75.64% of total lysine acetylation sites are taken from homo sapiens , a potential human lysine acetylation network ( hlan ) among hats , substrates and hdacs was constructed , with 1019 ppis among 199 proteins . interestingly , we revealed 1862 potential triplet relationships of hat - substrate - hdac , while at lease 13 were previously experimentally verified . taken together , the cpla database might be an integrated resource for protein lysine acetylation and provide useful information for further experimental or computational considerations . ( % ) homo sapiens2585540975.64salmonella typhimurium1902603.64mus musculus1875527.72escherichia coli1492593.62saccharomyces cerevisiae44851.19others1565868.20total33117151100sub . , number of substrates ; site , number of acetylation sites ; per . , percentiles of acetylation sites . to ensure the quality of cpla database , we searched the pubmed with a major keyword acetylation and collected experimentally identified lysine acetylated proteins with their sites from more than 18 500 published articles ( before 1 march 2010 ) . to avoid missing data after all substrates with unambiguous acetylation lysines were collected , we searched the uniprot knowledgebase ( 23 ) to obtain the corresponding protein sequences and associated annotation information . the theoretical pi ( isoelectric point ) and mw ( molecular weight ) were calculated for each protein ( http://www.expasy.org/tools/pi_tool.html ) ( 24,25 ) . in cpla database , we took experimental ppis from several major public databases ( on 10 april 2010 ) , such as hprd ( 20 ) , biogrid ( 26 ) , dip ( 27 ) , mint ( 28 ) and intact ( 29 ) . in addition , a well - known pre - predicted database of string ( 30 ) was also used . all proteins were mapped to the uniprot sequences by blast . for human , we collected a total of 59 481 experimental ppis in 12 221 proteins and 1 212 607 predicted ppis in 16 523 proteins , respectively . the cpla 1.0 database contains 7151 lysine acetylation sites in 3311 substrates ( table 1 ) . particularly , 1742 ( 24.4% ) acetylation sites in 726 proteins are collected from non - human species ( table 1 ) . the online service and local packages were implemented in php + mysql + javascript and java 1.5 ( j2se 5.0 ) , separately . moreover , the search option ( http://cpla.biocuckoo.org/search.php ) provides an interface for querying the cpla 1.0 database with one or several keywords such as gene / protein names , uniprot i d or cpla i d , etc . for example , if the keyword stat3 is inputted and submitted ( figure 1a ) , the result will be shown in a tabular format , with the features of cpla i d , uniprot accession and protein / gene names / aliases ( figure 1b ) . by clicking on the cpla i d ( cpla-000136 ) , the detailed information for human stat3 will be shown ( figure 1c ) . the acetylation information , including acetylated positions , flanking peptides , experimental reagents or upstream hats , and primary references are provided . the protein sequence , go annotation , domain organization , molecular weight , computed/ theoretical ip and ppi information are also presented . users could click on the cpla i d ( cpla-000025 ) to visualize the detailed information . users could click on the cpla i d ( cpla-000025 ) to visualize the detailed information . furthermore , we provided three additional advance options , including ( i ) advance search , ( ii ) browse and ( iii ) blast search ( supplementary figure s1 ) . ( i ) advance search : in this option , users could use relatively complex and combined keywords to locate the precise information , with up to two search terms . the interface of search - engine permits the querying by different database fields and the linking of queries through three operators of and , ( ii ) browse : instead of searching for a specific protein , all entries of cpla database could be listed by species name ( supplementary figure s1b ) . ( iii ) blast search : this option was designed for finding related information in cpla database quickly . the blastall program of ncbi blast packages ( 31 ) was included in cpla 1.0 database ( supplementary figure s1c ) . users can input a protein sequence in fasta format for searching identical or homologous proteins . recent progresses toward understanding the full functional content of acetylome have experimentally revealed several thousands of lysine acetylated substrates with their sites . besides experimental efforts , the current available computational resources were summarized and listed in supplementary table s2 . among these researches , database development is particularly important for integrating experimental data from heterogeneous sources , and providing a high quality benchmark for further experimental or computational designs . although several public databases ( 1922 ) have already maintained the acetylation information , the lysine acetylation is usually collected together with another less controlled n - terminal acetylation . in this work , we only focused on protein lysine acetylation and manually curated 7151 lysine acetylation sites in 3311 proteins . since a large proportion of acetylation sites were taken from homo sapiens , we had the opportunity to analyze abundance and functional diversity of lysine acetylation in an acetylomic level . we surveyed the go terms of 2585 acetylated proteins from uniprot annotations . using the human proteome as the background , we statistically calculated over - represented biological processes , molecular functions and cellular components in acetylome with the hypergeometric distribution ( p < 0.01 ) . the top five most enriched go entries in each category were shown in table 2 . for example , the three most abundant biological processes such as translational elongation , rna splicing and mrna processing suggest that acetylation predominantly regulates gene expression in a post - transcriptional manner ( table 2 ) . also , four most over - represented molecular functions such as atp binding , protein binding , rna binding and nucleotide binding suggest that acetylation modulates enzyme activity and protein interaction ability ( table 2 ) . in addition , the statistical analysis of cellular components revealed acetylated proteins to be highly enriched in distinct cellular compartments . for instance , 30 and 62% of cytosol and mitochondrial matrix proteins are acetylated , respectively ( table 2 ) . for more detailed information , the top 15 most over - represented go terms and interpro domains were shown in supplementary tables s3 and s4 . table 2.the top five most enriched go terms of biological processes , molecular functions and cellular components in human acetylomedescription of go termacetylomeproteomee - ratiop - valuen ( % ) n ( % ) the top five most enriched biological processes translational elongation ( go:0006414)69 ( 2.80)93 ( 0.52)5.443.35e-40 nucleosome assembly ( go:0006334)43 ( 1.75)79 ( 0.44)3.991.17e-17 rna splicing ( go:0008380)70 ( 2.84)188 ( 1.04)2.733.50e-16 interspecies interaction between organisms ( go:0044419)100 ( 4.06)333 ( 1.85)2.203.97e-15 mrna processing ( go:0006397)65 ( 2.64)193 ( 1.07)2.479.06e-13the top five most enriched molecular functions atp binding ( go:0005524)398 ( 16.17)1473 ( 8.17)1.984.67e-46 protein binding ( go:0005515)798 ( 32.06)3996 ( 22.16)1.459.00e-35 rna binding ( go:0003723)186 ( 7.56)557 ( 3.09)2.459.49e-34 structural constituent of ribosome ( go:0003735)71 ( 2.89)155 ( 0.86)3.361.99e-22 nucleotide binding ( go:0000166)85 ( 3.45)255 ( 1.41)2.445.75e-16the top five most enriched cellular components cytosol ( go:0005829)298 ( 12.11)1009 ( 5.60)2.164.71e-42 mitochondrial matrix ( go:0005759)83 ( 3.37)139 ( 0.77)4.386.33e-37 nucleoplasm ( go:0005654)124 ( 5.04)333 ( 1.85)2.731.12e-27 nucleolus ( go:0005730)133 ( 5.40)425 ( 2.36)2.291.84e-21 cytosolic small ribosomal subunit ( go:0022627)29 ( 1.18)36 ( 0.20)5.902.24e-19num . , number of proteins annotated ; per . the top five most enriched go terms of biological processes , molecular functions and cellular components in human acetylome num . the acetylation and deacetylation of proteins are carried out by hats and hdacs , which antagonistically and dynamically control protein function . combined with experimental and predicted ppis , we constructed a potential hlan among hats , substrates and hdacs , with 1019 ppis of 199 proteins ( supplementary table s5 ) . if only experimental ppis are considered , the core hlan contained 369 ppis among 77 proteins , including 12 hats and 12 hdacs ( figure 2 ) . from the whole hlan if a substrate is a hat or hdac , it should be acetylated or deacetylated by a different hat or hdac . we carefully surveyed scientific literature and found that at least 13 triplet interactions were experimentally identified ( supplementary table s6 ) . for example , gaughan et al . ( 32 ) observed that tip60 ( kat5 ) and histone deacetylase 1 ( hdac1 ) regulate the transcriptional activity of androgen receptor ( ar ) through changing its acetylation status , and form a kat5-ar - hdac1 relation ( supplementary table s6 ) . for instance , ep300 acetylates bcl6 at k379 and inhibits its function , while deacetylases were not clearly identified ( 33 ) . in our results , the ep300-bcl6-hdac5 , ep300-bcl6-sirt2 , ep300-bcl6-hdac11 , ep300-bcl6-hdac3 , ep300-bcl6-hdac2 and ep300-bcl6-hdac8 suggested that bcl6 might be deacetylated by multiple hdacs ( supplementary table s6 ) . moreover , human gcma / gcm1 was reported to be acetylated by cbp / crebbp at k367 , k406 and k409 ( 34 ) . in our results , the relations of crebbp - gcm1-hdac3 , crebbp - gcm1-hdac3 , crebbp - gcm1-hdac1 and crebbp - gcm1-hdac4 proposed that at least four hdacs might deacetylate gcm1 ( supplementary table s6 ) . green node : hat ; blue node : hdac ; yellow node : hat that is acetylated ; purple node : hdac that is acetylated ; pink node : substrate . a core hlan identified from experimentally identified ppi data . green node : hat ; blue node : hdac ; yellow node : hat that is acetylated ; purple node : hdac that is acetylated ; pink node : substrate . taken together the statistical analyses revealed functional diversity and enrichment of acetylation , while network studies generated a large number of potentially useful results for further experimental or computational researches . the cpla database will be routinely updated if new acetylated substrates are reported . funding for open access charge : national basic research program ( 973 project ) ( 2010cb945400 , 2007cb947401 ) ; national natural science foundation of china ( 90919001 , 30700138 , 30900835 , 30830036 , 31071154 ) ; chinese academy of sciences ( info-115-c01-sdb4 - 36 ) .

as a reversible post - translational modification ( ptm ) discovered decades ago , protein lysine acetylation was known for its regulation of transcription through the modification of histones . recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation . although acetylation is comparable with other major ptms such as phosphorylation , an integrated resource still remains to be developed . in this work , we presented the compendium of protein lysine acetylation ( cpla ) database for lysine acetylated substrates with their sites . from the scientific literature , we manually collected 7151 experimentally identified acetylation sites in 3311 targets . we statistically studied the regulatory roles of lysine acetylation by analyzing the gene ontology ( go ) and interpro annotations . combined with protein protein interaction information , we systematically discovered a potential human lysine acetylation network ( hlan ) among histone acetyltransferases ( hats ) , substrates and histone deacetylases ( hdacs ) . in particular , there are 1862 triplet relationships of hat - substrate - hdac retrieved from the hlan , at least 13 of which were previously experimentally verified . the online services of cpla database was implemented in php + mysql + javascript , while the local packages were developed in java 1.5 ( j2se 5.0 ) . the cpla database is freely available for all users at : http://cpla.biocuckoo.org .

advances in medical care and neonatal medicine have led to changes in survival pattern of high risk neonates ( 1 ) . one of the complications that have been found in these high risk neonates is lbw ( < 2500 grams ) . according to world health organization ( who ) statistics , the rate of lbw is 17% worldwide ( 6% in developed countries and 21% in developing countries ) . studies revealed that the rate of lbw is 10% in iran ( 2 ) ; and 8.4 % in yazd , a city in the center of iran ( 3 ) . one of the most common neonatal complications of prematurity and lbw is respiratory failures like respiratory distress syndrome ( rds ) which often needs using mechanical ventilation ( mv ) for improving the neonatal survival , especially for premature neonates born less than 30 weeks gestation with immature lung function ( 4 ) . studies showed that mv in lbw neonates , and especially extremely low birth weight ( elbw ) , is linked to poor neurodevelopmental outcomes ( 5 ) . although the main proportions of lbw neonates are born in developing countries ( 2 ) , the majority of studies on developmental outcomes of prematurity have been performed in developed countries . considering the effects of different cultures and socioeconomic status on development and the importance of conducting developmental evaluations in developing countries , close attention should be paid to such kinds of studies in these countries . there has been lack of research evidence about developmental outcomes of more complicated lbw neonates like neonates which used mv . in this study we prospectively investigated the development of gross and fine motor skills in infants with the history of lbw and over 14 days use of mv . the aim of this study was to compare gross and fine motor development of infants with the history of lbw with mv and without mv ( wmv ) over 14 days and healthy nbw infants with 8 to 12 months of age using peabody developmental motor scale-2 ( pdms-2 ) . this descriptive , prospective and cross sectional study was carried out in the occupational therapy clinic of aliasghar hospital , tehran , iran . the study was conducted on three groups of infants aged 8 to 12 months ( all mentioned ages for lbw infants are corrected for prematurity ) as follows : lbw group , birth weight < 2500 g , in form of mv and wmv groups and with normal birth weight ( nbw ) group . the sample size based on random sampling method was calculated to be 35 infants included 35 infants with the history of lbw using mv over 14 days ( n=35 ) , 35 infants with the history of lbw and wmv(n=35 ) and 40 healthy nbw infants ( n=40 ) . mv group consisted of infants with corrected age of 8 to 12 months admitted to nicu of aliasghar hospital who used mv for 14 days or more . 2 . using mv for 14 days or more after third days of birth with diagnosis of respiratory failures , pco2 more than 60 mmhg , po2 less than 50 mmhg and ph less than 7.20 . wmv group consisted of all infants with corrected age of 8 to 12 months who admitted to neonatal intensive care unit ( nicu ) of aliasghar hospital for 14 days or more . the data for lbw infants were obtained from neonatal files of infants in nicu . for nbw group , all infants aged 8 to 12 months referred to healthcare center of aliasghar hospital for vaccination were screened for eligibility to the study . any brain injury like intraventricular hemorrhage ( ivh ) or seizure , genetic disease , degenerative disease and /or other acquired problems which affect the development after discharge of nicu . any sensory problem like deafness and/or blindness . in this study because of our excluded criteria on any prenatal or perinatal brain complications we included all infants regardless of kind of delivery . also , because of not having access to all apgar scores we included infants regardless of their apgar scores . moreover , because of the methodology of our study which was prospective we could not completely monitor any complications that might be happened during intubation . however , a regular suctioning and tube control were mentioned for all mv group infants during intubation in their neonatal files . all infants were visited by a pediatrician for clinical and neurological examinations and if they fulfilled the above criteria were enrolled in the study . after screening and selecting all groups randomly , the informed consent form was given to their family and then the questionnaire containing medical and developmental history and demographic information of infants were completed by examiners . finally two occupational therapists blinded to the history of infants conducted pdms -2 for each infant . the pdms-2 is one of the most commonly - used assessments for measuring motor skills of infants and toddlers from birth through age 5 . for children with special needs , the pdms-2 is one of the most reliable testing instruments used by many professionals as a diagnostic tool for assessing of gross and fine motor skills . it has been used in a number of follow - up studies investigating motor skills in the preterm population . , most dysfunctions of motor skills will be identified . using the results of the pdms-2 , we can develop a more responsive learning and remediation program for the child with special needs . this test is composed of six subtests that assess related motor abilities that develop early in life : reflexes , stationary ( body control and equilibrium ) , and locomotion , object manipulation , grasping , and visual - motor integration . results from these subtests are used to generate the three composite scores : gross motor quotient , fine motor quotient , and total motor quotient with a mean of 100 and a standard deviation of 15 ( 10 ) . after collecting the perinatal variables and gross and fine motor scores , the motor quotients were determined and data were analyzed and compared using spss version 17 . spearman s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and qualitative variable and pearson s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and quantitative variable . this descriptive , prospective and cross sectional study was carried out in the occupational therapy clinic of aliasghar hospital , tehran , iran . the study was conducted on three groups of infants aged 8 to 12 months ( all mentioned ages for lbw infants are corrected for prematurity ) as follows : lbw group , birth weight < 2500 g , in form of mv and wmv groups and with normal birth weight ( nbw ) group . the sample size based on random sampling method was calculated to be 35 infants included 35 infants with the history of lbw using mv over 14 days ( n=35 ) , 35 infants with the history of lbw and wmv(n=35 ) and 40 healthy nbw infants ( n=40 ) . mv group consisted of infants with corrected age of 8 to 12 months admitted to nicu of aliasghar hospital who used mv for 14 days or more . 2 . using mv for 14 days or more after third days of birth with diagnosis of respiratory failures , pco2 more than 60 mmhg , po2 less than 50 mmhg and ph less than 7.20 . wmv group consisted of all infants with corrected age of 8 to 12 months who admitted to neonatal intensive care unit ( nicu ) of aliasghar hospital for 14 days or more . the data for lbw infants were obtained from neonatal files of infants in nicu . for nbw group , all infants aged 8 to 12 months referred to healthcare center of aliasghar hospital for vaccination were screened for eligibility to the study . any brain injury like intraventricular hemorrhage ( ivh ) or seizure , genetic disease , degenerative disease and /or other acquired problems which affect the development after discharge of nicu . any sensory problem like deafness and/or blindness . in this study because of our excluded criteria on any prenatal or perinatal brain complications we included all infants regardless of kind of delivery . also , because of not having access to all apgar scores we included infants regardless of their apgar scores . moreover , because of the methodology of our study which was prospective we could not completely monitor any complications that might be happened during intubation . however , a regular suctioning and tube control were mentioned for all mv group infants during intubation in their neonatal files . all infants were visited by a pediatrician for clinical and neurological examinations and if they fulfilled the above criteria were enrolled in the study . after screening and selecting all groups randomly , the informed consent form was given to their family and then the questionnaire containing medical and developmental history and demographic information of infants were completed by examiners . finally two occupational therapists blinded to the history of infants conducted pdms -2 for each infant . the pdms-2 is one of the most commonly - used assessments for measuring motor skills of infants and toddlers from birth through age 5 . for children with special needs , the pdms-2 is one of the most reliable testing instruments used by many professionals as a diagnostic tool for assessing of gross and fine motor skills . it has been used in a number of follow - up studies investigating motor skills in the preterm population . , most dysfunctions of motor skills will be identified . using the results of the pdms-2 , we can develop a more responsive learning and remediation program for the child with special needs . this test is composed of six subtests that assess related motor abilities that develop early in life : reflexes , stationary ( body control and equilibrium ) , and locomotion , object manipulation , grasping , and visual - motor integration . results from these subtests are used to generate the three composite scores : gross motor quotient , fine motor quotient , and total motor quotient with a mean of 100 and a standard deviation of 15 ( 10 ) . after collecting the perinatal variables and gross and fine motor scores , the motor quotients were determined and data were analyzed and compared using spss version 17 . mean motor quotients scores were compared using anova . spearman s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and qualitative variable and pearson s correlation coefficient was used to examine the relationship between the mean of gross and fine motor quotients and quantitative variable . study population was 70 lbw infants ( wmv group , n=35 ) and ( mv group , n=35 ) and 40 healthy nbw infants ( nbw group , n=40 ) . the study comprised 23 males and 12 females in mv group , 19 males and 16 females in wmv group , and 18 males and 22 females in nbw group . mean birth weight , gestational age and other perinatal characteristics of each group are summarized in table 1 . in this study we could not find any extremely low birth weight ( elbw ) infant matching our inclusion criteria . mv : with mechanical ventilation ; wmv : without mechanical ventilation ; nbw : normal birth weight in relation to mean fine motor quotient , there were significant differences between mv and wmv in comparison with nbw group . but , the mean fine motor quotient in mv and wmv group showed no significant differences ( p<0.05 ) . in relation to mean gross motor quotient , there were significant differences between mv and wmv in comparison with nbw group . in addition , the mean fine motor quotient in mv and wmv group showed significant differences ( p<0.05 ) . finally , in relation to mean total motor quotient , there were significant differences between mv and wmv in comparison with nbw group and there were no significant differences between mv and wmv group , in terms of the mean total motor quotient ( p<0.05 ) . comparison of three groups ( nbw and mv and wmv ) based on mean gross , fine and total motor quotient in two sub groups ( lbw , vlbw ) is shown in tables 2 - 4 . moreover , based on the multiple comparisons by tukey s hsd , statistically significant difference was found just in gross developmental motor quotient ( dmq ) when the dmqs of mv group compared with that of wmv group ( p < 0.05 ) . furthermore according to the guide to interpreting pdms-2 quotient scores in peabody examiner s manual , the mean of all motor quotients in mv sub groups ( lbw , vlbw ) except for fine motor quotient of vlbw were in the range of 80 - 89 . it means that motor development of all these groups except for fine motor of vlbw were below average ( 16.12% ) . the mean of fine motor quotient of vlbw was in the range of 70 - 79 that interpreted as poor ( 6.87% ) . on the other hand , in wmv group , the gross motor quotients of all sub groups ( lbw , vlbw ) like the nbw group , were in the range of 90 - 110 and considered as average ( 49.51% ) and the fine motor development is in the range of 80 - 89 and as a result , considered as below average ( 16.12% ) . the results of this study also showed that there was no correlation between motor quotients and gestational age , sex , length of stay in nicu and days of mechanical ventilation in lbw group ( p>0.05 ) . this study pursued to characterize the motor quotients and compare the gross and fine motor development of lbw infants with and without using mv with nbw infants at the age of 8 to 12 months . recent studies showed that lbw infants are prone to abnormal neurological signs in tone , coordination and reflexes due to their neonatal complications which lead to developing motor deficits and delays in these children at the age of 6 months or later ( 11 ) . in general , the shorter the gestational period or the lower the birth weight is , the greater the risk status for motor deficits in the premature infant ( 1 ) . it is reported that approximately 10% of the elbw ( < 1,000 grams ) preterm infants will develop cerebral palsy ( cp ) . also a 32% rate of cp is found in those infants weighting less than 1,500 grams ( 1 ) . grantham et al in1998 ( 12 ) showed that lbw infants had significantly lower scores in mental and psychomotor development index at 6 months of age and the difference in both of these scores increased by 12 months of age . in a study by datar in 2009 ( 13 ) , mental and motor development of vlbw and mlbw babies during the first two years of life was compared with those of normal birth weight ones . lbw had a small adverse effect on mental and motor development in the first two years of life . elbw infants are rarely admitted in aliasghar nicu . however , other studies evaluated the elbw infants or the infants born less than 29 weeks gestation which was not considered in our study . ( 9,14 ) in these studies the development of lbw infants without considering the effects of length of stay or days of mv was investigated and showed relatively poor outcome of growth and psychomotor development in these populations . moreover , , we reviewed a follow up study in iran in 2008 , in which fifty lbw preterm neonates admitted to shahid sadoughi hospital nicu were evaluated for developmental status at 6 and 12 months of age using persian version of ages and stages questionnaires ( asq ) . lbw and preterm infants admitted to the nicu showed degrees of developmental delay at the ages of 6 and 12 months , especially in the gross motor and personal - social developmental domains on the asq ( 15 ) . also , in yazd , iran , nbw and moderately low birth weight ( mlbw ) children of 60-month evaluated by asq . the results showed that frequency of developmental delay in gross motor , fine motor and problem solving domains were significantly higher in mlbw group and mean score in all developmental domains was statistically significant lower in case group . ( 16 ) as previous studies reported , the mv is often required by preterm infants with respiratory failure , and invasive form of this respiratory support can be related to lung injury and adverse neurologic outcomes ( 17 ) . in the study of mv in preterm infants in 1992 , graziani et al . ( 18 ) , described the relationships of prenatal factors , especially those associated with mechanical ventilation and hypocarbia , to the subsequent occurrence of neurosonographic and neurodevelopmental abnormalities in preterm infants . they suggested that prenatal and neonatal factors including the need for mechanical ventilation beginning on the first day of life and marked hypocarbia during the first 3 postnatal days are associated with an increased risk of damage to the periventricular white matter of some preterm infants and developmental delays . ( 5)mv in an elbw neonate has been linked to poor neurodevelopmental ( nd ) outcomes . ( 20 ) determined whether mv s effect on nd is a result of its relationship to bpd and other associated neonatal co morbidities , or whether mv alone is an independent risk factor for poor nd outcomes . they compared the potential impact of mv vs. continuous positive airway pressure ( cpap ) at 24 h of age on nd outcomes at 18 to 22 months corrected gestational age ( cga ) , and analyzed other important neonatal morbidities to ensure that any relationship between mode of ventilation and nd outcome was independent of co - morbid conditions . they found that ventilatory strategy at 24h of age independently predicts long - term neurodevelopmental outcome in elbw infants . the use of different inclusion criteria makes it difficult to compare the results of this study with previously reported outcome studies . most of these studies reported outcome according to birth weight and only included extremely preterm or elbw infants . nonetheless , in our study we could not find elbw infants matching our inclusion criteria . it seems that it may occur in the light of more adverse conditions of these infants in iranian nicu . as a result the results indicated that although dmq of both mv and wmv groups is significantly poorer than nbw group , this difference was more in mv group especially in fine motor quotient of vlbw infants in comparison to nbw group . these lower results in fine motor development and especially in vlbw were similar to cohort study by goyen and lui in 2002 . in this study there were no correlations between motor quotients and gestational age , sex , length of stay in nicu and days of mechanical ventilation in lbw group . this may be caused by small sample of infants or other possibilities that should be investigated later . in our study there were some limitations such as low parental cooperation and refusal to attend hospital for tests , fatigue and the children s need to rest frequently and the small sample size that interfere with our study.we just evaluated the motor development and more diagnostic evaluations in form of follow up study and in all aspects of development conduct on lbw infants with and without mv and in more days use of mv are suggested . the result of this study indicates the importance of special attention to developmental follow up of high risk and lbw infants , especially more complicated lbw infants . we hope for further studies in the field of developmental assessment and early rehabilitation of high - risk infants in our country . our results can be used by practitioners in their evidence based clinical works especially in developing countries . we would like to thank all lbw infants and their families and all who helped us in this study . this study was approved by the ethic committee of iran university of medical sciences , tehran , iran .

background : to determine whether using mechanical ventilation in neonatal intensive care unit ( nicu ) influences motor development of low birth weight ( lbw ) infants and to compare their motor development with normal birth weight ( nbw ) infants at the age of 8 to 12 months using peabody developmental motor scale 2 ( pdms-2 ) . methods : this cross sectional study was conducted on 70 lbw infants in two groups , mechanical ventilation ( mv ) group , n=35 and without mechanical ventilation ( wmv ) group , n=35 and 40 healthy nbw infants matched with lbw group for age . motor quotients were determined using pdms-2 and compared in all groups using anova statistical method and spss version 17 . results : comparison of the mean developmental motor quotient ( dmq ) of both mv and wmv groups showed significant differences with nbw group ( p < 0.05 ) . also , significant difference was found between the gross dmq of mv group and wmv group ( p < 0.05 ) . moreover , in mv group , both gross and fine motor quotients were considered as below average ( 16.12% ) . in wmv group , the gross motor quotient was considered as average ( 49.51% ) and the fine motor quotient was considered as below average ( 16.12% ) . conclusion : it seems that lbw infants have poor fine motor outcomes . the gross motor outcomes , on the other hand , will be significantly more influenced by using mechanical ventilation . in addition , more differences seem to be related to lower birth weight . very low birth weight ( vlbw ) infants are more prone to developmental difficulties than lbw infants with the history of using mechanical ventilation especially in fine motor development .

the alpha omega trial is a multicenter , double - blind , placebo - controlled trial with a 2 2 factorial design , which has been described in detail elsewhere ( 6,7 ) . in collaboration with cardiologists from 32 hospitals in the netherlands , we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . for the current study , the results of 1,014 patients with diabetes were analyzed ( supplementary fig . these patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . the patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha , 2 g / day ala , or the combination . the doses corresponded to recommended dietary allowances of those fatty acids . in trial margarines for active treatment groups , the margarines were developed by unilever research and development and could not be distinguished from each other in taste , odor , texture , and color . the trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . written informed consent was obtained from all patients . for logistical reasons , all patients used placebo margarine during the first 46 weeks after randomization , after which actual treatment started . after randomization , the patients received eight blinded margarine tubs of 250 g every 12 weeks . based on this information , the daily intakes of margarine and n-3 fatty acids were calculated . an objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . our definition of diabetes was based on a physician s diagnosis , use of antidiabetic drugs , and/or elevated blood glucose , defined as a casual plasma glucose level of 7.0 mmol / l ( 126 mg / dl ) for patients who had fasted > 4 h or a level 11.1 mmol / l ( 200.0 mg / dl ) in patients who did not fast . demographic factors , lifestyle , drug use , and medical history were assessed by questionnaire ( 6 ) . medication was coded according to the anatomical therapeutic chemical ( atc ) classification system ( world health organization , 2009 ) , including atc code a10 for antidiabetic medication . physical activity was assessed by the validated physical activity scale for the elderly ( pase ) questionnaire . a patient was called physically active when he or she had at least 5 days per week of > 30 min / day of physical activities of at least three metabolic equivalents of task . anthropometric measures , blood pressure , and heart rate were taken , and nonfasting blood was collected for determining serum lipids , plasma cholesteryl esters , and glucose ( 6 ) . examinations were repeated in a random sample of 159 ( 15.7% ) patients after 20 months and after 40 months in 476 patients ( 46.9% ) who completed the trial before 1 january 2009 . due to budget constraints , the remainder of the cohort filled out mailed questionnaires only . all patients were followed for clinical events , including those who discontinued margarine use during the trial . we monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . for fatal cases , death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . additional information on fatal events was obtained from hospitals , nursing homes , and family members . three members of the endpoint adjudication committee who were blinded for patient identity , treating physician , and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . in the cases of disagreement , information about the underlying causes of death was discussed until consensus was reached . in the original trial protocol , the primary end point was fatal chd , and one of the secondary end points was sudden death , a proxy for fatal arrhythmias ( 6 ) . due to the low mortality rate , the steering committee approved the switch from primary to secondary end point for fatal chd , and the extension with placement of cardioverter defibrillators for the definition of sudden death , as described in the statistical analysis plan ( 7 ) . fatal chd was defined as mortality from mi ( i20i25 ) , cardiac arrest ( i46 ) , and sudden death undefined ( r96 ) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest ( i46 ) , sudden death ( r96 ) , and implantable cardioverter defibrillator placement ( 6,7 ) . sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias ( 16,17 ) . self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . all indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . the definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest ( i46 ) and sudden death undefined ( r96 ) ( 7 ) . to prevent overlap in these definitions , we limited in the present analysis fatal chd to death from mi ( i20i25 ) . baseline characteristics were compared among the four treatment groups of patients using anova or tests , when appropriate . time - to - event data were analyzed with the kaplan - meier method and the log - rank test . the three treatment groups ( i.e. , epa - dha , ala , and epa - dha plus ala groups ) were compared with the placebo group for each outcome . hazard ratios ( hrs ) and 95% cis were computed for ventricular arrhythmia related events , fatal mi , and the combination of these two end points , using cox proportional hazard models . no adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias ( 5 ) . the alpha omega trial is a multicenter , double - blind , placebo - controlled trial with a 2 2 factorial design , which has been described in detail elsewhere ( 6,7 ) . in collaboration with cardiologists from 32 hospitals in the netherlands , we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . for the current study , the results of 1,014 patients with diabetes were analyzed ( supplementary fig . these patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . the patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha , 2 g / day ala , or the combination . the doses corresponded to recommended dietary allowances of those fatty acids . in trial margarines for active treatment groups , the margarines were developed by unilever research and development and could not be distinguished from each other in taste , odor , texture , and color . the trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . written informed consent was obtained from all patients . for logistical reasons , all patients used placebo margarine during the first 46 weeks after randomization , after which actual treatment started . after randomization , the patients received eight blinded margarine tubs of 250 g every 12 weeks . based on this information , the daily intakes of margarine and n-3 fatty acids were calculated . an objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . our definition of diabetes was based on a physician s diagnosis , use of antidiabetic drugs , and/or elevated blood glucose , defined as a casual plasma glucose level of 7.0 mmol / l ( 126 mg / dl ) for patients who had fasted > 4 h or a level 11.1 mmol / l ( 200.0 mg / dl ) in patients who did not fast . demographic factors , lifestyle , drug use , and medical history were assessed by questionnaire ( 6 ) . medication was coded according to the anatomical therapeutic chemical ( atc ) classification system ( world health organization , 2009 ) , including atc code a10 for antidiabetic medication . physical activity was assessed by the validated physical activity scale for the elderly ( pase ) questionnaire . a patient was called physically active when he or she had at least 5 days per week of > 30 min / day of physical activities of at least three metabolic equivalents of task . anthropometric measures , blood pressure , and heart rate were taken , and nonfasting blood was collected for determining serum lipids , plasma cholesteryl esters , and glucose ( 6 ) . examinations were repeated in a random sample of 159 ( 15.7% ) patients after 20 months and after 40 months in 476 patients ( 46.9% ) who completed the trial before 1 january 2009 . due to budget constraints , all patients were followed for clinical events , including those who discontinued margarine use during the trial . we monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . for fatal cases , death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . additional information on fatal events was obtained from hospitals , nursing homes , and family members . three members of the endpoint adjudication committee who were blinded for patient identity , treating physician , and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . in the cases of disagreement , information about the underlying causes of death was discussed until consensus was reached . in the original trial protocol , the primary end point was fatal chd , and one of the secondary end points was sudden death , a proxy for fatal arrhythmias ( 6 ) . due to the low mortality rate , the steering committee approved the switch from primary to secondary end point for fatal chd , and the extension with placement of cardioverter defibrillators for the definition of sudden death , as described in the statistical analysis plan ( 7 ) . fatal chd was defined as mortality from mi ( i20i25 ) , cardiac arrest ( i46 ) , and sudden death undefined ( r96 ) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest ( i46 ) , sudden death ( r96 ) , and implantable cardioverter defibrillator placement ( 6,7 ) . sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias ( 16,17 ) . self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . all indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . the definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest ( i46 ) and sudden death undefined ( r96 ) ( 7 ) . to prevent overlap in these definitions , we limited in the present analysis fatal chd to death from mi ( i20i25 ) . baseline characteristics were compared among the four treatment groups of patients using anova or tests , when appropriate . time - to - event data were analyzed with the kaplan - meier method and the log - rank test . the three treatment groups ( i.e. , epa - dha , ala , and epa - dha plus ala groups ) were compared with the placebo group for each outcome . hazard ratios ( hrs ) and 95% cis were computed for ventricular arrhythmia related events , fatal mi , and the combination of these two end points , using cox proportional hazard models . no adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias ( 5 ) . data were analyzed with spss 17.0 statistical software ( spss inc . , chicago , il ) . the definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels , physician - diagnosed self - report , and drug treatment , in 14.4% on elevated glucose levels only , in 9.9% on self - report only , and in 3.4% on drug treatment only . insulin was used by 273 ( 26.9% ) patients and 577 ( 56.9% ) used other antidiabetic medication , of whom 110 used both insulin and other antidiabetic medication . the proportion of women was lowest in the placebo group and highest in the ala group , and 5 ( 1.9% ) of the 267 women used hormone replacement therapy . the percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . baseline characteristics of the 1,014 patients with an mi and diabetes , * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . as data for a number of patients were missing from several variables ( bmi , 4 patients ; alcohol use , 1 patient ; and physical activity , 9 patients ) , some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician , was taking antidiabetic drugs , or had an elevated plasma glucose level ( 7.0 mmol / l [ 126 mg / dl ] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [ 200.0 mg / dl ] in the case of nonfasting patients ) . to convert the values for cholesterol to milligrams per deciliter , divide by 0.02586 . to convert the values for triglycerides to milligrams per deciliter , divide by 0.01129 . the bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task ( indicating at least moderate intensity for at least 30 min / day ) during 6 or 7 days / week . the mean intake ( sd ) of the trial margarine was 18.6 ( 5.2 ) g / day ; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 ( 4.2 ) g / day . patients in the two epa - dha groups received , on average , an additional amount of 223 ( 62 ) mg epa and 149 ( 42 ) mg of dha per day and those in the two groups of ala 1.9 ( 0.5 ) g / day . plasma cholesteryl esters were determined as a measure of compliance . at the final examination , the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo ( supplementary fig . 2 ) . ala supplementation increased ala by 64.2% and epa by 40.8% ; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9% ; and epa - dha plus ala supplementation increased plasma ala by 67.4% , epa by 118.4% , and dha by 44.0% . the median follow - up period was 40.7 months ( iqr 36.641.6 months ) , during which 3,195 person - years of follow - up were accumulated . during follow - up , 29 patients developed a ventricular arrhythmia related event , 2 died suddenly , 1 had a nonfatal cardiac arrest , 11 had a fatal cardiac arrest , and 15 had a cardioverter defibrillator implanted . indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% ( n = 13 ; 87% ) , resuscitated ventricular fibrillation ( n = 1 ) , and ventricular tachycardia with a left ventricular ejection fraction of 45% ( n = 1 ) . in addition , 27 patients died of mi , 23 from other vascular diseases , 34 from cancer , and 26 from other causes . the kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo ( fig . the lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . after adjustment for imbalances in age , sex , and current smoking , the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo ( cumulative incidence at median follow - up 0.9 vs. 5.6% , fig . similar effects were observed for the combined end - point cardiac arrest and sudden death ( 0.13 ; ci 0.021.09 ) and for placement of cardioverter defibrillators ( 0.19 : 0.021.55 ) . kaplan - meier curves of ventricular arrhythmia related events ( a ) , death from mi ( b ) , or both end points combined ( c ) . the cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . patients were randomly assigned to receive a margarine containing supplemental epa combined with dha , a margarine containing supplemental ala , a margarine containing both epa - dha and ala , or a placebo margarine . unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes , according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values , with the use of cox proportional hazards models . adjusted for age , sex , and smoking status . for fatal mi , the kaplan - meier curves did not differ significantly among the four groups ( fig . after adjustment for confounders , the strongest , although not significant , effect was observed for epa - dha plus ala ( hr 0.53 ; 95% ci 0.151.81 ; table 2 ) . the kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala ( fig . after adjustment , these three n-3 fatty acids together reduced this combined end point by 72% ( cumulative incidence at median follow - up 2.6 vs. 8.5% , fig . the definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels , physician - diagnosed self - report , and drug treatment , in 14.4% on elevated glucose levels only , in 9.9% on self - report only , and in 3.4% on drug treatment only . insulin was used by 273 ( 26.9% ) patients and 577 ( 56.9% ) used other antidiabetic medication , of whom 110 used both insulin and other antidiabetic medication . the proportion of women was lowest in the placebo group and highest in the ala group , and 5 ( 1.9% ) of the 267 women used hormone replacement therapy . the percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . baseline characteristics of the 1,014 patients with an mi and diabetes , * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . as data for a number of patients were missing from several variables ( bmi , 4 patients ; alcohol use , 1 patient ; and physical activity , 9 patients ) , some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician , was taking antidiabetic drugs , or had an elevated plasma glucose level ( 7.0 mmol / l [ 126 mg / dl ] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [ 200.0 mg / dl ] in the case of nonfasting patients ) . to convert the values for cholesterol to milligrams per deciliter , divide by 0.02586 . to convert the values for triglycerides to milligrams per deciliter , divide by 0.01129 . the bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task ( indicating at least moderate intensity for at least 30 min / day ) during 6 or 7 days / week . the mean intake ( sd ) of the trial margarine was 18.6 ( 5.2 ) g / day ; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 ( 4.2 ) g / day . patients in the two epa - dha groups received , on average , an additional amount of 223 ( 62 ) mg epa and 149 ( 42 ) mg of dha per day and those in the two groups of ala 1.9 ( 0.5 ) g / day . plasma cholesteryl esters were determined as a measure of compliance . at the final examination , the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo ( supplementary fig . 2 ) . ala supplementation increased ala by 64.2% and epa by 40.8% ; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9% ; and epa - dha plus ala supplementation increased plasma ala by 67.4% , epa by 118.4% , and dha by 44.0% . the median follow - up period was 40.7 months ( iqr 36.641.6 months ) , during which 3,195 person - years of follow - up were accumulated . during follow - up , 29 patients developed a ventricular arrhythmia related event , 2 died suddenly , 1 had a nonfatal cardiac arrest , 11 had a fatal cardiac arrest , and 15 had a cardioverter defibrillator implanted . indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% ( n = 13 ; 87% ) , resuscitated ventricular fibrillation ( n = 1 ) , and ventricular tachycardia with a left ventricular ejection fraction of 45% ( n = 1 ) . in addition , 27 patients died of mi , 23 from other vascular diseases , 34 from cancer , and 26 from other causes . the kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo ( fig . the lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . after adjustment for imbalances in age , sex , and current smoking , the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo ( cumulative incidence at median follow - up 0.9 vs. 5.6% , fig . similar effects were observed for the combined end - point cardiac arrest and sudden death ( 0.13 ; ci 0.021.09 ) and for placement of cardioverter defibrillators ( 0.19 : 0.021.55 ) . kaplan - meier curves of ventricular arrhythmia related events ( a ) , death from mi ( b ) , or both end points combined ( c ) . the cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . patients were randomly assigned to receive a margarine containing supplemental epa combined with dha , a margarine containing supplemental ala , a margarine containing both epa - dha and ala , or a placebo margarine . unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes , according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values , with the use of cox proportional hazards models . adjusted for age , sex , and smoking status . for fatal mi , the kaplan - meier curves did not differ significantly among the four groups ( fig . after adjustment for confounders , the strongest , although not significant , effect was observed for epa - dha plus ala ( hr 0.53 ; 95% ci 0.151.81 ; table 2 ) . the kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala ( fig . after adjustment , these three n-3 fatty acids together reduced this combined end point by 72% ( cumulative incidence at median follow - up 2.6 vs. 8.5% , fig . low - dose supplementation of n-3 fatty acids epa - dha plus ala significantly reduced ventricular arrhythmia related events in post - mi patients with diabetes . the combined supplementation of these three fatty acids also reduced the composite end - point ventricular arrhythmia related events plus fatal mi but not fatal mi alone . a limitation of the current study is the small number of patients who developed ventricular arrhythmias ( n = 29 ) or died of mi ( n = 27 ) . the study had enough power to detect significant effects of combined supplementation of epa - dha and ala on the end - point ventricular arrhythmia related events whether or not in combination with fatal mi . significant effects on these end points were not observed for either epa - dha or ala supplementation . the combined effects of epa - dha and ala on the two composite end points are compatible with additive effects of either epa - dha or ala . assuming an additive model , an hr of 0.47 0.58 = 0.27 is expected for ventricular arrhythmia related events and of 0.81 0.60 = 0.49 for the combination of ventricular arrhythmias and fatal mi . these results fit well with the 95% ci of these end points and are compatible with the hypothesis that all three n-3 fatty acids reduce the risk of ventricular arrhythmias . this is also in accord with the results of animal experiments suggesting that ala , epa , and dha have similar antiarrhythmic effects by influencing the na and l - type ca channels of cardiomyocytes ( 5 ) . in the gissi - prevenzione trial , patients with a recent mi ( <3 months ago ) were supplemented with 0.9 g of epa - dha per day , which reduced their risk of sudden death by 45% ( 8) . in later trials , recent meta - analyses quantified the effect of epa - dha supplementation on sudden death at a 19% ( hr 0.81 ; 95% ci 0.521.25 ) ( 2 ) and 13% ( 0.87 ; 0.760.99 ) reduction ( 3 ) . an explanation for the smaller effect of epa - dha in the more recent trials could be improved drug treatment of cardiovascular risk factors ( 18 ) . this is supported by the recently published results of the omega trial in which patients were randomized 314 days after acute mi . in these high - risk patients , a supplement of 0.8 g epa - dha per day did not reduce sudden cardiac death ( 19 ) . in this trial and in the alpha omega trial , almost all patients received antithrombotic drugs , antihypertensive drugs , and statins ( 7 ) . the patients in the gissi - prevenzione trial were also well treated with antithrombotic and antihypertensive drugs but only 29% received statins ( 8) . the alpha omega trial started in 2002 , the omega trial in 2003 , and the gissi - prevenzione in 1993 . one of the secondary end points in the alpha omega trial was ventricular arrhythmia related events . ventricular arrhythmias are the major cause of sudden death and cardiac arrest and cardioverter defibrillators were generally implanted in post - mi patients with a low ejection fraction who are at high risk for these arrhythmias ( 16,17 ) . the protective effect of the combined supplementation of the three n-3 fatty acids on this end point ( hr 0.16 ; 95% ci 0.040.69 ) contrasts with the absence of an effect of epa - dha in cardiac patients with implanted cardioverter defibrillators ( who all had had a severe arrhythmic event before placement ) ( 2 ) . these discrepant results suggest that n-3 fatty acids may reduce primary arrhythmias in post - mi patients with diabetes with a low ejection fraction but not secondary arrhythmias in cardiac patients with cardioverter defibrillators . epa - dha supplementation reduced fatal mi by 47% but this reduction was not significant , possibly due to the small number of events . a possible protective effect of epa - dha on fatal mi would be in accord with the results of a prospective study carried out in postmenopausal women ( 20 ) . in that study , smaller increases in stenosis were observed in diabetic women who consumed greater than two servings of fish or greater than one serving of fatty fish compared with diabetic women who consumed less fish . an intervention study carried out in japan showed that diabetic patients who were supplemented with 1.8 g epa per day in contrast with the control group did not show an increase in intima - media thickness during 2.1 years of follow - up ( 21 ) . these results suggest that in diabetic patients , fish and epa supplementation may reduce progression of atherosclerosis and the eventual risk of fatal mi . diabetes alters smooth muscle function through atherosclerotic lesion formation , plaque instability , and cardiovascular events . basic research shows that peroxisome proliferator activated receptor ( ppar- ) plays a critical role in the regulation of insulin sensitivity . to become functional , ppar- should bind to an appropriate ligand . cell culture studies provide evidence that epa and dha are potential ligands for ppar- activation ( 22 ) . recently published results of an in vivo experiment suggested that fish oil increases the expression of glucose uptake genes , leading to reduced glucose levels ( 23 ) . experiments in obese mice showed that activation of the g protein coupled receptor 120 ( gpr120 ) by epa and dha inhibited multiple inflammation cascades and reversed insulin resistance caused by a high - fat diet ( 24 ) . these mechanistic findings support an important role for n-3 fatty acids in the etiology of diabetes , a major risk factor of fatal mi . in conclusion , low doses of n-3 fatty acids reduced the risk of ventricular arrhythmia related events in post - mi patients who also have diabetes . ongoing trials in diabetic patients , e.g. , origin ( outcome reduction with an initial glargine intervention ; clinical trial reg . nct00069784 ) ( 25 ) and ascend ( a study of cardiovascular events in diabetes ; nct00135226 ; http://www.ctsu.ox.ac.uk/ascend/ ) , evaluate the effect of epa - dha on sudden death and fatal chd . the results of more trials on the effect of different n-3 fatty acids are needed before definitive conclusions can be drawn on their role in the etiology of ventricular arrhythmias and fatal chd .

objectivewe carried out a secondary analysis in high - risk patients with a previous myocardial infarction ( mi ) and diabetes in the alpha omega trial . we tested the hypothesis that in these patients an increased intake of the n-3 fatty acids eicosapentaenoic acid ( epa ) , docosahexaenoic acid ( dha ) , and -linolenic acid ( ala ) will reduce the incidence of ventricular arrhythmias and fatal mi.research design and methodsa subgroup of 1,014 post - mi patients with diabetes aged 6080 years was randomly allocated to receive one of four trial margarines , three with an additional amount of n-3 fatty acids and one placebo for 40 months . the end points were ventricular arrhythmia related events and fatal mi . the data were analyzed according to the intention - to - treat principle , using multivariable cox proportional hazards models.resultsthe patients consumed on average 18.6 g of margarine per day , which resulted in an additional intake of 223 mg epa plus 149 mg dha and/or 1.9 g ala in the active treatment groups . during follow - up , 29 patients developed a ventricular arrhythmia related events and 27 had a fatal mi . compared with placebo patients , the epa - dha plus ala group experienced less ventricular arrhythmia related events ( hazard ratio 0.16 ; 95% ci 0.040.69 ) . these n-3 fatty acids also reduced the combined end - point ventricular arrhythmia related events and fatal mi ( 0.28 ; 0.110.71).conclusionsour results suggest that low - dose supplementation of n-3 fatty acids exerts a protective effect against ventricular arrhythmia related events in post - mi patients with diabetes .

for more than 40 years , metal ceramic restorations ( mcrs ) have been widely used in the fabrication of fixed partial dentures ( fpds ) and still represent the gold standard nowadays.1 the success of these mcrs depends on the effective bonding between the veneering ceramic and metal substructure , which is believed to develop from chemical bond of the metal oxides.2 however , although the conventional mcrs have demonstrated superior fracture resistance , metal - free restorations have gained importance and their development has been accelerated by increasing interest in aesthetic dentistry.3 the introduction of zirconium dioxide as a framework structure or coping for ceramic restorations has initiated lengthy discussions on the design and limitations of these restorations . furthermore , the bonding mechanism between zirconia framework and veneering ceramic is not yet well understood since no documented evidence of the bonding mechanism between these materials is available.4 however , a few studies have looked into methods to increase the surface roughness of the zirconia.56 as the surface roughness increases , the bonding between zirconia coping and veneering ceramic increases , thus leading to success of the restorations . various surface treatments ( e.g. sandblasting , acid etching , glazing , heat treatment , and application of liner onto coping materials ) have been recommended to enhance the bonding efficiency between veneering ceramic and coping material . however , none of these treatments have been determined to produce the highest bond strength . airborne - particle abrasion or sandblasting , an important treatment procedure for achieving strong adhesion of veneering ceramics , works by increasing surface roughness and providing undercuts.7 for ceramic surface treatment , acid reacts with the glassy matrix that contains silica and forms hexafluorosilicates.8 as a result , the surface of the ceramic becomes rough , which is advantageous for micromechanical retention on the ceramic surface . combination of surface treatments , such as sandblasting with alumina oxide - particle and acid etching may substantially increase the surface area for micromechanical retention.9 this will subsequently increase the bond strength . therefore , the purpose of this study is to investigate shear bond strength ( sbs ) of veneering ceramic to different types of coping material ( metal alloy , zirconia oxide , and lithium disilicate ) after various pre - surface treatments such as sandblast and acid etch . the null hypothesis is that the shear bond strength of these veneering ceramic will not be different after various pre - surface treatments . ninety - six disc specimens measured 14 4 0.2 mm were prepared from three different type of coping materials ( n = 32 ) : i ) metal alloy ( ma ) , ii ) zirconia oxide ( zo ) , and iii ) lithium disilicate ( ld ) pre - cast wax patterns were prepared using silicone mold ( metrosil , metrodent , huddersfield , england ) and were invested with investment materials . , ramat - gan , israel ) pre - heated to 850 with alumina plunger for 90 minutes . for ma group , the mold was filled with molten ni - cr alloy ( 4all , ivoclar vivadent ag , schaan , liechtenstein ) using a casting machine ( fornax , bego gmbh , bremen , germany ) . for ld group , ips e.max press ( ivoclar vivadent ag , liechtenstein ) ingots were softened at 920 and were automatically pressed into the mold in a furnace ( ep 600 , ivoclar - vivadent , liechtenstein ) . after pressing and cooling to room temperature finally , the specimens were cleaned in an ultrasonic bath with distilled water for 15 minutes and air - dried for 10 seconds . for zo group , the specimens were machined out of a block of pre - sintered zirconia ( cercon , degudent gmbh , hanau - wolfgang , germany ) . the specimens were milled using cercon brain expert ( degudent gmbh , germany ) machine in an enlarged proportion according to the data installed from manufacturer 's software . the milled specimens were then sintered in the cercon heat plus furnace ( degudent gmbh , germany ) at 1,350 for six hours , producing final specimens sized 14 4 mm . the specimens from each coping material group were randomly divided into 4 subgroups with 8 specimens in each subgroup ( n = 8) . four pre - surface treatments were prepared for each subgroup ; a ) no treatment or control ( c ) , b ) sandblasted ( sb ) , c ) acid etched ( ae ) , and d ) combination of sandblasted and acid etched ( sbae ) . the specimens in subgroup c were grounded with diamond disc with no further treatment applied to the surface . the specimens in subgroup sb were treated with 50 m alumina ( al2o3 ) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen . the specimens in subgroup ae were etched with 5% hydrofluoric acid ( hf ) ( ips ceramic etching gel , ivoclar vivadent ag , liechtenstein ) for 20 seconds . the specimens in subgroup sbae were sandblasted with 50 m alumina ( al2o3 ) particles at 0.2 mpa for 10 seconds and at the 10 mm distance from the nozzle to the specimen . all specimens were cleaned in an ultrasonic bath containing acetone , alcohol , and distilled water for 10 minutes . two thin layers of opaque veneering ceramic in paste - liquid form were applied to the prepared surfaces of the specimens from group ma ( ips inline , ivoclar vivadent ag , liechtenstein ) and zo ( ceram kiss , degudent gmbh , germany ) . the specimens were fired in the furnace ( programart p500 , ivoclar vivadent ag , liechtenstein ) according to the recommendations of the manufacturers . a silicone mold ( duplicone , shofu inc . , kyoto , japan ) was fabricated with a slit measured 4 4 mm for the placement of veneering ceramic . the silicone mold was placed on top of the specimen disc . veneering ceramic for specimens ma ( ips inline , ivoclar vivadent ag , liechtenstein ) , zo ( ceram kiss , degudent gmbh , germany ) and ld ( ips emax ceram , ivoclar vivadent ag , liechtenstein ) in the study were manipulated as recommended by the manufacturer . liquid was added to veneering ceramic powder until paste consistency was obtained and condensed in the mold . after condensation was completed and excess moisture was removed with absorbent paper , the silicone was carefully removed . after the first firing cycle , another layer of dentin ceramic was applied and excess water was removed using absorbent paper . the specimens were fired for the second time to compensate for the shrinkage generated during the first cycle . at the end of these firing cycles , the veneering ceramic surface was ground flat and parallel to the coping surface using diagen turbo grinder ( bredent gmbh , senden , germany ) . a universal testing machine ( utm ) ( autograph ag - x , shimadzu , kyoto , japan ) was used for the sbs test at a crosshead speed 0.5 mm / min . a shear load was applied until failure occurred and failures fractures were determined from the chart recorder . the data were compiled and analyzed using the statistical package for social science ( spss ) software version 20.0 ( ibm inc . , armonk statistical analyses using two - way anova and tukey 's multiple comparisons tests were used . the mean sbs values for all groups of coping materials ranged from 11.61 4.84 mpa to 27.89 5.64 mpa ( table 1 ) . shapiro - wilk normality test showed that the data had a normal distribution ( p > .05 ) . two - way anova revealed that there were statistically significant interactions for the types of coping material groups , pre - surface treatment groups , and among the groups ( p < .05 ) ( table 2 ) . furthermore , there was a statistically significant interaction between the effect of coping materials and pre - surface treatments on shear bond strength , as shown by non - parallel lines within the graph ( p < .05 ) ( fig . tukey 's multiple comparisons showed a statistically significant difference in sbs of veneering ceramic within the coping material groups and the pre - surface treatment groups as shown in table 3 and table 4 ( p < .05 ) . among various types of coping materials tested , group zo produced significantly higher sbs than group ma , while group ma produced significantly higher sbs than group ld . on the other hand , the specimens treated with sbae showed significantly higher sbs than the specimens treated with sb alone or with no treatment . the specimens treated with ae were also showed significantly higher shear bond strength than the specimens treated with sb . for relationship between coping material and pre - surface treatment , tukey 's comparisons test showed a statistical significant interaction between the groups with p < .05 ( table 5 ) . in group ma , the specimens treated with ae showed significantly higher shear bond strength than other pre - surface treatments , while , in group zo and ld , the specimens treated with sbae showed significantly higher shear bond strength than other pre - surface treatments . international standard of organization ( iso ) has standardized the bond strength measurement of metal ceramic system through the schiwickerath crack initiation test ; the mean debonding strength should be greater than 25 mpa . however , due to the brittleness of all - ceramic coping materials , this test can not be applied to all - ceramic multilayered system.10 to date , there is no standardized test or minimum bond strength requirement for all - ceramic system.11 according to some authors , sbs values of 10 mpa is the minimum value for clinical flaw to happen between metal and ceramic.1213 therefore , sbs values more than 10 mpa indicate excellent bonding clinically . assuming that shear stresses are generally responsible for the clinical failure of the coping - ceramic interface , sbs test was adopted in this present study . in addition , this test required simple preparation of specimens and the testing could be performed easily . important aspects should be considered , including storage condition , type of substrate , specimen preparations , rate of load application , cross - sectional surface area , and experience of the researcher.11 in the present study , when the ceramic was veneered to ma , sbs values ranged from 14 to 24 mpa with mean value of 19.00 6.39 mpa . this finding was lower than sbs of base metal group recorded by al - dohan et al .. 11 the specimen 's size could be accounted for the differences . they used smaller diameter of veneering ceramic , which could have given higher bond strength value as the formula was calculated by dividing the maximum applied force by the bonded cross - sectional area . in other studies , sbs between nickel - chromium or cobalt - chromium metal alloy were reported higher than the sbs finding in the present study.141516 however , it is difficult to compare the results of the present study with those obtained in other studies because different methods were used to evaluate the sbs . furthermore , some authors suggested that the failure of the bonded interface occurred when a crack propagated from a flaw of a considerable size found in an area subjected to high tensile stresses.10 the sbs values reported for zo group in this study ranged from 22 to 27 mpa with mean value of 24.45 5.14 mpa . this finding was slightly lower than the sbs values obtained from other similar studies.1718 the other studies used the circular interface test , which was different from the sbs test in the present study . guess et al.19 found the mean sbss of veneering ceramic to zr to be ranged from 9.4 to 12.5 mpa , which were lower than the values in the present study . the different findings were attributed to different testing method , particularly related to the size and form of the specimens tested . the mean sbs value for ld group was 13.62 5.12 mpa , which the lowest sbs value obtained among all coping materials . the present study is in agreement with study done by umer et al.20 the finding of the current study showed that application of veneering ceramic onto the coping material lowered the strength of the bilayer specimens . however , several authors have reported that the mean sbs value for ld group was higher than zirconia and base metal groups in their studies , which contradicted the current finding.1121 there are several methods used for surface grinding , which include grinding using an abrasive paper or wheels ( sic or al2o3 ) , particle air - abrasion using al2o3 or other abrasive particles ranging in size from 50 to 250 m , and grinding using a diamond bur.22 the highest mean sbs value for sandblasting treatment was obtained from group zo , followed by ma and ld groups . this finding is in agreement with other studies.2324 the other studies reported that sbs value for sandblasting group was higher than the other groups and concluded that sbs of veneering ceramic on zirconia treated with sandblasting was significantly higher than that subjected to other pre - surface treatments . contrary to the current finding above , some researches have shown that surface grinding techniques have no significant effect on increasing the bond strength of zirconia to veneering ceramic.25 another possible problem with sandblasting is that it can create surface microcracks that can initiates bigger crack . these cracks later can decrease strength and cause fracture toughness of the material.26 since acid etching was first suggested as a ceramic presurface treatment for resin bonding , many different etching periods have been advocated and used . the most profound ceramic surface roughness and the highest bond strength data at the ceramic resin interface have been obtained by 2-minute acid etching , as reported by chen and suh in 1998.27 the present study found that the mean of sbs value for acid etching treatment was highest in ma group , followed by zo and ld group . the application of hf acid to metal was capable of roughening the surfaces , therefore increasing mechanical retention . the hf acid can also cause diminishing of oxide layer to the degree that would not affect the bonding.28 the statement might explain why the sbs value of ma group was still the highest after acid etching treatment was done . on the contrary , smielak and klimek found that etching zirconia copings with 5% hf showed no significant difference to the surface roughness , as the nature of the material was not glass - like.5 the present study found that the mean of sbs value for the combined treatment of sandblasting and acid etching produced the highest sbs value in ma and ld group compared with other pre - surface treatment . sandblasting with 50 m alumina particles changed the surface by increasing the number of pits per unit area . application of hf acid following acid etch was able to remove the glass matrix and the second crystalline phase , thus creating irregularities within the ld crystals.29 zirconia had the highest shear bond strength value , while lithium disilicate had the lowest shear bond strength value among the coping materials tested . combination of sandblasting and acid etching produced the highest sbs value in zirconia and lithium disilicate .

purposepre - surface treatments of coping materials have been recommended to enhance the bonding to the veneering ceramic . little is known on the effect on shear bond strength , particularly with new coping material . the aim of this study was to investigate the shear bond strength of veneering ceramic to three coping materials : i ) metal alloy ( ma ) , ii ) zirconia oxide ( zo ) , and iii ) lithium disilicate ( ld ) after various pre - surface treatments.materials and methodsthirty - two ( n = 32 ) discs were prepared for each coping material . four pre - surface treatments were prepared for each sub - group ( n = 8) ; a ) no treatment or control ( c ) , b ) sandblast ( sb ) , c ) acid etch ( ae ) , and d ) sandblast and acid etch ( sbae ) . veneering ceramics were applied to all discs . shear bond strength was measured with a universal testing machine . data were analyzed with two - way anova and tukey 's multiple comparisons tests.resultsmean shear bond strengths were obtained for ma ( 19.00 6.39 mpa ) , zo ( 24.45 5.14 mpa ) and ld ( 13.62 5.12 mpa ) . there were statistically significant differences in types of coping material and various pre - surface treatments ( p<.05 ) . there was a significant correlation between coping materials and pre - surface treatment to the shear bond strength ( p<.05).conclusionshear bond strength of veneering ceramic to zirconia oxide was higher than metal alloy and lithium disilicate . the highest shear bond strengths were obtained in sandblast and acid etch treatment for zirconia oxide and lithium disilicate groups , and in acid etch treatment for metal alloy group .

the ministry of health , labour and welfare of japan has recommended shortening of the length of hospital stays1 . in general , shortening the length of stay contributes to a reduction in the physical and mental stress of patients and rapidly returns them to their jobs and communities . actually , in a previous study , it was shown that a shorter length of stay resulted in not only a financial benefit but also a patient benefit2 . the patient s benefit is spending less time out of their home and reduction of the possibility of contracting nosocomial infections during a stay in the hospital2 . in stroke patients , stroke - related impairment , medical complications , family support , and discharge destination predict the length of stay3 . in another previous study , impairment , activities of daily living ( adl ) , unplanned discharges , and discharge to facilities affected the length of stay4 . on the other hand , similarly , patient - related factors were found to affect rehabilitation outcome , whereas illness- and intervention - related factors did not6 . this behavior pattern includes impatience , urgency , aggressiveness , and particularity about details . the type a behavior pattern is regarded as an independent risk factor of cardiovascular diseases7 . however , patients with type a behavior pattern characteristics may have better compliance with rehabilitation and other forms of medical and social support than those with non - type a behavior . if patients with the type a behavior pattern have better compliance , they would have a shorter length of hospital stay than those with non - type a behavior . to examine this hypothesis , in this study , we compared the length of stay of patients with the type a behavior pattern with that of patients with a non - type a behavior pattern . we hypothesized that length of stay of patients with the type a behavior pattern would be shorter than that of patients with a non - type a behavior pattern . fifty - seven patients participated in this study . all patients had stayed in the comprehensive rehabilitation unit of omaezaki municipal hospital between april and december 2013 . the exclusion criteria were as follows : transfer to another hospital , medical facility , or welfare facility during the research term and refusal to provide informed consent . type a behavior pattern was assessed by an abbreviated set of 12 questions developed by maeda8 . hardly were scored as 2 , 1 , and 0 points , respectively , for nine questions , and the points were doubled for three questions . a total score of 17 or greater was defined as type a , and the other subjects were defined as non - type a. after the subjects were discharged , we counted the number of days between admission and discharge to evaluate the length of stay . to evaluate the ability to perform adl at discharge , we measured barthel index for all patients . we used the student s t - test to examine statistical differences in length of stay and barthel index at discharge between subjects with type a behavior and those without type a behavior . we analyzed these measurements by statistical package for social sciences ( spss ) version 19 . ultimately , 55 subjects ( 75.311.7 years old , 18 males and 37 females ) were classified into either the type a group ( n = 26 ) or the non - type a group ( n = 29 ) , as shown in table 1table 1.characteristics of the type a and non - type a behavior patternscharacteristicstype a groupnon - type a grouppatients ( n)2629male ( n)108female ( n)1621cause of admissionstroke ( n)810fractures ( n)1310disuse syndrome ( n)23others ( n)36values represent number of subjects . others causes of admission include spinal code injury , spinal canal stenosis , and total knee arthroplasty .. the characteristics of subjects ( sex and cause of admission ) were similar in the two groups . age was not significantly different between the two groups . also , the barthel index at discharge was not significantly different between the two groups . however , length of stay was significantly higher in the non - type a group compared with the type a group ( p < 0.05 ) ( table 2table 2.difference in length of stay between the type a group and non - type a groupscharacteristicstype a groupnon - type a groupage ( years)77.271.4discharge bi97.5897.08length of stay ( days)66.287.4 * * p < 0.05 vs. type a group , values represent averages . others causes of admission include spinal code injury , spinal canal stenosis , and total knee arthroplasty . our results support the hypothesis that patients with the type a behavior pattern have better compliance with forms of medical support than patients with a non - type a behavior pattern . patients with the type a behavior pattern had a shorter length of hospital stay than patients with a non - type a behavior pattern . length of hospital stay can be affected by multiple factors , including medical complication , family support , discharge destination3 , impairment , adl4 , and rehabilitation9 . patient - related factors5 , especially psychological factors6 , affect rehabilitation outcomes . the subjects in this study were patients who stayed in a comprehensive rehabilitation unit . patients who stay in a comprehensive rehabilitation unit do not require much medical care and are able to focus on rehabilitation . hence , our data suggest that psychological factors of the patients , such as type a behavior , affect rehabilitation compliance . length of stay , ability to perform adl at discharge , and patient age influence rehabilitation outcome and whether patients can return home or not10 , 11 . there was no significant difference in barthel index or patient age between patients with the type a behavior pattern and those with a non - type a behavior pattern . thus , it appears likely that ability to perform adl at discharge , patient age , and destination at discharge do not influence length of stay . the type a behavior pattern includes impatience , urgency , aggressiveness , and particularity about details . patients who have higher levels of physical activity during rehabilitation were associated with a shorter length of stay12 . because patients with the type a behavior pattern might have good compliance with rehabilitation and increase their physical activity the type a behavior pattern is well known to be an independent risk factor of cardiovascular diseases7 . also in the japanese , the type a behavior pattern is associated with an increased risk of acute myocardial infarction , especially in women13 . so , the type a behavior pattern may be considered not a good characteristics in general . in contrast , another article reported that the type a behavior pattern reduced the risk of coronary heart diseases in japanese men14 . japanese men who do not express their anger may have an increased risk of high blood pressure15 . this previous article indicates that non - type a japanese men may have a higher hypertension risk than type a japanese men . in the japanese , the type our results also show that patients with the type a behavior pattern may be able to return home early . finally , the cause of a short length of stay may not be rehabilitation compliance . in conclusion , our results suggest that the type a behavior pattern shortens the length of hospital stay . the data show that we should consider the patient s characteristics in rehabilitation to protect the patient and for financial benefit .

[ purpose ] the aim of this study was to describe the importance of patient - related factors in rehabilitation . we focused on the type a behavior pattern . if individuals with the type a behavior pattern have better compliance , they would have a shorter length of hospital stay than those with non - type a behavior . we compared the length of stay of patients with the type a behavior pattern with that of patients with a non - type a behavior pattern . [ subjects and methods ] fifty - seven patients staying in a comprehensive rehabilitation unit participated in this study . type a behavior pattern , length of stay , and barthel index were assessed . we use the student s t - test to examine the statistical differences in length of stay and barthel index at discharge between subjects with type a behavior and those without type a behavior . [ results ] age and barthel index at discharge were not significantly different between the two groups . however , length of stay was significantly higher in the non - type a group compared with the type a group . [ conclusion ] patients with the type a behavior pattern had a shorter length of hospital stay than patients with a non - type a behavior pattern . in conclusion , our results suggest that the type a behavior pattern shortens the length of hospital stay . those data show that we should consider the patient s characteristics in rehabilitation to protect the patient and for financial benefit .

its effect is mediated via interactions with several receptors in the central nervous system and results from a combination of antidopaminergic , anticholinergic , antihistaminic , and weak antiadrenergic actions . the therapeutic effects of chlorpromazine are frequently accompanied by unwanted side effects that include sedation , autonomic , endocrine , and neurological effects . to date tinnitus is a common adverse reaction ( adr ) to several drugs and may occur during long - term therapies or after a single drug administration . even though not life threatening , tinnitus may be discomforting ; it may also be irreversible despite drug withdrawal . to date , over 130 drugs have been described to be potentially ototoxic , among which the most common inducers of tinnitus are aminoglycosides and other antimicrobials . we report on a suspect adr to chlorpromazine that occurred in a 12-year - old boy , affected by severe generalized anxiety disorder . he received initially a benzodiazepine therapy , which was switched to chlorpromazine ( 6.25 mg / day orally ) because of the absence of a significant clinical response . ten days after treatment with chlorpromazine , the patient experienced an enhanced sensitivity to sounds accompanied by perception of noises of the buzzing or ringing type . information about the patient 's medical history did not report conditions that may have predisposed to the onset of the disturbance manifested . moreover , the patient was in overall good health and had never suffered from hearing disorders . in view of the medical history , the inability to discontinue therapy with chlorpromazine resulted in an objective worsening of the patient 's symptoms , which are still present to date . the naranjo adr probability scale identified the relationship between the patient 's development of adr and the drug as possible . this is the first report on a case of tinnitus related to the administration of chlorpromazine . chlorpromazine is an antagonist of several dopamine cochlear receptors that play an important role in the sensory process by modulating afferent auditory nerve activity . dopamine , released from the terminals of lateral olivocochlear efferent fibers , is protective against acoustic trauma , hypoxia , and ototoxicity . in this context , the dopamine antagonist activity of chlorpromazine may result in a higher risk of ototoxicity . it controls the cochlear blood flow , acting on the precapillary sphincters and increasing the microcirculatory flow . clinical evidence indicates that h1 histamine agonists are effective in reducing tinnitus via improving vestibular compensation of the microcirculation . chlorpromazine antagonism on h1-receptors may thus play a role in counteracting the vessel modulatory effect of histamine and by this means have contributed to tinnitus development in our patient . acetylcholine is the major neurotransmitter in the olivocochlear efferent pathway , which is a feedback control system to the inner ear comprising a medial olivocochlear pathway projecting to outer hair cells and a lateral olivocochlear pathway projecting to dendrites of cochlear nerve fibres . in this context , the anticholinergic effects of chlorpromazine may have inhibited efferent signalling via the 9/10 nicotinic acetylcholine receptor complex in the outer hair cells which is known to be protective against acoustic injury . another action of chlorpromazine that may have contributed to generate tinnitus in our patient is its antagonism of serotonergic receptors . serotonin is one of the neurotransmitters acting on the auditory pathways ; in particular it is involved in sound detection , location , and interpretation . serotonin is currently believed to be one of the most important neurotransmitter involved in the perception of tinnitus . indeed , serotonin reuptake inhibitor drugs reduce the intensity of tinnitus acting directly on nerve conduction of the auditory stimulus , particularly in the central auditory pathways . in this scenario , it is thus conceivable that antagonism at serotonin receptor levels caused by chlorpromazine causes auditory disorders leading to tinnitus . finally , a role for an action of chlorpromazine on gamma amino butyric acid ( gaba ) can not be excluded . gaba inhibits auditory system and systemic administration of a gaba transaminase inhibitor improves tinnitus by suppressing hyperactivity in the auditory system . the neurotransmitter serotonin , involved in a large variety of physiological functions , behaves as a neuromodulator by strengthening the gaba system . chlorpromazine , by decreasing the availability of serotonin , may lead to decreased gabaergic activity and this action may have contributed to tinnitus development . we can not establish which among the actions of chlorpromazine described above has been predominant in the tinnitus - inducing action we observed , and the most likely possibility is that tinnitus resulted from a synergism among these different actions . a predisposition of the patient to develop tinnitus following chlorpromazine can not also be ruled out . receptors for serotonin , histamine , dopamine , and gaba are polymorphic and the presence of specific single nucleotide polymorphisms that acted as predisposing factors may be present in this specific patient . in addition chlorpromazine is substrate of cytochrome p4502d6 ( cyp2d6 ) , a highly polymorphic isoform of cytochrome . genetically determined functional variations of this cytochrome may be present in the patient and may also have contributed to the onset of tinnitus . to our knowledge , this is the first report on the development of tinnitus following chlorpromazine administration . although there is no information on dechallenge and rechallenge , the inability to discontinue therapy with chlorpromazine resulted in an objective worsening of the patient 's symptoms . this clinical case is of great clinical interest as chlorpromazine is not currently included among potentially ototoxic drugs ; paradoxically phenothiazines can be prescribed to alleviate symptoms related to disorders of the vestibular system .

chlorpromazine is a well - known antipsychotic agent that binds with a variety of receptors in the central nervous system . to date , chlorpromazine has never been associated with onset of hearing disorders and tinnitus . we report on an unexpected suspect adverse reaction to chlorpromazine that occurred in a 12-year - old boy , affected by severe generalized anxiety disorder . after treatment with chlorpromazine , the patient experienced an enhanced sensitivity to sounds accompanied by perception of noises of the buzzing or ringing type . this clinical case is of great clinical interest as chlorpromazine is not currently included among potentially ototoxic drugs .

investigating factors that influence the stability of the secondary structure of peptides and proteins , such as confinement , competitive solvation in a heterogeneous environment , and peptide functional group modifications , is important to our understanding of why proteins misfold and aggregate . reverse micelles ( rms ) are a convenient environment in which to encapsulate and probe biological molecules . the water cores of rms are similar to cavities found in biological systems , such as water molecules near the water membrane interface of a cell . because the size of rms is determined by the water to surfactant ratio ( water loading , w0 = [ h2o]/[surfactant ] ) , rms represent a environment for the study of the effects of limited hydration and confinement on protein folding , aggregation , and function . a significant effort has been made to explore the properties of rms using computer simulation , beginning with the influential work of ladanyi and coworkers . that work provided the foundation for subsequent studies of rms employing all - atom and coarse - grained models . subsequent studies explored the nature of water dynamics in rm environments , where it has been shown that water rotational relaxation is dramatically slowed relative to water in the bulk . the seminal experimental work of mukherjee et al . explored the structure of capped alanine - rich peptides akan ( ace - ygakaaaa-(kaaaa)ng - nh2 ) in aot rms ( using nonpolar solvent isooctane = 2,2,4-methylpentane ) and in bulk water . sodium bis(2-ethylhexyl ) sulfosuccinate ( aot ) is a widely used anionic surfactant that forms monodisperse rms in nonpolar solvents . using cd and ir spectroscopy , they observed that upon encapsulating the peptides in rms of low w0 the helical content increased significantly as compared with that measured in bulk water or a buffer . tian and garcia performed molecular dynamics simulations of self - assembling rms and encapsulated zwitterionic aka4 peptides . they observed nonspherical shape fluctuations of the rms and also found that the peptides preferred to reside at the water abel and coworkers obtained similar results for previous simulations of zwitterionic octa - alanine peptides in rms . martinez and coworkers performed simulations on capped aka2 peptide in bulk water and in rms of w0 = 6 that were both spherically restrained and unrestrained . for reference rms of w0 = 6 , the results indicated that the peptides were more helical in rms than in bulk water . additionally , the shape of unrestrained rms fluctuated significantly from an initial spherical geometry , facilitating interaction between the dehydrated peptide and the aot aliphatic tails that appears to be important in stabilizing the peptide s partial helical character . these results are in agreement with experiments as well as previous computational studies . it is known that the treatment of n- and c - termini with neutral caps can influence the peptide structure . however , while experimental studies of alanine - rich peptides in rms were performed with capped peptides , simulations studies by abel and coworkers and tian and garcia have employed zwitterionic termini , and those by martinez and coworkers have employed capped termini . the experimental observables in the original studies of mukherjee et al . were cd and ir spectra . however , no simulation studies have computed spectra to be directly compared with experiment . this study explores the effects of capped versus zwitterionic n- and c - termini on the helix stability of these same peptides in rms using two force fields . we performed simulations of the aka2 peptide in two forms , capped ( ace - ygakaaaa-(kaaaa)2g - nh2 ) and zwitterionic ( nh3 + -ygakaaaa-(kaaaa)2g - coo ) , in spherically restrained and unrestrained rms of w0 = 6 . the results demonstrate that capped aka2 peptides form more stable helices than zwitterionic aka2 peptides . the ir spectra of the amide i bond of the peptides were computed and directly compared with experiment . the computed peptide amide i ir spectra and water rotational anisotropy decay were found to be largely insensitive to the treatment of the peptide termini , while showing a distinct dependence on shape fluctuations in the rm environment . overall , our results provide insight into the nature of peptide confinement and hydration in a rm environment and the importance of considering shape fluctuations in characterizing the rm ensemble . the rms each contained a peptide monomer , and the starting structure for the peptides was an -helix . table 1 contains a summary of the simulation details for all systems , including the number of all molecules ( surfactant , ions , water , and solvent ) used and simulation times . the composition of the rms was determined from the saxs experiments of amararene et al . this composition differs from that suggested by the experiments of eicke and rehak . in a separate study , we have explored the structure and dynamics of alternative interpretations of the water loading for comparison . rm systems were generated using the charmm32 package with the charmm27 all - atom force field for proteins and lipids and the tip3p water model for charmm . charmm parameters for aot and isooctane were taken from the work of abel et al . to construct the rms the helical structure generated for the peptide was solvated with a sphere of water , and random water molecules were replaced with 76 sodium cations ( one for each aot molecule to be added ) and three chlorine anions ( one for each lys ) to create a neutral system . a spherical aot shell was added to the rm and was centered in a truncated octahedron of isooctane . for the spherically restrained rms , a massless dummy atom was fixed in the center of the rm , and a harmonic restraint was placed on the sulfur atom of each aot molecule to keep it within 11.25 and 15.25 of the dummy atom . the distance restraints were chosen to agree with experimental measurements of the solution density . the cutoff for the short - range electrostatics calculations was set to be 12 , and particle - mesh ewald was used to treat the long - range electrostatics . the temperature was held constant at 300 k , and the pressure was held constant at 1 atm using the langevin piston . each trajectory was run for 50 ns using a 1 fs time step and saving data every 0.1 ps . the same systems were also run using gromacs and the gromos96 53a6 united atom force field . the electrostatics were treated as in the charmm simulations . the temperature was held constant at 300 k , and the pressure was held constant at 1 atm using the berendsen piston . each trajectory was run for 50 ns using a 2 fs time step and saving data every 0.1 ps . analysis of all systems was performed using charmm , gromacs , mdanalysis , and vmd . consistent with experimental observations by halle and theoretical considerations by chandler , recent results from simulation and experiment suggest that unrestrained rms showed significant shape fluctuation . figure 1 shows representative structures taken from unrestrained charmm simulations after 50 ns of simulation time . the water and aot molecules are found to pucker around the peptide , allowing for significant interaction of the aot tail groups and at times the isooctane with the nonpolar surface of the peptide . these observations of nonspherical rm shape fluctuations and contacts between the peptide and the environment were also observed by tian and garcia in their simulations of similar systems . to characterize these structural fluctuations , we calculated the average elliptical radii and the radius of gyration for each rm system . the results ( not shown ) indicate an elliptical geometry for the unrestrained rms that are consistent with the results of previous experimental and simulations studies . helical peptides form dipoles with a partial positive charge at the n - terminus and a partial negative charge at the c - terminus . capping the termini eliminates the charge repulsion present when the terminal residues are charged and tends to stabilize the helix . figure 2 shows the secondary structure progression with respect to time for the residues of each of the aka2 peptides in rms for the charmm trajectories . the structure of the capped aka2 peptides changes very little during the simulation with the peptides remaining almost entirely -helical . the structure of the terminal residues of the zwitterionic aka2 peptides shows significant fluctuations , especially in the case of the unrestrained rms , suggesting that the zwitterionic termini contribute to the destabilization of the helical secondary structure . similar results were found for the gromacs trajectories ( data shown in supporting information ) . the secondary structure definitions used are those of kabsch and sander s dssp method . charmm unrestrained reverse micelles with capped ( left ) aka2 and zwitterionic ( right ) aka2 peptides . the components of the rm are colored as follows : aot tail groups , white surface ; sulfonate head groups , yellow and red ; sodium ions , dark green ; water molecules , blue ; peptide , bright green . secondary structure progression with respect to time of aka2 peptides in unrestrained ( top ) and spherically restrained ( bottom ) reverse micelles for the charmm systems : capped aka2 ( left ) and zwitterionic aka2 ( right ) . the secondary structure is indicated as helix ( blue ) , turn ( yellow ) , and coil ( white ) . another way to evaluate secondary structure stability is the root - mean - square deviation ( rmsd ) with respect to time . figure 3 presents a histogram of the observed peptide rmsd , demonstrating that the two capped aka2 peptides have a more stable backbone structure and undergo smaller fluctuations compared with the zwitterionic peptides . comparison of the dssp and rmsd plots suggests that fluctuations in rmsd for the capped aka2 peptide , in the unrestrained rm , in particular , result from the changes in the secondary structure in the terminal residues . significant interaction of the aka2 peptides with the surrounding environment of the rm is observed in all systems studied . in the unrestrained rms , however , in addition to the interaction between the peptides and water molecules , there is significant contact between the peptides and the surfactant molecules , especially the aliphatic aot tail groups . distribution of the peptide backbone rmsd for the last 30 ns of simulation for the aka2 peptides in rms for the charmm systems . data are shown for capped / zwitterionic peptides in restrained ( blue / yellow ) and unrestrained ( pink / green ) rms . fluctuations of the capped peptides are substantially smaller than for the zwitterionic peptides . figure 4 shows the average number of aot tail groups and water molecules within 4 of each residue of the aka2 peptides in rms for the charmm systems . the core residues of the peptides in the restrained rms are significantly more hydrated than the residues of the peptides in the unrestrained rms , which are in contact with more aot tail groups . average number of aot tail groups ( top ) and water molecules ( bottom ) within 4 of each aka2 residue in the charmm rms . data are shown for capped / zwitterionic peptides in restrained ( blue / yellow ) and unrestrained ( pink / green ) rms . figures 5 and 6 show the number of aot tail groups and water molecules within 4 of each of the aka2 peptides . the distributions show good agreement between peptides in the same environment ( restrained or unrestrained rm ) regardless of nature of the n and c termini . the distribution of the hydration values of the two peptides in the restrained rms is almost identical . peptides in the unrestrained rms have more contact with the tail groups than peptides in the restrained rms . data are shown for capped / zwitterionic peptides in restrained ( blue / yellow ) and unrestrained ( pink / green ) rms . data are shown for capped / zwitterionic peptides in restrained ( blue / yellow ) and unrestrained ( pink / green ) rms . the level of hydration of the peptides is similar in each environment with peptides in the restrained rms being more hydrated than peptides in the unrestrained rms . figure 7 shows snapshots of charmm aka2 in restrained and unrestrained rms with the surrounding water and aot tails after 50 ns of simulation . the peptide in the restrained rm is almost completely hydrated , while the peptide in the unrestrained rm is in contact with aot aliphatic tails . tian and garcia noted similar behavior in their simulations of aka4 peptides in rms . they observed significant contact between the nonpolar peptide core and the aot / water interface as well as some contact with isooctane molecules . water molecules bound to the interface and may increase the entropy of the system . the lysine residues , in particular , were either well - hydrated or in contact with the aot head groups . similar results were obtained for systems modeled using the gromos force field with the exception that , in general , the aka2 peptides in the rms are less hydrated than those for the charmm systems . capped aka2 in a restrained ( left ) and unrestrained ( right ) rm . the snapshot on the left shows the peptide in pink , and the snapshot on the right shows the peptide in green . each peptide is shown with the surrounding water molecules in blue and aot tails groups in gray . infrared spectra for the amide i modes of capped and zwitterionic aka2 peptides encapsulated in rm were calculated and compared with experimental data measured for capped aka2 peptides in rms of w0 = 6 at room temperature . simulated spectra were computed using a vibrational exciton model , in which the fundamental frequencies ( ffs ) and couplings are expressed as a function of electric field and van der waals forces on the atoms of the peptide bonds . in that recently developed approach , the ff for a particular amide i mode is derived from a map parametrized as1 in terms of the components of the electric field , ei , and van der waals force , fi , at atom i ( which includes the o , c , n , and h atoms of the peptide bond ) , where 0 is a static frequency ( which may be taken to be the gas phase value ) and ci and di are fitting coefficients as a function of the sum over parameters . all forces were computed using the full interaction between peptide bonds and the surrounding environment , the same forces that inform the molecular dynamics . the proposed maps successfully reproduced experimental data for a set of proteins with a wide range of sizes and secondary structures as well as model peptides in polar and nonpolar solvents . this broad range of applicability is essential for any method that aspires to compute accurate amide i vibrational spectra for peptides in the heterogeneous rm environment such as a rm , that includes polar , nonpolar , and charged domains . the carbonyl stretch makes the primary contribution to the amide i vibration , with minor contributions from the c -helices typically display a peak at 1655 cm , which shifts to lower frequency with increasing helix length . several other factors , including hydrogen bonding and solvation , contribute to the peak location . typically , hydrogen bonding lowers the frequency of bond stretching , and solvated helices may have peaks up to 20 cm lower than nonsolvated helices . calculated ir spectra for capped and zwitterionic aka2 peptides in restrained and unrestrained reverse micelles , with w0 = 6 at room temperature , compared with experiment . in our simulations , we observed that systems with more helical structures were associated with red - shifted spectra . we further observed that capped systems exhibited red - shifted spectra when compared with zwitterionic systems . the most helical peptide , capped aka2 in a restrained rm , was associated with a main peak at 1637 cm , while the peptide with the least helical content , zwitterionic aka2 in an unrestrained rm , produced a major peak with the highest frequency at 1646 cm . in fact , in our simulated spectra the positions of the main peak for all systems , with the exception of capped aka2 in a restrained rm , were nearly identical . additionally , the two peptides in the restrained rms were observed to be more hydrated than those in the unrestrained rms . as a result , the amide i spectra were found to be broader and peaked at lower frequencies , 1637 cm for the capped aka2 and 1645 cm for the zwitterionic aka2 . other features present in our calculations include additional small peaks , both red- and blue - shifted with respect to the main peaks . to explain the origin of these peaks , we examined the ffs of each peptide bond and included contributions from peptide , water , counterions , and aot surfactant molecules . figure 9 shows histograms of ff for each peptide bond , sums of the ffs , and the calculated spectra . our analysis demonstrates that the small peak at 1610 cm for the capped aka2 in a restrained rm can be associated with the 18th peptide bond , resulting from strong interactions with counterions . similarly , the appearance of a small peak at 1613 cm for zwitterionic aka2 in an unrestrained rm resulted from strong interactions of the fourth peptide bond with counterions . for the same system , the peak at 1663 cm is associated with large couplings of the 13th peptide bond with the nearest peptide bonds . histograms of the environmentally induced ff shift contributions provide additional insight into the role of the environment in inducing spectral heterogeneity . comparison of histograms of ffs of each peptide bond ( low lying histograms ) , sum of ffs ( green lines ) , and the calculated spectrum ( orange lines ) for each studied system . red - shifted ffs are shown as thin lines , and blue - shifted ffs are shown as dotted lines . the orientational dynamics of water confined in rms has been shown to be qualitatively different than the dynamics of bulk water . however , in an rm environment there are clear signs of stretched exponential and power - law decay resulting from the heterogeneous environment of water molecules under nanoscale confinement , as observed in other nanoconfined water systems . less is known about the how the presence of peptide in a rm influences the water dynamics , through direct interactions with the peptide , disruption of the water nanopool , and impact on the water h bond can be described through the autocorrelation function2where p2 is the second legendre polynomial and u(t ) is the unit vector along the o h bond at time , t. figure 10 shows c2(t ) reorientational correlation functions for water in six distinct rm environments , including restrained and unrestrained rms with capped aka2 peptide , aka2 peptide in a zwitterionic form , and rms without peptide . data for rms in the absence of peptide show faster relaxation than either peptide system . it is likely that this is due to the fact that the water rotational anisotropy decays faster for bulk water , and there is the largest percentage of bulk - like water in the rm with no peptide . table 2 contains parameters for fits of the rotational anisotropy decay to a piecewise continuous function formed from a short - time stretched exponential decay and long - time power - law decay . these parameters provide further evidence that the rotational anisotropy decay of water is slowed by the presence of peptide . rotational anisotropy decay autocorrelation functions for restrained and unrestrained rms with capped aka2 peptide , aka2 peptide in a zwitterionic form , and rms without peptide . for rms encapsulating aka2 peptide , water in the restrained rm systems shows slightly faster relaxation than water in the unrestrained rm up to 30 ps . this may be due to the fact that the aka2 peptides are more hydrated in the restrained rm system than in the unrestrained rm . peptides in the unrestrained rm are found to be in contact with only half the number of water molecules compared with water in the restrained rm simulations . in the unrestrained rm , there is a corresponding increase in the contact between the peptide and aot tail groups . however , this observed difference in the rotational anisotropy decay may not be statistically significant . at longer times , the difference in dynamics for these systems is on the order of the noise . for the aka2 peptide solvated in aot rms , no significant difference is observed in the rotational anisotropy decay of water interacting with the capped or zwitterionic peptides . this is consistent with the observation that the treatment of the n- and c - termini of the aka2 peptide does not dramatically impact interactions of the peptides with the water or surfactant environment . we have studied the effects of capped versus zwitterionic termini on the secondary structure of aka2 peptides encapsulated in spherically restrained and unrestrained rms . our results using two force fields demonstrate that capped aka2 peptides form more stable helices than zwitterionic aka2 peptides . we found that the cap employed on the peptide termini did not significantly alter the interactions between the peptides and their environment . calculation of the ir spectra of aka2 peptides in the charmm systems also suggests that our results are in agreement with the work of mukherjee et al . , with all four systems exhibiting a major peak in the amide i vibrational spectra within a few wavenumbers of the experimental peak . the observed rotational anisotropy decay shows distinct differences in water dynamics induced by the presence of the aka2 . this difference may be detectable in experiment , providing a means of assessing the differing dynamics of water directly involved in peptide solvation . overall , our results provide support for the presence of significant fluctuations in the rm structure away from an ideal spherical geometry as well as the importance of surfactant surface fluctuations in facilitating hydrophobic and hydrophilic interactions . the existence and potential significance of such shape fluctuations away from an idealized spherical geometry continue to be debated , with computational predictions forming a consensus view that is only dependent on force field and interpretations of water loading in terms of absolute numbers of water and surfactant molecules in a characteristic rm . ultimately , these questions must be resolved by experimental studies that directly assess rm structure and allow for direct , critical comparison of observables with the predictions of simulation studies . our simulation results demonstrate the amide i vibrational spectral lineshapes as well as the rotational anisotropy decay of water are sensitive to differences in restrained spherical rms and rms undergoing significant nonspherical fluctuations . as such , measurement of these experimental observables provides one way to critically assess the structure of rms , in both the absence and presence of solvated peptide .

the propensity of peptides to form -helices has been intensely studied using theory , computation , and experiment . important model peptides for the study of the coil - to - helix transition have been alanine lysine ( aka ) peptides in which the lysine residues are placed on opposite sides of the helix avoiding charge repulsion while enhancing solubility . in this study , the effects of capped versus zwitterionic peptide termini on the secondary structure of alanine - rich peptides in reverse micelles are explored . the reverse micelles are found to undergo substantial shape fluctuations , a property observed in previous studies of aot reverse micelles in the absence of solvated peptide . the peptides are observed to interact with water , as well as the aot surfactant , including interactions between the nonpolar residues and the aliphatic surfactant tails . computation of ir spectra for the amide i band of the peptide allows for direct comparison with experimental spectra . the results demonstrate that capped aka2 peptides form more stable helices than zwitterionic aka2 peptides in reverse micelles . the rotational anisotropy decay of water is found to be distinctly different in the presence or absence of peptide within the reverse micelle , suggesting that the introduction of peptide significantly alters the number of free waters within the reverse micelle nanopool . however , neither the nature of the peptide termini ( capped or charged ) nor the degree of peptide helicity is found to significantly alter the balance of interactions between the peptides and the environment . observed changes in the degree of helicity in aka2 peptides in bulk solution and in reverse micelle environments result from changes in peptide confinement and hydration as well as direct nonpolar and polar interactions with the water surfactant interface .

one of them is that development is the increase in production , promoting the health and therapeutic services level , and eliminating problems . therefore , development as it is considered by people and national institutions is a complicated and multidimensional process that requires changes in social structure , accelerating economic growth , reducing inequality , eradicating poverty , establishing social justice and equality , and environment stability . therefore , in order to achieve development , identifying developed and undeveloped regions is of great importance in the first step ( 1 ) . codifying and establishing development and success strategies in implicational and executive plans , studying and recognizing capabilities and shortcomings and deficiencies , determining development levels in regions based on a set of appropriate indexes are inevitable requirements through which executive managers will become enable to identify development strategies based on needs and specific conditions in each and propose plans consistent with the conditions in each region ( 2,3 ) . assessing and evaluating and determining priorities help executive managers meet necessary needs more quickly . health and therapy section is one of the sections in which prioritizing for meeting needs regarding resources limitation is essential ( 4 ) . inequality and its dimensions are indexes of underdevelopment because in fact countries are considered developed that have high economic and social indexes and the distribution of incomes and revenues and facilities is rather equal in them while in undeveloped countries the amounts of these indexes are low and there is inequality in their distributions ( 5 ) . numerous indexes are considered for assessing development level with the limitations in a time interval or period and place range . there has been a lot of efforts to define and design an appropriate instrument which is an indicator of the socioeconomic status of people in a society ( 7 , 8) . for example , australian researchers believe that using an index such as the validity of residency place for determining the socioeconomic status of individuals is very questionable . therefore , it is necessary to use different indexes for determining the socioeconomic status . among these different development indexes , health care index is one of the most important indexes of progress and development in any country due to its significant role in providing the society s health and the success of national development programs depends highly on achieving these aims ( 9 ) . a transient look at the health indexes in the country in the past decade shows the fast growing trend of the promotion of the indexes on one hand and inequalities in some indexes in different regions and provinces in the country on the other hand . it is necessary that in iran like in any developing country , special attention should be paid to the development of health and therapy section in order to improve the country s development status and position among other countries in the world since development in this section is the prerequisite for the development in other sections of the society . nowadays , the issue of development is a big concern in many countries . in other words , development is nothing more than making the living status and conditions more satisfying for people ( 10 ) . a lot of studies have been done about ranking the level of health indexes of development using statistical methods and mathematical patterns . in a research , regions of portugal were assessed and ranked regarding development using factor and cluster analysis techniques in the time intervals of 1991 and 1995 ( 11 ) . in studies by soares et al . ( 11 ) in portugal and yanis and andriant ( 12 ) , regions are ranked using health and economy indexes by factor analysis and clustering and fuzzy logic models . despite all criticisms to using quantitative models in city issues since 1970 , mathematic models can give a clearer picture and understanding of city phenomena if they are established and codified in simple frameworks with limited number of variables . standardized score and moriss s models are among quantitative models for assessing and ranking regions . standardized score model is used for comparing indexes and obtaining a single index from the combinatory results of the indexes . in fact , the standardized score model can uncover main differences among regions regarding the determined indexes ( 13 ) . morris model is also introduced as a model for ranking regions in terms of development by civil program of the united nations organization which is the most recent official model applied at an international level and is capable of being developed , replaced , and applied in the planning environments with various different scales . therefore , for achieving this goal and establishing social equality , it is necessary to rank regions and identify their degree or level of development regarding health and therapy indexes , determining capabilities , shortcomings , and deficiencies . the present study addresses the analysis of development levels in cities in tehran province regarding health infrastructural indexes using standardized score and morris inequality models . the present study is applied or pragmatic based on its aim and descriptive based on its nature . in this study , the cities in tehran were ranked based on health indexes in 2012 using standardized score and morris models . the studied geographic scope was tehran province and the statistical population was 14 cities in tehran . after studying the experts articles and opinions , 10 indexes were selected as health indexes including the ratio of active medical and therapeutic institutions to a population of one thousand individuals , the ratio of beds at active medical and therapeutic institutions to a population of one thousand individuals , the ratio of health therapeutic and medical centers to a population of one thousand people , the ratio of governmental public health and medical institutions to active health and medical institutions , the ratio of daily medical and therapeutic centers to active health medical and therapeutic centers , the ratio of 24-hour health medical and therapeutic centers to active health medical and therapeutic centers , the ratio of laboratories to a population of one thousand individuals , the ratio of pharmacies to a population of one thousand individuals , the ratio of radiology centers to a population of one thousand individuals , and the ratio of rehabilitation centers to a population of one thousand individuals . the required data were gathered from the statistics center and tehran university of medical sciences using a researcher - made information list including items and questions about the name of the cities , the number of active medical and therapeutic institutions , available beds , the number of health and medical therapeutic centers , the number of governmental public health medical and therapeutic centers , the number of daily health medical and therapeutic centers , the number of 24-hour health medical and therapeutic centers , the number of laboratories , the number of pharmacies , the number of radiology centers , the number of rehabilitation centers , and the population of the cities . after the completion of the lists , the development in cities was ranked and calculated using morris inequality and standardized score models in excel in 2010 . the calculations in the two methods are as follows : first , the indexes were standardized based on the city using standardized score model , as follows : which , ssij is standardized score of index i for city j , xij is amount of index i for the city j , x is mean of indexes and is standard deviation of the index i. then , the standardized score of each of the studied indexes in each city are added up together and the result is divided by the total number of indexes . the obtained score is the average of the standardized score or the development index of any city that makes the comparison regarding development status possible as a single index : which , ssi is the index for city j and n is the number of considered indexes . in morris model , the development status or position of each region among other regions is determined based on the selected indexes using the gathered information for each region . this method is used for reporting human resource development by the united nations and the obtained index is the evidence showing countries ranking regarding human resource development . in this method , the deviation of the numerical values of the index i in any region or area from the minimum of the index i among the regions or areas is divided by the range of the changes of that index , and the degree of the inconsistency or inequality of the numerical value of the index in relation to the dispersion index of its changes or variation range is computed ( r = x ( max ) - x ( min ) i ) . ( 3)miiij = xijx(min)ix(max)ix(min)i which , miiij is the value of morris inequality index for i index in the region or neighborhood j , xij is the numerical value of the index i in the region or neighborhood j , x(min)i is the lowest value of the index i and x(max)iis the highest value of the index i. in the next step , the mean of the numerical values of morris inequality index for indexes in each region or neighborhood is applied as the criteria for determining the ranking or development status from the highest value ( the first ranking ) to the lowest ( the last ranking ) : which , miii is development index for region or neighborhood j , and n is the number of considered indexes . in addition , for determining the developmental gap in health and therapy section in cities , 5 classifications of developed , rather developed , intermediately developed , less developed , and undeveloped were considered . then , for determining the distance between cities in 5 levels , first the range of changes in scores was obtained using a formula and then the distance between levels was calculated by b formula and as a result the cities were classified in 5 groups . based on the standardized score model , rey city has the best , and pishva city has the worst condition in terms of health indexes ( table 1 ) . based on the standard score model , 28% of the cities were placed in developed group and 43% of the cities were in undeveloped group ( table 2 ) . based on morris inequality model , rey has the best state and pishva has the worst state ( table 3 ) . based on morris inequality model , 28% of the cities were placed in developed group and 43% of the cities were placed in undeveloped group ( table 4 ) . the first step for developing health and therapy section and reducing the gap regarding health among different regions is to achieve a thorough understanding of the condition of the healthcare sector in the region . indexes of development in developing countries are not distributed equally in the regions and geographical zones . one of the main indexes of development is health index or in other words , the degree to which a society benefits from the health and therapeutic services and facilities . the developmental gap of these indices can be easily observed in the provinces and cities in the country . the findings showed that regarding the rate of development in the health sector , there is a deep development gap among the cities in tehran and the distribution of health services and facilities in the cities of the province seems unbalanced and unequal.for determining the development of cities in the standardized model , five levels of highly developed , developed , developing , deprived , and very deprived were considered.based on standardized score and morris inequality , only 28% of the cities were placed in the developed countries and this is in line with the results obtained by tofighi et al ( 14 ) and mirvis ( 15 ) . therefore , planners and policy makers should focus their efforts to find the cause of the gap in development.given the values of tables ( 1 ) and ( 3 ) , rey city has maintained its superiority over other cities and was considered as the developed city and pishva got the last ranking for the development of health indexes.the noticeable point is that tehran is in the third ranking despite the fact that it is the capital city which indicates that the geographical location can not be considered as the factor influencing the development of a city and this was confirmed in other studies ( 14).bahadori et al . ( 16 ) also investigated the ranking of the health structural indexes in golestan province using scalogram method.the results showed that there is a large gap regarding the benefits of the health structural indexes among the cities in golestan province.aqqala city with 97 points enjoyed the most and azad city with 41 points enjoyed the lowest level of the benefits of health structural indexes respectively . in this study , in the studies by nastaran ( 17 ) and zarabi in isfahan ( 18 ) , amini in all the provinces in the country ( 19 ) and taghvaee in all the provinces in the country ( 20 ) , similar results were obtained about the gap in enjoying the health structural indexes . therefore , it is suggested that officials , authorities , and planners pay attention to eliminating the mentioned problems , achieving growth and equal regional development , balancing the pattern of the distribution of health services and facilities , and decentralization of them in some cities ( 21 ) . the distribution of health services and facilities should be done based on the level or degree of underdevelopment in cities . in general , ranking the country s cities and provinces in health sector makes it possible for the related authorities to make more accurate planning , identify strengths and weaknesses , and prioritize resources adapted to the needs of each city and province . in summary , the results showed that statistical methods are effective tools for ranking and determining the status of development in the health sector . the results of this study concerning the allocation of the health sector resources would be useful for health planners and policy makers . structural indexes have always been considered as one of the main factors influencing health status and life.thus , to achieve a fair , balanced , and equal state of health in the province , it is suggested to make plans and take actions based on facts and the development situation of provinces and cities in order to reduce the gap in accessing and enjoying health facilities and services among the cities.furthermore , it is suggested that in the first stages of city development , authorities need to focus on short term policies and equity in access and pay attention to the development of necessary services in developing and deprived cities over a medium and long term plan .

background : one of the main indexes of development is health index or the degree to which a society enjoys health and therapeutic services . the present study was done with the aim to analyze development levels in cities in tehran regarding health infrastructural index using the standardized score and morris model . methods : this is a descriptive and pragmatic study which ranks 14 cities in tehran province using the standardized score and morris models based on 10 selected health indexes . the required data were gathered using a researcher - made information list and the information gathered from the statistics center and tehran university of medical sciences . the data were analyzed using excel software . results : the development coefficient in the studied cities varies from 0.595 to -0.379 so that rey city has the highest level of development and pishva city has the lowest level of development among the studied cities . the more number of the cities ( 43% ) was among the rather undeveloped group and none of the cities ( 0% ) was in the rather developed group . conclusion : regarding the findings , there is a big gap and difference regarding enjoying health and therapeutic infrastructural indexes among the cities in tehran province . therefore , it is suggested that development - oriented plans consistentent with development levels should be implemented in these cities .

hepatolithiasis , that is , the formation of stones in the intrahepatic biliary tree , is most prevalent in east asia , including china , japan , and korea ( 1 ) . the goals of primary treatment are to resolve ongoing infections and to prevent recurrent cholangitis , subsequent hepatic fibrosis , and progression to cholangiocarcinoma ( 2 ) . however , complications associated with hepatolithiasis , such as recurrent cholangitis , hepatic cirrhosis , and cholangiocarcinoma , frequently occur even after effective treatment . long - term complications are usually considered to result from inadequate stone retrieval , concurrent biliary stricture , or long standing cholangitis ( 3 , 4 ) . however , few studies have evaluated the risk factors of these complications after hepatolithiasis treatment . moreover , previous studies have only evaluated the risk factors of recurrent cholangitis after initial surgical and/or endoscopic treatment ( 5 , 6 ) . furthermore , the results of previous articles are inadequate to identify the risk factors responsible for hepatobiliary complications after treatment . therefore , we sought to identify the risk factors and long - term hepatobiliary outcomes with respect to recurrent cholangitis , liver abscess , secondary biliary cirrhosis , and cholangiocarcinoma , in patients with hepatolithiasis after treatment . the medical records of 260 patients with hepatolithiasis that were surgically and/or endoscopically managed at our institution between december 1996 and august 2005 were retrospectively reviewed . we excluded 175 patients due to concurrent malignancy , underlying biliary cirrhosis , a congenital anomaly , incomplete medical records , or postoperative mortality ( table 1 ) . hepatolithiasis was confirmed by abdominal ultrasonography , computed tomography , endoscopic retrograde cholangiopancreatography , magnetic resonance cholangiopancreatography , and/or surgery . further examinations depended on the ultrasonographic findings , severity of associated symptoms , combined clinical problems , and the intended mode of treatment . when the presence of intrahepatic duct stones , a liver mass , or a liver abscess was suspected based on sonographic findings , further examinations , such as abdominal ct , magnetic resonance cholangiopancreatography , and/or biopsy , were undertaken . definite indications for hepatectomy were stones localized in a unilateral lobe , bile duct stricture associated with stones , or atrophy of the affected liver segments or lobe . hepatectomy was performed at the lobe with intrahepatic stones . however , if treatment provided by endoscopic methods was inadequate in patients with stones in both lobes , a surgery strategy was adopted . on the other hand , patients at high operative risk , including the elderly , and those that refused operative treatment were treated endoscopically . follow - up was defined as time from date of discharge after initial treatment to the detection of subsequent cholangitis , liver abscess , secondary biliary cirrhosis , or cholangiocarcinoma . a residual stone was defined as a stone detected within 6 months by any diagnostic method . bile duct stricture was defined as definite narrowing of bile duct documented by cholangiography , cholangioscopy , or surgery . recurrent cholangitis was defined as a condition accompanied by presence of abdominal pain , fever , and/or jaundice without any other infection focus outside the hepatobiliary system requiring antibiotic administration . univariate analysis of individual and total hepatobiliary complications was performed using the chi - square test , fisher 's exact test , and the t - test . were approved by institutional review board of inha university hospital ( iuh - irb 13 - 1906 ) . univariate analysis of individual and total hepatobiliary complications was performed using the chi - square test , fisher 's exact test , and the t - test . the study protocol and amendments were approved by institutional review board of inha university hospital ( iuh - irb 13 - 1906 ) . there were 21 men and 64 women , whose ages ranged from 37 to 83 yr ( mean , 59.4 yr ) . of the patients , 65 were surgically treated and 20 underwent percutaneous transhepatic cholangioscopic lithotripsy ( ptcsl ) . residual stone rates were 35% in patients treated with ptcsl ( 7/20 ) and 26.2% in patients treated surgically ( 17/65 ) . biliary stricture was found in 35 patients , and 29/35 patients were manipulated by endoscopic dilation or segmental hepatectomy . however , surgery was performed in patients with stones located in bilateral lobes and liver atrophy in only one lobe , to prevent cholangiocarcinoma . similarly , partial segmentectomy was performed in patients with segmental atrophy of the liver and inadequate liver function . recurrent cholangitis occurred in 14 ( 16.5% ) , liver abscess in 3 ( 3.5% ) , secondary biliary cirrhosis in 5 ( 5.9% ) , and cholangiocarcinoma in 2 ( 2.4% ) . patients with recurrent cholangitis were treated by peroral transpapillary endoscopic lithotripsy ( n=6 ) , ptcsl ( n=4 ) , surgery ( n=1 ) , or systemic antibiotics only ( n=3 ) . univariate analysis showed that the presences of bile duct stricture and residual stones were significant predictors of hepatobiliary complications ( p = 0.001 , p = 0.009 , respectively ) . the rate of hepatobiliary complication was 34.3% in patients with a biliary stricture and 37.5% in patients with a residual stone . age , sex , and treatment methods were not found to be risk factors of hepatobiliary complications ( table 3 ) . multivariate analysis included age , sex , the presence of a residual stone , the presence of bile duct stricture , and treatment method . age ( hr , 1.046 ; ci , 1.006 - 1.089 ) , bile duct stricture ( hr , 4.894 ; ci , 1.295 - 18.495 ) , and residual stone ( hr , 3.482 ; ci , 1.214 - 9.981 ) were found to be independently correlated with the risk of developing a hepatobiliary complication ( table 4 ) . 1 demonstrates the cumulative risks of hepatobiliary complications according to the presence of bile duct stricture or a residual stone , respectively . although hepatolithiasis is a benign condition , the clinical progression of the disease may lead to liver parenchymal destruction due to recurrent cholangitis ( 7 ) . furthermore , hepatolithiasis can subsequently result in biliary cirrhosis and even cholangiocarcinoma ( 8) , which usually result from inadequate stone removal or stricture treatment ( 2 ) . residual stones are the most troublesome problem after treatment ( 9 ) . in a previous study , the overall recurrence rate of hepatolithiasis and/or cholangitis in patients with a residual stone after initial treatment was 57% ( 39/69 ) . on the other hand , the recurrence rate of hepatolithiasis and/or cholangitis in patients without a residual stone was only 17% ( 27/167 ) . in addition , it has been reported that recurrent biliary symptoms were more common in patients with a residual stone than in patients without a residual stone ( 6 ) . another retrospective study of 193 patients with hepatolithiasis , who had been newly diagnosed and treated , reported similar results ( 4 ) . in the study , cholangitis developed more commonly in patients with retained stones after hepatolithiasis treatment and cholangitis also developed more in patients with recurrent calculi in intrahepatic bile ducts than in patients without residual or recurrent stones ( 60% and 55% vs 29% and 9% , respectively ) . the results are similar to the present study , in which the complication rate in patients with a residual stone after hepatolithiasis treatment was 37.5% but the rate in patients without a residual stone was only 9.8% . furthermore , multivariate analysis showed that the presence of a residual stone was independently related to the risk of developing a hepatobiliary complication . in our experience , hepatolithiasis patients who retained residual stones even after aggressive treatment often had a good prognosis without any hepatobiliary events during long - term follow - up . we speculate that the good prognosis in this subset of patients with a residual stone , was due to the tendency of residual stones to locate to the peripheries of intrahepatic ducts and not to migrate into main ducts . nevertheless , the presence of a residual stone was found to be an independent risk factor of a long - term hepatobiliary complication . intrahepatic biliary stricture is a major cause of treatment failure for intrahepatic stones and the main cause of stone recurrence . biliary stricture often gives rise to bile stasis , cholangitis , and stone formation , and if the diseased ducts are not removed , the possibility of stone recurrence is high ( 2 ) . one retrospective study reviewed 74 patients treated for hepatolithiasis . at 1- to 23-yr follow - ups , patients with a bile duct stricture were found to have a higher rate of incomplete clearance ( 30% [ 11/37 ] vs 5% [ 2/37 ] ; p furthermore , the recurrence rate of hepatolithiasis was higher in patients with a bile duct stricture ( 69% [ 18/26 ] vs 37% [ 13/35 ] ; p , biliary stricture was found to be associated with a recurrent hepatobiliary complication after treatment , and the rate of stone and/or cholangitis recurrence was greater in patients with an intrahepatic duct stricture than in those without ( 31% [ 41/133 ] vs 47% [ 16/34 ] ; p = 0.075 ) . furthermore , multivariate analysis showed that an intrahepatic duct stricture was an independent risk factor of stone recurrence , cholangitis , and residual stones after treatment ( 4 ) . in the present study , hepatobiliary complication rate was 34.3% in patients with a biliary stricture , and by multivariate analysis , bile duct stricture was independently correlated with the risk of developing a hepatobiliary complication ( table 4 ) . these findings show that complete stone clearance and the elimination of intrahepatic duct strictures , is necessary to prevent recurrent hepatobiliary complications in patients with hepatolithiasis . stricture dilation with a balloon catheter has been reported to decrease the risk of recurrent cholangitis in hepatolithiasis concurrent with biliary stricture ( 10 ) . however , biliary stricture has also been reported to be an independent risk factor of long - term hepatobiliary complications in patients with hepatolithiasis , regardless of stricture dilation ( 5 ) . generally , the prevalence of intrahepatic stones increases with age ( 11 ) . however , we were unable to find any study that investigated the relations between recurrent hepatolithiasis and age . in the present study , patient age was found to be an independent risk factor for a long - term hepatobiliary complication . an age threshold was used to evaluate its influence on the rate of recurrent hepatobiliary complications . univariate analysis showed that age was not a significant risk factor of a hepatobiliary complication , but cox proportional hazards analysis showed that age was significantly associated with a long - term hepatobiliary complication . a previous study concluded that hepatectomy is more effective than non - hepatectomy surgical treatment for hepatolithiasis ( 3 ) . hepatectomy seems to be the most definitive approach because it allows stone and biliary stricture removal simultaneously , and thus , reduces the risk of intrahepatic stone recurrence ( 12 ) . however , other surgical methods , such as , choledochoenterostomy and t - tube drainage are available ( 13 ) . sometimes surgery may not be an option in those at high surgical risk who refuse surgery , or those that have undergone biliary surgery , or when stones are distributed in multiple segments ( 14 ) . the endoscopic treatment of hepatolithiasis is considered relatively safe ( 11 ) , but access is limited by strictures , ductal angulation , and the degree of stone impaction ( 12 ) . the cholangioscopy was introduced to visualize stones , blood clots , air bubbles , and duct strictures and to improve stone clearance success rates ( 15 ) . the first use of percutaneous transhepatic cholangioscopic lithotripsy for the treatment of hepatolithiasis was described in 1981 , and it is now common used to remove intrahepatic duct stones non - surgically . however , almost 40% of cases of hepatolithiasis patients have intrahepatic bile duct strictures , which make stone extraction difficult ( 2 ) . furthermore , bile duct strictures prevent the definitive removal of sclerotic damage to intrahepatic biliary ducts , and this predisposes the recurrence of septic complications and the need for repeated treatments ( 16 ) . in our study , treatment method was not found to be a significant risk factor of hepatobililary complications after initial treatment for hepatolithiasis by statistical analysis . however , this result could have been caused by selective bias , because when decisions were made regarding surgical and endoscopic methods , patient condition and location of stones in the biliary tract were considered . thus , when stones were deemed not amenable to endoscopy , a surgical method was chosen . first , it is inherently limited by its retrospective nature and particularly by the absence of clear clinical information . second , the study involved a relatively small number of patients , and it was performed at a single medical center , and thus , our results may not be representative of the general population . more specifically , the incidence of cholangiocarcinoma and the residual stone rate could have been affected by bias , as is suggested by a lower incidence of cholangiocarcinoma and a higher residual stone rate than have been previously reported . in conclusion , the present study shows the presence of a bile duct stricture is the strongest predictor of subsequent complications after initial treatment for hepatolithiasis . furthermore , hepatobiliary complications , such as , recurrent cholangitis , liver abscess , secondary biliary cirrhosis , and cholangiocarcinoma may occur frequently even after multidisciplinary treatment . therefore , close observation is required especially in hepatolithiasis concomitant with bile duct stricture or residual stones , and in the elderly , even after constructive treatment .

hepatobiliary complications , such as stone recurrence , recurrent cholangitis , liver abscess , secondary biliary cirrhosis , and cholangiocarcinoma may occur after treatment for hepatolithiasis . however , few previous studies have addressed the risk factors and long - term outcomes after initial treatment . eighty - five patients with newly diagnosed hepatolithiasis , actively treated for hepatolithiasis , constituted the cohort of this retrospective study . patients were treated by hepatectomy or nonoperative percutaneous transhepatic cholangioscopic lithotomy . long - term complications , such as recurrent cholangitis , liver abscess , secondary biliary cirrhosis , and cholangiocarcinoma , and their relationships with clinical parameters were analyzed . the mean follow - up period was 57.4 months . the overall hepatobiliary complication rate after the treatment was 17.6% . multivariate analysis of suspected risk factors showed that complications were associated with age ( hr , 1.046 ; ci , 1.006 - 1.089 ) , bile duct stricture ( hr , 4.894 ; ci , 1.295 - 18.495 ) , and residual stones ( hr , 3.482 ; ci , 1.214 - 9.981 ) . in conclusion , several long - term hepatobiliary complications occur after hepatolithiasis treatment , and regular observation is necessary in patients with concomitant biliary stricture or residual stones .

tuberculosis of oral cavity is a rare entity . even before antitubercular chemotherapy era , oral lesions occurred less commonly in patients with pulmonary tuberculosis . a 55-year - old labourer was referred to the outpatient department of our centre as a case of suspected gingivobu - ccal carcinoma for further evaluation and management . he had a non - healing ulcer in the oral cavity for one and a half months , which was associated with pain . he had been well until six weeks earlier when he noticed a lesion in his oral cavity . he visited the local doctor for this and was given symptomatic treatment and vitamin supplements . on enquiry , he had no systemic symptoms of chronic disease like fever , weight loss , and loss of appetite or weight . he also had no history of tuberculosis or contact with a tuberculosis patient . on clinical examination , the patient had an ulcer in the right lower gingivobuccal sulcus of size about 21 cm and was covered with slough . there was also mild fullness and soft tissue induration in the cheek below the lip . the patient 's height was 167 cm ; weight , 62 kg ; blood pressure , 124/76 mm hg ; and pulse , 74 beats per minute . on further investigation , haemoglobin was 13.9 gm% , total white blood cell count was 6,400 cells / dl , and erythrocyte sedimentation rate was 13 mm at the end of one hour . these are usually accompanied by associated history like a stressor event or the presence of a sharp tooth , etc , pointing to diagnosis . . the most common cause of a non - healing ulcerative lesion in the oral cavity is malignancy . the history is usually that of a rapidly growing lesion that does not respond well to any form of management . in most cases it is also usually accompanied by weight loss and features of chronic disease . in a developing country every year , approximately 2.2 million people develop tuberculosis , of which about one million are new - smear positive highly infectious cases and about 5 lakh people die of tuberculosis every year . this should be considered in the differential diagnosis , particularly in a non - healing lesion that does not respond to the usual therapy . actinomycosis is a rare , chronic , spreading , suppurative , granulomatous and fibrosing infection characterised by the formation of multiple abscess , draining sinuses and the release of characteristic sulphur granules . peak incidence occurs between 15 and 30 years , and males are more frequently infected than females . the majority of examples of the clinical disease are cervicofacial ( 55% ) , with only 20% occurring in an abdo - minopelvic form and 15% as a thoracopulmonic form . . are anaerobic or aerotolerant ( facultatively anaerobic ) , non - sporulating , gram - positive bacteria that tend to form branching rods and filaments and have a fermentative type of carbohydrate metabolism . are not regarded as virulent human pathogens and are best considered as opportunistic pathogens , as they are normally present in healthy individuals as commensals of the mouth cavity and the upper respiratory tract . their presence in the oral and respiratory specimens does not necessarily signify clinical disease and might often not be reported . are polymicrobial . the copathogens are most commonly colonisers of the respective organ systems involved . they act synergistically by inhibiting host - defense mechanisms or reducing the oxygen tension in the affected tissue , which promotes the growth of actinomyces spp . syphilis is an infectious disease caused by a spirochete called treponema pallidum ( tp ) , which has a tropism for several organs and tissues in the body , causing complex clinical manifestations . transmission of syphilis occurs mostly through sexual intercourse and sites of inoculations are usually genitals , though it can also be extragenital . it is characterised by several different dermatological lesions , involving both skin and mucous membrane . in the first stage of the disease ( primary syphilis ) , oral chancres syphilitic chancre is a solitary , painless , indurated , hard in consistency , reddish ulcer , accompanied by regional lymphadenopathy , which is localised at the site of tp inoculation and usually resolves after approximately one month without scarring . in secondary syphilis , the oral manifestations are characteristic lesions , which are multiple mucous patches that are slightly raised and covered by grayish , white pseudomembranes and surrounded by erythema . oral lesions are often painful and snail track ulcers result when multiple mucous patches become confluent . the diagnosis is confirmed by the specific serology and the demonstration of tp in the lesion . tp has a slender , coiled morphology and when examined by dark - field microscopy , it was seen moving in a drifting rotary motion ( corkscrew ) . naat is better than dark - field microscopic examination in the case of oral lesions because of the possible presence of saprophytic tp in the mouth . chest x - ray revealed an ill - defined patchy opacity and reticulo - nodular opacities in the upper and mid zone of both lungs [ figure 2 ] . sputum was positive for the presence of acid - fast bacilli 1 + in two consecutive samples . these are usually accompanied by associated history like a stressor event or the presence of a sharp tooth , etc , pointing to diagnosis . . the most common cause of a non - healing ulcerative lesion in the oral cavity is malignancy . the history is usually that of a rapidly growing lesion that does not respond well to any form of management . in most cases it is also usually accompanied by weight loss and features of chronic disease . in a developing country every year , approximately 2.2 million people develop tuberculosis , of which about one million are new - smear positive highly infectious cases and about 5 lakh people die of tuberculosis every year . this should be considered in the differential diagnosis , particularly in a non - healing lesion that does not respond to the usual therapy . actinomycosis is a rare , chronic , spreading , suppurative , granulomatous and fibrosing infection characterised by the formation of multiple abscess , draining sinuses and the release of characteristic sulphur granules . peak incidence occurs between 15 and 30 years , and males are more frequently infected than females . the majority of examples of the clinical disease are cervicofacial ( 55% ) , with only 20% occurring in an abdo - minopelvic form and 15% as a thoracopulmonic form . are anaerobic or aerotolerant ( facultatively anaerobic ) , non - sporulating , gram - positive bacteria that tend to form branching rods and filaments and have a fermentative type of carbohydrate metabolism . are not regarded as virulent human pathogens and are best considered as opportunistic pathogens , as they are normally present in healthy individuals as commensals of the mouth cavity and the upper respiratory tract . their presence in the oral and respiratory specimens does not necessarily signify clinical disease and might often not be reported . they act synergistically by inhibiting host - defense mechanisms or reducing the oxygen tension in the affected tissue , which promotes the growth of actinomyces spp . syphilis is an infectious disease caused by a spirochete called treponema pallidum ( tp ) , which has a tropism for several organs and tissues in the body , causing complex clinical manifestations . transmission of syphilis occurs mostly through sexual intercourse and sites of inoculations are usually genitals , though it can also be extragenital . it is characterised by several different dermatological lesions , involving both skin and mucous membrane . in the first stage of the disease ( primary syphilis ) , oral chancres syphilitic chancre is a solitary , painless , indurated , hard in consistency , reddish ulcer , accompanied by regional lymphadenopathy , which is localised at the site of tp inoculation and usually resolves after approximately one month without scarring . in secondary syphilis , the oral manifestations are characteristic lesions , which are multiple mucous patches that are slightly raised and covered by grayish , white pseudomembranes and surrounded by erythema . oral lesions are often painful and snail track ulcers result when multiple mucous patches become confluent . the diagnosis is confirmed by the specific serology and the demonstration of tp in the lesion . tp has a slender , coiled morphology and when examined by dark - field microscopy , it was seen moving in a drifting rotary motion ( corkscrew ) . naat is better than dark - field microscopic examination in the case of oral lesions because of the possible presence of saprophytic tp in the mouth . chest x - ray revealed an ill - defined patchy opacity and reticulo - nodular opacities in the upper and mid zone of both lungs [ figure 2 ] . sputum was positive for the presence of acid - fast bacilli 1 + in two consecutive samples . primary tuberculosis of the oral cavity is rare , as it occurs in young individuals and is associated with cervical lymphadenopathy.[258 ] secondary tuberculosis of the oral cavity usually occurs following pulmonary tuberculosis . it is more common than the primary variety and occurs more often in old age.[257 ] tuberculosis of the oral cavity is rare because the intact mucosa is resistant to tuberculous infection and saliva has a protective effect . other possible causes for the relative rarity of oral tuberculosis include the presence of saprophytes in the oral cavity and resistance of stratified muscles to bacterial invasion . there are reports in which dentists with pulmonary tuberculosis have infected patients during dental manipulation . nasolabial infection occurred in a physician who administered mouth - to - mouth resuscitation to a tuberculosis patient . other sites include the gingiva , soft palate , lip , buccal mucosa , floor of the mouth , and gingivobuccal sulcus . oral tuberculosis may present as single or multiple , painful or painless ulcers with irregular border . it can also present as nodules , fissures , vesicles , or peri - apical granulomas . the oral tuberculous ulcers have some characteristic clinical features including chronicity , hyperemic changes , and multiple or satellite lesions , which lead to the suspicion of a tuberculous lesion . oral tuberculosis occurs in 0.4 - 1.5% of patients with pulmonary tuberculosis . at present , with improved community health and anti - tubercular medication , the incidence of tuberculosis has decreased . oral tuberculosis is not considered in the differential diagnosis of patients with oral ulcer by many clinicians ; hence , it is missed or the treatment is delayed . the purpose of this paper is to alert clinicians to consider oral tuberculosis in patients with non - healing oral ulcer . on consultation with the physician , anti - tubercular therapy was initiated with isoniazid ( 10 mg / kg of body weight ) , rifampicin ( 10 - 20 mg / kg of body weight ) , and pyrazinamide ( 10 - 20 mg / kg of body weight ) and ethambutol for two months , followed by isoniazid and rifampicin for the following four months . during the period , the patient was instructed not to undergo any surgical procedure within the oral cavity and was warned of transmitting the disease to others . tuberculous ulcer of the oral cavity secondary to pulmonary tuberculosis . from the department of head and neck oncology , tata memorial hospital , mumbai .

tuberculosis of the oral cavity is a rare condition . a 55-year - old labourer was referred as a case of oral cancer for further management . the patient had no systemic symptoms . biopsy of the lesion revealed caseating granulomatous inflammation . chest x - ray and sputum revealed evidence of asymptomatic pulmonary tuberculosis . the purpose of this paper is to sensitize clinicians to consider oral tuberculosis as a differential diagnosis in patients with an non - healing oral cavity ulcer .

a sandwich or laminate technique is one of the methods proposed for composite resin restorations , which was introduced for the first time by mclean , et al . in 1985 . the basic idea behind this technique is to use two different restorative materials for a single restorative procedure so that the maximum physico - mechanical and esthetic properties of these two materials can be exploited simultaneously . generally , the first component is a layer of conventional or resin - modified glass - ionomer cement which has drawn attention due to its capacity to form an inherent bond with tooth structures and the resultant better seal and decrease in microleakage ( particularly in dentinal walls ) ; it can also release fluoride and decrease the odds of carious lesions . the second component is a layer of composite materials ( including composite resin , compomer , and ormocer ) , which is used to compensate for the limitations and disadvantages of glass - ionomer cements , including weak mechanical properties and inappropriate esthetic appearance . it has been reported that the combination of these two materials have different property results in the clinical success of restorations . the success of the laminate technique depends on the strength of the bond between the glass - ionomer and the resin composite materials in addition to the strength of the bond between the glass - ionomer cement and dentin . however , there is a relatively weak bond strength between conventional glass - ionomers and composite resin materials , mainly because of the lack of a chemical bond between these two materials and the low cohesive strength of glass - ionomer.[35 ] several studies have evaluated different surface preparation techniques for glass - ionomers , such as the use of different bonding systems and surface etching procedures in order to increase the bond strength between these two materials in the laminate technique . etching the surface with phosphoric acid has yielded different results . in a study carried out by sheth , et al . etching the surface of glass - ionomer had no effect on bond strength increase ; however , an increase in bond strength after acid etching has been reported in another study . it has also been reported that premature etching of glass - ionomer cement ( before its initial setting reaction ) and failure to use an adhesive resin between the glass - ionomer cement and composite resin increases the odds of restoration failure . in another study , it was demonstrated that the use of a self - etch adhesive system on half - set glass - ionomer cement ( before its initial setting ) increases the bond strength between the glass - ionomer cement and composite resin more than that observed with the use of total - etch systems ( its application after the initial setting of the cement ) . it has also been reported that the use of a glass - ionomer - based adhesive system applied after the cement 's initial setting improves the bond strength between the glass - ionomer cement and composite resin compared to that with the use of total - etch adhesive systems under similar conditions . a new group of composite resin materials , giomers , have been introduced in less than a decade , which consist of reacted glass - ionomer fillers in a resin matrix ; they are used in cavities in a manner similar to composite resins with the application of an adhesive system . in addition to appropriate esthetic results , easy polishing , fluoride recharging potential and strength , these materials release fluoride which may enhance their antibacterial effects . however , no studies to date have evaluated the bond strength between glass - ionomers and giomers ; therefore , the aim of the present study was to evaluate the effect of three surface preparation methods on the shear bond strength of giomer to different surface treated conventional glass - ionomer . sixty cylindrical specimens were used in the present in vitro study . in order to prepare the samples , a plastic mold ( with an inner diameter of 6 mm and a height of 4 mm ) was placed on a glass slab . then conventional glass - ionomer ( gc fuji ii , gc corporation , tokyo , japan ) was packed into the plastic mold after mixing according to the manufacturer 's instructions . another glass slide was used on the other side of the mold to make the free surface of the conventional glass - ionomer smooth . then the samples were randomly divided into three groups of 20 . according to the manufacturer , the initial setting of gc fuji ii conventional glass - ionomer lasts in 5 minutes and a half , and the net setting time is 2 minutes and a half . three groups were compared ; a total - etch adhesive resin or a self - etch giomer - based adhesive resin on set glass - ionomer ; or the self - etch giomer - based adhesive resin on the unset glass - ionomer ( before completion of the initial setting ) . in group 1 the surfaces of the specimens were etched with 37% phosphoric acid ( 3 m espe dental products , st . paul , mn , usa ) for 15 seconds after 5 minutes and 30 seconds from the mixing procedure ; the initial setting reaction was confirmed with the use of a sharp dental explorer . it should be pointed out that a complete setting of conventional glass - ionomer takes 24 - 72 hours . after surface etching , a single bond total - etch adhesive system ( 3 m espe dental products , st . paul , mn , usa ) was applied according to the manufacturer 's instructions and light cured [ table 1 ] . a second transparent mold , with a diameter of 3 mm and a height of 2 mm , was placed on the conventional glass - ionomer specimen and a giomer restorative , beautifil ii a3-shade ( shofu dental corporation , osaka , japan ) , was packed into the transparent mold and cured using a halogen light - curing unit ( astralis 7 ; ivoclar vivadent , fl-9494 schaan , liechtenstein ) . the light - directing probe had a diameter of 8 mm and directed a light ray at an intensity of 700 mw / cm perpendicular to the surface , barely touching the surface , for 40 seconds . a light intensity of 700 mw / cm was confirmed using a radiometer before the start of each experimental session . after transparent mold removal , the specimens were cured for another 20 seconds from each direction . then the samples were kept in humidity chamber at 37c for 1 hour before being immersed in distilled water at 37c for the next 23 hours . in the next stage , a 500-cycle thermocycling procedure was carried out at 5c/55c 5c with a dwell time of 30 seconds and a transfer time of 10 seconds . chemical composition and application mode of adhesives used in group 2 , the procedures were similar to those in group 1 except that fl - bond ii ( shofu dental corporation , osaka , japan ) self - etch adhesive resin was used on set glass - ionomer surface according to the manufacturer 's instructions and light cured [ table 1 ] , without acid etching . in group 3 , all the procedures were similar to those in group 2 except that fl - bond ii adhesive was applied according to the manufacturer 's instructions right after the initial setting and before hardening of glass - ionomer ( 2 minutes and a half after the initiation of mixing , which is equal to the cement 's net setting time ) . it should be pointed out that during that time the surface hardness of the cement was sufficient to place the second mold without damaging the cement . in order to measure the shear bond strength , the samples were mounted in cold - cured acrylic resin from the glass - ionomer side and the shear bond strength values of the samples were measured in newton using a universal testing machine ( h5k - s model , hounsfield test equipment , surrey , uk ) at a crosshead speed of 1 mm / min using a 0.5 mm - wide chisel . then the shear bond strength values were calculated in mpa by dividing the force ( in newton ) by the surface area ( mm ) of the samples . data were analyzed by one - way analysis of variance ( anova ) and pairwise comparisons were made by a tukey test using spss/ win.15 . the failure modes were determined under a stereomicroscope ( smz1500 , nikon , tokyo , japan ) at 20 . the failure modes were classified as follows : adhesive failure : failure at giomer glass ionomer cement interface . in addition , to evaluate the interface between the conventional glass - ionomer and giomer in the groups under study , two additional specimens were prepared in each group and subsequent to longitudinal sectioning , they were evaluated under an scanning electron microscope [ sem ] ( tescan , vega ii xmu , brno , czech republic ) [ figure 1 ] . scanning electron micrographs of conventional glass - ionomer and giomer interface in the study groups ( mag 500 ) . arrows indicate margins of adhesive resin between glass - ionomer and giomer . in the three scanning electron micrographs , the materials which are placed on the right and left sides of the adhesive resin indicate glass - ionomer and giomer , respectively shear bond strengths in mpa ( means and standard deviations ) for the study groups are represented in table 2 . there were statistically significant differences in shear bond strength values between the study groups ( p < 0.0005 ) . pairwise comparisons by a tukey test revealed that there were statistically significant differences in shear bond strengths between group 2 and the two other groups ( p < 0.0005 ) , whereas the bond strength difference between groups 1 and 3 was not statistically significant ( p = 0.609 ) . means and standard deviations ( sd ) of the shear bond strengths ( mpa ) measured in study groups moreover , all the cohesive failures were within glass - ionomer cement and there were no cohesive failures inside the giomer . failure modes of the study groups sem photomicrographs of the glass - ionomer and giomer interface in the study groups are represented in figure 1 . in the sem photomicrograph in group 1 [ figure 1a ] the hybrid zone ( the area of adhesive resin penetration into glass - ionomer ) is almost homogeneous but has irregular borders on the glass - ionomer cement side . in the sem photomicrograph in group 2 [ figure 1b ] the hybrid zone is regular and homogenous and in the sem photomicrograph in group 3 [ figure 1c ] the hybrid zone is irregular and non - homogenous . moreover , the use of self - etch adhesive resin ( fl - bond ii ) in groups 2 and 3 resulted in the thicker hybrid zone than total - etch adhesive resin ( single bond ) in group 1 . an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of the sandwich technique . in the present study shear bond strength of conventional glass - ionomer to giomer was evaluated using three different surface preparations for glass - ionomer ( use of self - etch adhesive on glass - ionomer with or without complete initial setting reaction and use of total - etch adhesive on glass - ionomer after completion of the initial setting reaction ) . in designing the present study , the use of total - etch adhesive on glass - ionomer without the completion of the initial setting reaction was not considered , because in total - etch adhesives , rinsing after etching and contamination with moisture before completion of the initial setting reaction of glass - ionomer influences the surface integrity of the cement . the results of the present study showed that shear bond strength of glass - ionomer to giomer depends on surface preparation . in this context , in the group in which glass - ionomer was set and self - etch adhesive was used , the highest shear bond strength was recorded compared to two other groups , which is consistent with the results of a study carried out by gopikrishna , et al . on glass - ionomer - based adhesives ; in the case of complete setting reaction the bond strength was higher than that in incomplete setting reaction . contrary to the results of the present study , knight , et al . reported no statistically significant differences in the bond strength before and after initial setting of glass - ionomer in the co - cure technique , where conventional glass - ionomer , resin - modified glass - ionomer ( rmgi ) , and composite resin were placed in consecutive layers before light - curing procedure . the differences in the results of that study and the present study might be attributed to different etching times in the two studies . the etching time was 15 seconds in the present study , while in the study done by knight , et al . it appears that longer etching time paves the way for greater destruction of glass - ionomer surface by the acid and decreases the bond strength . in addition , a different technique was used in the above - mentioned study , i.e. , a layer of rmgi was used over conventional glass - ionomer before placement of composite resin and the curing process was carried out for rmgi and composite resin simultaneously . it is well - known that the ph of glass ionomer cements is strongly acidic upon mixing , and it will increase with time , with the most rapid increase during the first 5 - 10 minutes of setting , regardless of the composition of the glass ionomer material . in comparison of groups 2 and 3 , it appears that the acidic ph of unset glass - ionomer prevented complete polymerization of the giomer - based self - etch adhesive and therefore decrease the bond strength . in addition , this acidic ph might have an adverse effect on the polymerization of giomer itself . in a study carried out by mohamed - tahir , et al . another study has shown that surface hardness of composite resin placed on polyalkenoate glass ( set for 4 minutes ) significantly decreased . moreover , sem observation of the interface in group 3 revealed an inhomogeneous structure and occasionally breakdown of the glass - ionomer surface , which may have affected the bond strength . polyacrylic acid prevents polymerization of composite resin and results in composite resin softening ; apparently this effect was intensified in incompletely set glass - ionomer . in comparison of groups 1 and 2 , it appears that etching the glass - ionomer surface immediately after the initial setting in group 1 in the total - etch system resulted in the destruction of cement surface , crack formation and decrease in bond strength . this speculation was confirmed by the sem micrographs of the interface . the irregular interface in group 1 may be an indication of damage and weakening of the glass ionomer surface rather than improved micromechanical interlocking , when compared to group 2 which was treated by a comparatively mild acid , i.e. , the self - etching primer . several studies have demonstrated a decrease in bond strength of glass - ionomer to composite resin as a result of etching the glass - ionomer surface . it has been reported that etching during the initial setting reaction of glass - ionomer leads to dissolution of weak calcium - polyacrylate rings , with the resultant deterioration of its physical properties . in the total - etch procedure however , another reason for a higher bond strength in the self - etch group compared to the total - etch group is the fact that the acidic monomers in the self - etch primer can chemically bond to the calcium in glass - ionomer and increase bond strength . in addition , a more appropriate compatibility of giomer with the giomer - based self - etch adhesive might be another reason for a higher bond strength in group 2 ; it has been suggested that the use of adhesives compatible with resin - based restorative materials can decrease deleterious chemical interferences . moreover in the self - etch adhesive group , resin penetration occurs simultaneous with the etching process and it is probable that the discrepancy between these two processes is eliminated or minimized . another factor that needs to be taken into account is the difference in composition and mechanical properties of the two adhesives ; fl - bond ii is a two - step self - etching adhesive with filler particles while single bond is a mixture of hydrophilic monomers and solvents that contains no fillers . it has been suggested that the two - step self - etching adhesives that incorporate a hydrophobic resin as a separate bonding agent may have enhance mechanical properties compared to simplified adhesives . moreover , apart from the composition of resin matrix , addition of filler particles enhances the mechanical strength of the bonding layer and contributes to bond strength . in the present study , no differences were observed in bond strength between groups 1 and 3 , and bond strength in both groups was significantly less than that in group 2 . regarding failure mode in the present study , the majority of failures were of the cohesive type in glass - ionomer , which is consistent with previous studies . this phenomenon might be attributed to lower mechanical properties of glass ionomer cements when compared to resin - based materials and the presence of numerous air inclusion bodies inside glass - ionomer , which act as stress concentration points and probably increase the odds of cohesive failure . a disadvantage of sandwich technique is the absence of a chemical bond between conventional glass - ionomer and superficial resin materials ; therefore , researchers have focused on the establishment of a chemical bond between them . considering the results of the present study , use of giomer - based self - etch systems not only decreases the time needed for the clinical application but also can result in the establishment of a chemical bond between giomer and conventional glass - ionomer . taking into account the limitation of this in vitro study , etching the surface of set glass - ionomer with a total - etch system or placement of self - etch adhesive on the surface of glass - ionomer with incomplete initial setting compromised bonding of giomer to glass - ionomer .

background : an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of sandwich technique . the aim of the present in vitro study was to evaluate the effect of three surface treatments of conventional glass - ionomer on its shear bond strength to giomer.materials and methods : sixty cylindrical specimens of a conventional glass - ionomer ( gc fuji ii ) were prepared and randomly divided into three groups ( n = 20 ) . the specimens in groups 1 and 2 were treated with total - etch adhesive resin ( single bond ) along with acid etching , and self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer , respectively . specimens in group 3 were treated with self - etch adhesive resin ( fl - bond ii ) before initial setting of the glass - ionomer was complete . then a giomer restorative ( beautifil ii ) was added to the specimens . subsequent to thermocycling , the specimens were subjected to shear bond strength test . failure modes were evaluated under a stereomicroscope . data were analyzed by one - way analysis of variance and a post hoc tukey test at a significance level of p < 0.05.results:there were statistically significant differences in bond strengths between the groups ( p < 0.0005 ) . differences in bond strengths between group 2 and other groups were significant ( p < 0.0005 ) while the differences between groups 1 and 3 were not significant . failures were predominantly of the cohesive type in all the groups.conclusion:based on the results of this study , the use of self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer yielded the highest bond strength in the glass - ionomer / giomer sandwich technique .

it is well known that the stress transfer between an implant device and a bone is not homogeneous when young 's moduli of the implant device and the bone are different ; this is defined as stress shielding . in such conditions , bone atrophy occurs and leads to the loosening of the implant and refracturing of the bone . therefore , it is desirable if the stiffness ( young 's modulus ) is not too high compared to that of bone . implant devices are mainly made from metallic biomaterials such as stainless steels , co - cr alloys , and titanium ( ti ) and its alloys . young 's moduli of these metallic biomaterials are generally much greater than that of the bone . young 's moduli of the most widely used stainless steel for implant devices , sus316l stainless steel and co - cr alloys , are around 180 gpa and 210 gpa , respectively . young 's moduli of ti ( pure titanium ) and its alloys are generally smaller than those of stainless steels and co - cr alloys . for example , ti and its alloy , ti-6al-4v eli , which are widely used for constructing implant devices , have a young 's modulus of around 110 gpa . however , this value is still higher than that of the bone , that is , 1030 gpa . ti alloys are grouped into - , ( + )- , and -type alloys . young 's moduli of - and ( + )-type titanium alloys such as ti and ti-6al-4v eli are higher than those of -type titanium alloys . therefore , -type titanium alloys are advantageous for the development of titanium alloys with low young 's modulus for biomedical applications . researchers have been focusing on reducing young 's moduli of -type titanium alloys for use in biomedical applications because they are composed of toxicity- and allergy - free elements . a number of -type titanium alloys mainly composed of toxicity- and allergy - free elements and with low young 's moduli have been developed or are still being developed . their young 's moduli are approximately below 80 gpa in solution - treated conditions . young 's modulus of a material can be different depending on the type of measurement methods used , such as tensile tests , three - point bending tests , and free resonance methods . the lowest value of young 's modulus reported for the polycrystal -type titanium alloy , ti-35nb-4sn , or ti-24nb-4zr-7.9sn , subjected to severe cold working , is around 40 gpa . the authors have also developed a -type titanium alloy , ti-29nb-13ta-4.6zr , referred to as tntz , that is composed of toxicity- and allergy - free elements and that has a low young 's modulus . young 's modulus of tntz subjected to solution treatment and measured by a resonance method has been found to be around 60 gpa . this value is lowered to around 55 gpa by severe working such as severe cold rolling and cold swaging . the strength as well as young 's modulus of titanium alloys is a very important factor for their long - term use in implants for biomedical applications . in particular , dynamic strength such as fatigue strength is highly important . developing the fatigue strength and simultaneously lowering young 's modulus is somewhat difficult because they are opposite natures when the fatigue strength of -type titanium alloys used in biomedical applications with maintaining the young 's modulus as low as possible is currently being developed . concerning young 's modulus , the level of the value of young 's modulus , which is effective to prevent the stress shielding between the implant made of the low - young 's modulus -type titanium alloy and bone should be proved . the effect of young 's modulus on bone atrophy has previously been investigated using implants made of titanium alloys with different young 's moduli . this paper chiefly describes the simultaneous improvement of the dynamic strength and lowering young 's modulus of the -type titanium alloy , tntz . the effect of young 's modulus on bone atrophy and bone remodeling has also been discussed in this paper . improving static strength such as tensile strength can be achieved by employing strengthening mechanisms such as work hardening , grain refinement strengthening , precipitation strengthening , and dispersion strengthening . one of the best ways to increase tensile strength while maintaining a young 's modulus low is to generally introduce many dislocations by ordinal severe cold working such as severe cold rolling and swaging and special severe cold working such as high pressure torsion ( hpt ) , accumulative roll - bonding ( arb ) , and equal channel angular pressing ( ecap ) . figure 1 shows the relationships between the tensile properties and working ratio of tntz subjected to cold working by general cold rolling or swaging . the relationships between young 's modulus and working ratio of tntz subjected to severe cold working by general severe cold rolling or swaging are shown in figure 2 . the tensile strength and 0.2% proof stress increase with an increase in the working ratio and become almost equal to those of ti-6al-4v eli ( having tensile strength of around 800 mpa ) with good elongation of tntz subjected to both cold rolling and cold swaging . young 's modulus of tntz subjected to cold rolling or cold swaging is almost constant with increasing working ratio . young 's modulus of tntz subjected to cold rolling tends to decrease when the working ratio is high because the trend of the formation of the texture becomes significant . figure 3 shows the tensile properties of tntz subjected to hpt as a function of the number of rotations , n. it also shows the tensile properties of tntz subjected to solution treatment and severe cold rolling . the tensile strength under hpt increases significantly with an increase in the number of rotations , in contrast , the elongation decreases with an increase in the number of rotations . young 's modulus of tntz subjected to hpt is almost constant with increasing the number of rotations although it decreases a little with increasing the rotation number . as shown in figure 4 , the dynamic strength , that is , fatigue strength of severe cold worked tntz is not high as compared to that of tntz subjected to solution treatment . the fatigue strength is significantly improved by conducting aging treatment after solution treatment or thermomechanical processing including severe cold working and aging treatment . the phase or the phase precipitates in the matrix phase by aging treatment . therefore , the fatigue strength can be significantly improved by precipitation strengthening caused by the precipitation of the or phase . however , as seen in figure 5 , young 's modulus increases by the precipitation of the or phase because young 's moduli of these phases are much higher than that of the matrix phase . the phase precipitation significantly increases the strength and young 's modulus of the alloy as compared to the phase precipitation although the phase enhances its brittleness . therefore , a small amount of phase precipitation is expected to improve the fatigue strength of tntz while maintaining its young 's modulus fairly low . for this purpose , short - time aging at a fairly low temperature , which enhances phase precipitation by a small amount , is effective . figure 6 shows young 's moduli of tntz subjected to solution treatment ( st ) , severe cold rolling ( cr ) , and aging after solution treatment at a temperature of 573 k(at ) as a function of the aging time . up to an aging time of around 10.8 ks , young 's modulus is below 80 gpa , which is a tentative target value for low young 's modulus . figure 7 shows fatigue properties ( s - n curves ) of tntz subjected to solution treatment ( st ) , severe cold rolling ( cr ) , and aging for 3.6 ks ( at3.6 ) and 10.8 ks ( at10.8 ) at a temperature of 573 k. the fatigue strength of tntz is improved by aging treatment for 10.8 ks ( at10.8 ) , whereas young 's modulus is lower than 80 gpa . thus , employing the proper precipitation method for the phase , that is , short - time aging at a relatively low temperature improves the fatigue strength of tntz while keeping young 's modulus low . the addition of a small amount of ceramic particles in the matrix is also expected to improve the fatigue strength of -type titanium alloys while maintaining low young 's modulus . figure 8 shows young 's modulus of tntz with y2o3 additions subjected to severe cold rolling as a function of y concentration . young 's modulus is nearly constant at around 60 gpa with increasing y concentration . figure 9 shows the s - n curves of tntz with 0.2 mass% and 0.5 mass% ( tntz-0.2ycr and 0.5ycr , resp . ) subjected to severe cold rolling after solution treatment and the s - n curves of tntz subjected to solution treatment or cold rolling after solution treatment . relationships between tensile strength and elongation of tntz added with different amounts of y2o3 subjected to severe cold rolling ( 0.05ycr : tntz-0.05ycr , 0.1ycr : tntz-0.1ycr , 0.2ycr : tntz-0.2ycr , 0.5ycr : tntz-0.5ycr , and 1.0ycr : tntz-1.0ycr ) and tntz subjected to severe cold rolling after solution treatment ( tntcr ) are shown in figure 10 . the balance of the tensile strength and elongation of tntz with y2o3 additions is excellent . while using low - modulus titanium alloys , some surgeons specializing in spinal diseases , such as scoliosis , spondylolisthesis , and spine fracture , pointed out that the amount of spring back in the implant rods should be small so that the implant offers better handling ability during surgeries . the implant rods undergo bending when they are manually handled by surgeons within the small space inside the patient 's body for in situ spine contouring . it is considered that the amount of spring back depends on both the strength and young 's modulus of the implant rod . if two implant rods having the same strength but with different young 's moduli are used , the implant rod having lower young 's modulus shows greater spring back . implant rods made of low modulus titanium alloys exhibit lower young 's modulus , resulting in greater spring back . thus , low young 's modulus , which is one of the key features of -type titanium alloys such as tntz as a metallic biomaterial , is obviously a desirable property for patients but becomes an undesirable property for surgeons . titanium alloys , which satisfy the requirements of both surgeons and patients with regard to young 's modulus of the implant rod , are currently being developed . the amount of spring back is considered to be small for the alloy having higher young 's modulus as compared to that of one having low - young 's modulus as schematically shown in figure 11 . therefore , the low - young 's modulus -type titanium alloy , whose young 's modulus varies and becomes high only at the deformed part , is considered to reduce spring back and satisfy the low - young 's modulus condition as well . this concept is called self - adjustment of young 's modulus . in general , the young 's modulus of metals and alloys does not drastically change by deformation . however , in the case of certain metastable -type titanium alloys , nonequilibrium phases such as , , and phases appear in the matrix during deformation . if young 's modulus of the deformation - induced phase is higher than that of the original phase , young 's modulus of only the deformed part of the implant rod increases , whereas that of the nondeformed part remains low . in orthopedic operations performed for the treatment of spinal diseases , the implant rod is bent by the surgeons so that it corresponds to the curvature of the spine . therefore , if a suitable titanium alloy is employed as the implant rod material , spring back can be suppressed by the deformation - induced phase transformation that occurs during bending in the course of operation , while low young 's modulus can be retained for patients . in general , young 's modulus of phase is much greater than those of the , , , and phase . among these phases , the , , and phase one of the candidate alloys with self - adjustable young 's modulus for biomedical applications has been reported to be ti-12cr . figure 12 shows young 's moduli of ti-12cr subjected to solution treatment ( ti-12cr - st ) and severe cold rolling ( ti-12cr - cr ) along with those of tntz subjected to solution treatment ( tntz - st ) and severe cold rolling ( tntz - cr ) . ti-12cr - st exhibits low young 's modulus of < 70 gpa ; this value is comparable to that of tntz - st , which has been developed as a biomedical -type titanium alloy having low young 's modulus . tntz - cr also shows low young 's modulus almost equal to that of tntz - st . however , in the case of ti-12cr , young 's modulus increases by cold rolling and that of ti-12cr - cr is > 80 gpa . the deformation - induced phase was detected in ti-12cr , but no induced phase was detected in tntz . therefore , the increase in young 's modulus of ti-12cr is probably the deformation - induced phase transformation . figure 13 shows the tensile properties of ti-12cr - st , ti-12cr - cr , tntz - st , and tntz - cr . the tensile strengths of both ti-12cr - st and tntz - st show an increase , but the elongation due to cold rolling tends to decrease . further , the tensile strengths of ti-12cr - st and ti-12cr - cr can be higher than those of tntz - st and tntz - cr , respectively . moreover , the elongations of ti-12cr - st and ti-12cr - cr are > 10% and ~10% , respectively . high strength is an essential requirement from the viewpoint of practical application , although such high strength could lead to undesirable spring back . therefore , the fundamental composition of ti-12cr makes it one of the preferred candidates for use in spinal fixation devices as a biomedical titanium alloy with the ability to self - adjust its young 's modulus . in the case of some types of internal fixation devices implanted into the bone marrow such as femoral , tibia , and humeral marrow , in the case of screws used for bone plate fixation , and in the case of implants used for children , which otherwise would grow into the bone , it is essential to remove the internal fixation device after surgery owing to certain specific indications ; these indications include significant local symptoms such as palpable hardware , wound dehiscence / exposure of hardware , or athletes returning to contact sport [ 16 , 17 ] . the assimilation of removable internal fixation device with the bone due to precipitation of calcium phosphate might cause refracture of the bone during the removal of the fixation device . therefore , in these cases , it is essential to prevent the adhesion of the alloys with the bone tissues . hence , considering this requirement , it is essential to inhibit the precipitation of calcium phosphate . it is reported that zr , which is nontoxic and allergy - free element , has the ability to prevent precipitation of calcium phosphate and ti alloys with zr contents exceeding 25 mass% prevent the formation of calcium phosphate , which is the main component of human bones . thus , ti-30zr - mo has been proposed as low young 's modulus titanium base biomaterials for use in removable implants . figure 14 shows young 's moduli of ti-30zr - xmo alloys subjected to solution treatment and those of the alloys considered for comparison . young 's modulus of ti-30zr - xmo is lower than that of the alloys considered for comparison except tntz . young 's modulus of 6mo shows a minimum value of around 60 gpa , and tntz also shows low young 's modulus . in orthopedic applications , ideal biomedical implant materials are required to have high strength and low young 's modulus . the elastic admissible strain , defined as the strength - to - modulus ratio , is a useful parameter considered in orthopedic applications . the higher the elastic admissible strain is , the more suitable are the materials for such applications . figure 15 shows the distribution of the as - solutionized ti-30zr - xmo alloys and the alloys considered for comparison in the plot of elastic admissible strain against elongation . 6mo and 7mo exhibit larger elongation and higher elastic admissible strain than those of the other ti-30zr - xmo and sus316l , cp ti , ti64 eli , and tntz . figure 16 shows the density of the cell cultured in 7mo and the alloys considered for comparison , for 24 h. 7mo has the highest value of cell density . therefore , 6mo and 7mo show promising potential to be new candidates for use in biomedical applications . it is very important to prove that the implant has young 's modulus similar to that of the bone , which will inhibit bone atrophy and good bone remodeling [ 1 , 4 ] . of course , the geometry of implant is another factor to control young 's modulus . studies have been conducted on the implantation of intramedullary rods and bone plates made of low - young 's modulus -type titanium alloy ( tntz ) , conventional practical ( + )-type titanium alloy ( ti-6al-4v eli ) , and conventional stainless steel ( sus316l ) into fracture models made into the tibiae of rabbits [ 8 , 22 ] . young 's moduli of tntz , ti-6al-4v eli , and sus316l stainless steel used for intramedullary rods , which were measured by three - point bending tests , were 58 , 108 , and 161 gpa , respectively . in both cases , the implantation of intramedullary rod and the implantation of bone plate , bone atrophy , and bone remodeling have been reported to be the least and the best , respectively , for tntz . figure 17 [ 8 , 22 ] shows the x - ray photographs of the fracture models implanted with intramedullary rods made of tntz and sus316l stainless steel at 24 weeks after implantation . bone atrophy can be observed at the upper rear portion of the tibia for the intramedullary rod made of sus316l stainless steel , but no bone atrophy is seen for the intramedullary rod made of tntz . large bone formation can be observed at the front part of the tibia for the intramedullary rod made of sus316l stainless steel , but very small bone formation can be observed at the front part of the tibia for the intramedullary rod made of tntz . therefore , low young 's modulus is effective in inhibiting bone atrophy and leads to excellent bone remodeling . further study on the effect of young 's modulus on the bone remodeling has been done by implanting bone plates made of tntz , ti-6al-4v eli , and sus316l stainless steel into the fracture models made in tibiae of rabbits . only for the case of the bone plate made of tntz , the increase in the diameter of the tibia and the double - wall structure in the intramedullary bone tissue has been reported to be observed as shown in figure 18 . for figure 18 , the inner wall bone structure is the original cortical bone , namely , the remained old cortical bone and the outer wall bone structure is newly formed one . this is the possible result of the bone remodeling with the low - young 's modulus bone plate . furthermore , it is necessary to understand the level of young 's modulus , which is effective for inhibiting bone atrophy and enhancing bone remodeling . figure 19 shows the profiles of the extracted bone plates made of tntz subjected to solution treatment ( tntz - st ) , tntz subjected to aging after solution treatment ( tntz - at ) , and sus316 l stainless steel ( sus316l ) attached to the tibiae of rabbits at 52 weeks after implantation . young 's moduli of tntz - st , tntz - at , and sus316l , which were measured by three - point bending tests , were 58 , 78 , and 161 gpa , respectively . the upper and side surfaces of each bone plate made are covered by newly formed bone , but a fairly large amount of newly formed bone can also be observed on the heads of the screws made of tntz - st and tntz - at , which are surrounded by circle marks . figure 20 shows the optical micrographs of the bone state beneath the bone plates made of tntz - st , tntz - at , and sus316l . bone atrophy can be observed in all cases but becomes more distinct with increasing young 's modulus ; it is the highest for sus316l , followed by tntz - at and tntz - st . it is expected that titanium alloy with even lower young 's modulus will be advantageous in inhibiting bone atrophy and lead to much better bone remodeling . titanium base alloys with low young 's modulus have been proved to be effective for inhibiting bone atrophy and enhancing bone remodeling by conducting animal tests in rabbits . therefore , low young 's modulus titanium alloys are expected to be useful in practical applications such as implant devices used for replacing failed hard tissue . however , considering the ease of the operation , other properties such as small spring back , which is effective in maintaining the bending shape of the implant in the body , and low yielding stress and high ultimate strength , which lead to ease in achieving permanent deformation of the implant in the narrow space in the body , are also important . metallic biomaterials , which satisfy the demands of both patients and surgeons , are highly required .

-type titanium alloys with low young 's modulus are required to inhibit bone atrophy and enhance bone remodeling for implants used to substitute failed hard tissue . at the same time , these titanium alloys are required to have high static and dynamic strength . on the other hand , metallic biomaterials with variable young 's modulus are required to satisfy the needs of both patients and surgeons , namely , low and high young 's moduli , respectively . in this paper , we have discussed effective methods to improve the static and dynamic strength while maintaining low young 's modulus for -type titanium alloys used in biomedical applications . then , the advantage of low young 's modulus of -type titanium alloys in biomedical applications has been discussed from the perspective of inhibiting bone atrophy and enhancing bone remodeling . further , we have discussed the development of -type titanium alloys with a self - adjusting young 's modulus for use in removable implants .

cervical spondylosis is a common cause of chronic neck pain , radiculopathy and/or myelopathy resulting in significant disability . when patients do not respond to conservative therapy , anterior cervical discectomy and fusion ( acdf ) usually is used to achieve neural decompression , maintain cervical lordosis , and provide segmental stabilization . in our practice , it was often found that patients with cervical spondylosis tended to have concomitant cervical vertigo symptoms such as tinnitus , headache , blur vision , and palpitation . recent studies have indicated that cervical spondylosis patients with cervical vertigo symptoms could be managed successfully with acdf . in this study , we report 2 cases of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with acdf . this study obtained the approval of the medical ethics committee of our hospital ( general hospital of armed police force , beijing , china ) . case 1 , a 49-year - old female , was admitted with 2 years of history of chronic neck pain , headache , and episodic vertigo and tinnitus . episode occurred 1 to 2 times every day , and lasted 1 to 2 h each time . she felt numbness of both upper extremities and weakness of all 4 extremities for 1 year . physical examination showed that limited neck motion , slightly diminished sensation in both arms , and brisk deep tendon reflexes . radiographic examination of the cervical spine showed mild diminution in the height of the c5/6 disc space ( figure 1 ) . magnetic resonance imaging ( mri ) scan showed cord compression by a large central herniation of c5/6 disc with signs of myelomalacia ( figure 2 ) . lateral radiograph of cervical spine before operation showed slight narrowing of the c5/6 disc space . t2-weighted sagittal magnetic resonance imaging ( mri ) revealed a large herniation of c5/6 disc before operation , with marked compression of cervical cord , resulting in myelomalacia . the patient had a 2-year history of hypertension , blood pressure was 160/100 mmhg at onset , and then controlled in the normal range ( 110130/7080 mmhg ) by oral medications ( betaloc , 100 mg , once daily ; enalapril , 10 mg , once daily ) . case 2 , a 60-year - old male , was admitted with 10 years of history of chronic neck pain and stiffness . he also experienced vertigo , palpitation , nausea and vomiting with noted numbness of upper limbs , and heaviness of 4 limbs for over 6 months before admission . radiographic examination of the cervical spine revealed large anterior and posterior spur formation at the c5/6 and c6/7 disc levels , severe diminution in the height of the c5/6 disc space , and moderate diminution in the height of the c6/7 disc space . mri scan showed cervical canal stenosis with marked cord compression secondary to spondylotic changes in c5/6 and c6/7 discs . the patient had a 1-year history of hypertension ( maximum at 180/120 mmhg ) , which was controlled within the normal range by oral medications ( irbesartan , 150 mg , once daily ; nifedipine sustained release tablets , 20 mg , once daily ; amlodiping besylate , 5 mg , once daily ) . cervical spondylotic myelopathy was diagnosed and subsequently cervical anterior surgery was recommended for the 2 patients preoperatively based on clinical findings consistent with imaging study . case 1 underwent acdf at the c5/6 disc level ( figures 3 and 4 ) . after surgery , strength in all 4 limbs was significantly recovered , and symptoms of neck pain , headache , vertigo , and tinnitus disappeared . interestingly , her blood pressure also dropped to within the normal range after surgery even without oral medications . lateral radiograph of cervical spine 3 months after operation showed anterior cervical plate fixation with cage fusion at c5/6 disc level . t2-weighted sagittal magnetic resonance imaging ( mri ) 7 days after operation showed disappearance of cord compression at c5/6 disc level . his symptoms of cervical vertigo completely disappeared , numbness of both upper limbs significantly released , and muscle strength of 4 limbs obviously increased . similarly to case 1 , her cervical vertigo never recurred , and her blood pressure remained normal without the use of antihypertensive medications . case 2 has undergone a follow - up of 14 months , the patient never had cervical vertigo anymore , and his blood pressure remained normal throughout ( 120130/7585 mmhg ) . clinical results indicated that acdf can eliminate the concomitant vertigo symptoms in the 2 patients . however , the exact mechanisms of resolution of symptoms can not be elucidated precisely . because cervical spondylosis is a common disease , vertigo occurred in patients with cervical spondylosis is of particular importance . one previous study showed that vertigo was present in 50% of patients with cervical spondylosis , whereas another study identified cervical spondylosis as the cause of dizziness in 65% of elderly patients . it has been shown that the patients with cervical spondylosis complaining of vertigo have significant lower blood flow parameters than non - vertigo patients with cervical spondylosis . insufficient blood supply to posterior circulation is called vertebrobasilar insufficiency . the most common complaint in patients with vertebrobasilar insufficiency insufficient blood supply does not necessarily cause symptoms if there is sufficient collateral circulation , whereas a full range of symptoms commonly occur as a result of an insufficient terminal vessel . the vascular supply to the vestibulocochlear organ , being an end artery , makes this organ more susceptible to vertebrobasilar insufficiency . neurons , axons , and hair cells in the vestibulocochlear system are known to respond to ischemia by depolarizing , causing transient hyperexcitability with ectopic discharges , manifesting as tinnitus , vertigo , and dizziness . it was hypothesized that vertebral artery insufficiency secondary to cervical spondylosis could result in vertigo . however , the mechanisms of vertigo caused by cervical spondylosis are not clear so far . the mechanical compression on vertebral artery from spurs of the luschka joint was considered as a main mechanism . the reduction of vertebral artery flow can be more obvious with the rotation and hyperextension of the head . in our current study in addition , mechanical compression can not explain some other symptoms such as palpitation , nausea , vomiting , etc . recent clinical studies by hong et al and li et al demonstrated that the stimulation of the sympathetic nerve fibers other than the compression of the vertebral artery induces these symptoms such as vertigo , tinnitus , nausea , and vomiting . current study clearly indicates that hypertension can be associated with cervical spondylosis , as a secondary condition . we speculate that the mechanisms of hypertension induced by cervical spondylosis could be the same as that of cervical vertigo . it is well known that cervical disc , dura mater , and posterior longitudinal ligament are rich in sympathetic fibers . sympathetic nerve fibers distributed around the vertebral artery have been implicated in the autoregulation of vertebrobasilar artery blood flow and cerebral blood flow . it has been evidenced that sympathetic nerve activity withdraws the blood flow , whereas sympathectomy can increase it . a previous study investigated sympathetic and parasympathetic changes incited by the stimulation of nerves around the proximal vertebral artery in cat model and found subsequent papillary changes , pulse and blood pressure changes . elevation in levels of inflammatory cytokines has been detected in painful discs of humans , and thought to be related to degeneration and pain . the pathogenic change in the painful disc is featured with the formation of zones of vascularized granulation tissue and extensive innervation extending from the outer layer of the annulus fibrosus into the nucleus pulposus . degenerative changes in the disc , such as loss of the normal structure and a mechanical load , can lead to abnormal motion , which can provoke mechanical stimulation . mechanical stimuli , which are normally innocuous to disc nociceptors can , in certain circumstances such as inflammation , generate an amplified response termed peripheral sensitization . the vertebral arteries are mainly innervated by nerve fibers from the cervical sympathetic ganglia ( 13 ) . innervation in cervical disc is analogous to that in the lumbar spine , receiving innervation posteriorly from the sinuvetebral nerves , laterally from the vertebral nerve , and anteriorly from the sympathetic trunks . recently , an animal study revealed a reciprocal neural connection between cervical spinal and sympathetic ganglia . it seems likely that stimulation of sympathetic nerve fibers in the pathologically degenerative disc and surrounding tissues produce sympathetic excitation , and induce a sympathetic reflex to cause vertebrobasilar insufficiency and hypertension . improvement of neurological function , resolution of vertigo , and recovery of blood pressure following surgery could be attributed to the excision of degenerative and herniated cervical disc , the decompression of the dura mater and posterior longitudinal ligament , and the stabilization of degenerated segment . chronic neck pain is one of the major symptoms in patients with cervical spondylosis , whereas some studies have indicated that resting blood pressure levels may be elevated in patients with persistent pain . in healthy individuals , these interactions are believed to reflect a homeostatic feedback loop that helps restore arousal levels in the presence of acutely painful stimuli . a role for baroreceptors in this feedback loop appears likely , with pain triggering sympathetically driven blood pressure increases , resulting in increased stimulation of baroreceptors , which in turn activates descending pain inhibitory pathways . previous studies suggested that blood pressure / pain regulatory relationship may be substantially altered in chronic pain conditions . a retrospective study by bruehl et al suggested that chronic pain could be associated with increased risk of hypertension . their study found that over 39% of patients with chronic pain were diagnosed with clinical hypertension , compared with only 21% of pain - free internal medicine patients . they thought that chronic pain - related impairments in overlapping systems modulating both pain and blood pressure would result in a higher prevalence of clinically diagnosed hypertension in patients with chronic pain than in comparable pain - free patients . our study indicates a possible relation between chronic neck pain and hypertension . if chronic neck pain could lead to hypertension through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms , early treatment for resolution of chronic neck pain may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis we report here 2 patients of cervical spondylosis with concomitant cervical vertigo and hypertension that were treated successfully with anterior cervical discectomy and fusion . stimulation of sympathetic nerve fibers in pathologically degenerative disc could produce sympathetic excitation , and induce a sympathetic reflex to cause cervical vertigo and hypertension . in addition , chronic neck pain could contribute to hypertension development through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms . early treatment for resolution of symptoms of cervical spondylosis may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis .

abstractcervical spondylosis and hypertension are all common diseases , but the relationship between them has never been studied . patients with cervical spondylosis are often accompanied with vertigo . anterior cervical discectomy and fusion is an effective method of treatment for cervical spondylosis with cervical vertigo that is unresponsive to conservative therapy . we report 2 patients of cervical spondylosis with concomitant cervical vertigo and hypertension who were treated successfully with anterior cervical discectomy and fusion . stimulation of sympathetic nerve fibers in pathologically degenerative disc could produce sympathetic excitation , and induce a sympathetic reflex to cause cervical vertigo and hypertension . in addition , chronic neck pain could contribute to hypertension development through sympathetic arousal and failure of normal homeostatic pain regulatory mechanisms.cervical spondylosis may be one of the causes of secondary hypertension . early treatment for resolution of symptoms of cervical spondylosis may have a beneficial impact on cardiovascular disease risk in patients with cervical spondylosis .

the worldwide incidence of strabismus varies from 3% to 5%,1,2 whereas hu and colleagues3 identified strabismus in 2% to 4% of white populations , and abrahamsson and colleagues4 reported strabismus in 0.6% of asians3 and africans.5 in a study conducted by dana and colleagues5 in sydney , the incidence of strabismus was established in 48 patients ( 2.8% of total population ) . in another study carried out by donnelly and colleagues , the prevalence was 3.98%.6 in an analysis conducted amongst afghan immigrants in pakistan , strabismus was noticed in 1.4% of patients.7 another study conducted at peshawar in 2004 confirmed that the total frequency of squint was 2%.8 the clinical treatment of squint includes evaluation and correction of multiple errors of refraction , management of amblyopia , and surgical treatment . the first surgery on squint was executed in 1839 by a general surgeon , johann dieffenbach.9 the ocular realignment of visual axis by surgical intervention becomes necessary when conservative management is unsuccessful . surgical interventions are able to improve diplopia , rectify three dimensional ( 3d ) vision , broaden the visual field , and improve psychological status10 and cosmesis.11 this is a retrospective clinical analysis conducted on the patients of both sexes with horizontal strabismus presenting for the first time from june 2004 to december 2007 . all patients suffering concomitant monocular horizontal squint ( esotropia and exotropia ) , with deviation under 60 prism diopters ( pd ) devoid of any coupled vertical deviations were included in the study . all subjects with history of paretic or limited extra ocular muscle component , more than 60 pd angle deviations , nystagmus , past history of any squint surgical procedure , and repeated deviation , initial primary inspection of all patients was performed in our outpatient department by two qualified experienced ophthalmologists and four medical officers . after obtaining informed consent , the subjects were assessed for the following : general history : including age , sex , citizenship , occupation , any history of corrective lenses , and which eye was affected . photos of patients at up to the age of ten years were requested from patients and assessed to rule out any congenital anomaly . visual acuity of dominant ( fixating ) eye and squinting ( nonfixating ) eye , was evaluated with the help of snellen s chart for educated subjects and an e - chart for uneducated children . slit lamp biomicroscopy , and applanation tonometry . the divergence was calculated in patients with fine bilateral visual acuity by a prism and cover test for near ( 33 cm ) and far ( 6 m ) distance using a fixation object . the modified krimsky test was used to measure deviation in patients with intense amblyopia , limited vision , or children younger than the age of five years . in children , refraction under cycloplegia using cyclopentolate 1% eye drops was undertaken and any accommodative element of more than 2.0 d was excluded preoperatively . scheduled investigations at admission were examined thoroughly and included a complete blood picture , bleeding and clotting times , detailed urine analysis , and chest x - rays . all the patients and their attendants were fully informed of the postoperative results and probability of a subsequent surgery . a prophylactic antibiotic and ophthalmic drops , eg , ofloxacin and chloramphenicol , were administered every three to four hours the day surgery . a complete surgical procedure was carried out under general anesthesia after approval by consultation with a visiting physician and qualified anesthetist . the surgical procedure consisted of monocular recession and resection of horizontal recti muscles of the nonfixing eye . the muscle was then separated from its attachments by round - edge curved conjunctival scissors and destabilized by a muscle hook . two 60 poly gelactin 910 absorbable sutures were used for two whip stitches ; one at the upper border and other at the lower boundary of muscle in close proximity to its insertion point during recession , and far from the insertion point in the muscle cone during muscle resection . at the time of recession the muscle was incised in close proximity to its insertion point and during muscle resection , then it was incised far from its insertion point , finally the muscle was allowed to retract and draw back . sutures were carried out of the conjunctival incision and left unfastened with one edge at the 12 oclock position with the other in the opposite position . recession was measured with a caliper from posteriorly at the beginning of the muscle insertion point and afterwards the muscle was sutured directly on the sclera by piercing the sclera with both the upper and lower suture needles opposite each other . both needles were passed gently up to half the width of the sclera under resistance without penetrating deeply into uveal tissue . the uveal penetration was confirmed when the needle passed very easily through the sclera without resistance . as the sutures were tied , the retracted muscle was lifted and brought forward to be fastened to the attachment site , and it was sutured at its normal anatomical insertion point in case of resection . absorbable sutures were used to close the conjunctiva . an antibiotic / steroid eye ointment ( neomycin with betamethasone ) was applied and the eye bandaged for 24 hours . on every postoperative outpatient follow - up visit , a complete orthoptic assessment was performed , including visual acuity and a photograph of the patient to measure the angle of deviation . the final best - corrected visual acuity and angle of deviation was documented on a sixth month post - treatment follow up . secondary surgical procedures in the fixating eye for consecutive squint over 15 pds were performed six months after initial surgery . out of approximately 7000 ophthalmic patients , 87 ( 1.24% ) patients presented with strabismus during the study period . out of these patients , seven subjects refused to undergo surgical procedure , while the remaining 39 patients ( esotropia = 24 [ 61.5% ] and exotropia = 15 [ 38.5% ] ) were selected for surgery . the successful surgical outcome was considered as an angle deviation of 15 pds or less at the six - month postoperative follow - up . table 2 presents group 1 ( esotropia ) patient data including preoperative angles of deviation ( figure 1 ) , surgical procedures and postoperative results ( figure 2 ) . table 3 presents group 2 ( exotropia ) patient data including all details ( figures 3 and 4 ) . in group 1 ( esotropia ) , five patients ( 19.0% ) presented with residual deviation of more than 15 pds . similarly , in group 2 ( exotropia ) , patients presented with orthophoria ( 73.3% ) , while four ( 26.7% ) patients demonstrated residual deviation . three patients in both groups developed suture - related foreign body granuloma formation , which resolved within a few weeks during the course of treatment . ocular movements , including convergence , were normal in all patients except two ( 13.4% ) in group 2 , who underwent 10 mm recession of lateral rectus and experienced limitation in ocular movements . 12 patients were lost to follow - up , while the remaining 27 completed six months postoperative follow - up . of nine patients ( 23.0% ) in both groups who exhibited consequent strabismus after six months , six patients did not agree to a second surgery and only three patients underwent successful secondary surgical intervention . this procedure minimizes the handling of the dominant eye , thereby reducing surgical duration.12,13 occasionally it becomes difficult to visually align the eyes by operating only on one eye because of larger deviations of more than 60 pds . at this stage , clinical circumstances for a second surgery on the fixing eye or binocular squint surgery concerning more than two horizontal rectus muscles is the main and extensive used clinical procedure . this technique also avoids limitations in ocular movement.1416 in our study , we acquired excellent outcomes through a surgical procedure carried out on the fixating eye in subjects who had developed residual deviation after the initial surgery on the nondominant , fixating eye . monocular squint surgery can be safely performed in adults under peribulbar anesthesia , with rapid recovery and less complications.17,18 although there are some disadvantages of regional anesthesia including : retrobulbar hemorrhage , optic nerve injury , central retinal artery occlusion , and ptosis.19 in this study all the subjects in both groups were operated under general anesthesia . scott and colleagues define the accuracy of squint surgery as a residual deviation of 10 pds or less . scott also states that for larger angles , the surgical objective should be a small residual deviation rather than straight eyes.20 following this concept of undercorrection , the residual deviation of 15 pds and less was considered accurate in this study . restricted ocular movements are an occasional clinical problem following huge recessions . multiple authors strongly recommend not exceeding a 7 mm recession on medial rectus and 8 mm recession on lateral rectus muscle to avoid diminished restricted ocular movements.2123 in this study only two ( 13.4% ) subjects in group 2 , who underwent a 10 mm recession on lateral rectus muscle suffered postoperative limitation in ocular movements .

background and objectivesquint surgery is frequently performed successfully in hyderabad . however , no study in any detail has been performed on the outcome of monocular surgery for horizontal squint in the region . this study aims to determine the results of monocular surgery for horizontal trabismus.designretrospective/observational study.subjects and methodsthe study was conducted on patients aged under 45 years , presenting with horizontal strabismus and undergoing monocular squint surgery . anterior segment slit lamp examination , and if possible , posterior segment examination with 90 diopter ( d ) and 78 d fundoscopes was performed . angle of deviation was measured . patients were divided into two groups ( esotropia and exotropia ) . investigations were performed . surgery was done under general anesthesia . a second surgical procedure was performed after six months for any residual deviations.resultsafter squint surgery , patients in group 1 ( 79% ) and in group 2 ( 73.3% ) had residual deviation of less than 15 prism diopters ( pd ) . the preoperative deviations of 60 pd or less illustrated excellent domino effects with residual deviation of fewer than 15 pd . three patients with successive deviation underwent a second surgery with excellent outcomes.conclusionfew extraocular muscles can be prevented due to monocular squint surgery when multiple surgeries are needed .

sepsis is the systemic inflammation caused by infection with bacteria , viruses , fungi , parasites , or toxic products , and has become one of the leading causes of mortality in children . in developed countries , the mortality rate of pediatric sepsis is about 210% and is as high as 50% in developing countries . pediatric sepsis usually leads to tachycardia , tachypnea , peripheral vasodilation , fever or hypothermia , and even multiple organ dysfunction syndrome ( mods ) . frequently - used applications in pediatric sepsis treatment concentrate on maintaining metabolic , circulatory , and respiratory stabilities , and antimicrobial agents , as well as surgical and adjunctive therapies such as nitric oxide and corticosteroids , which show great benefits in the outcome of pediatric sepsis treatment . acute kidney injury ( aki ) is a severe consequence of sepsis and mods , and about 1527% of aki in children is generated from pediatric sepsis . the pathophysiology of septic aki is related to the altered global renal blood flow , as well as the comprehensive effects of inflammation , microvasculature blood flow , and bioenergetics factors . for example , glomerular endothelial injury during aki progression in a lipopolysaccharide - induced mouse sepsis model is closely related to tumor necrosis factor and receptor 1 . due to the increased risk of mortality caused by septic aki , it is imperative to explore effective therapies for controlling aki in pediatric sepsis . baicalin is one of the flavonoid constituents in scutellaria baicalensis georgi , a kind of perennial herb with protective effects against diseases like hydrogen peroxide - induced oxidative stress and cerebral ischemia injury [ 911 ] . specifically , baicalin has been revealed to protect against oxidants , anxiety , acute hepatic injury , experimental periodontitis , and sepsis . it also alleviates reperfusion - induced damage to the myocardium , the mechanism of which may be associated with its anti - apoptotic roles . moreover , a recent study found that baicalin can inhibit inflammation and cell apoptosis , which allows it to protect against ischemia - reperfusion injuries in the kidney . however , few studies have investigated the role of baicalin in septic aki or pediatric sepsis . this study aimed to uncover the effects of baicalin in treating aki in pediatric sepsis patients . the baicalin adjunctive therapy was tested in pediatric sepsis patients , after which the effect was compared based on the renal function assessment from bun and serum creatinine ( cr ) . we also performed experiments in the cecal ligation and puncture ( clp)-induced mouse sepsis model to investigate the effects and potential mechanisms of baicalin in septic aki . this study lays the foundation for future application of baicalin in adjunctive therapies for aki in pediatric sepsis . a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) . the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure . the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients . no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) . patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions . the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report . thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin . the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity . then the cecum was relocated to the abdominal cavity , and the abdomen was closed . the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) . mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling . cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions . briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark . then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min . slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) . total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse , 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) . data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control . protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions . the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c . after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature . ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals . comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) . a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) . the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure . the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients . no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) . patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions . the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report . thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin . the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity . then the cecum was relocated to the abdominal cavity , and the abdomen was closed . the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) . mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling . cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions . briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark . then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min . slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) . total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse , 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) . data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control . protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions . the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c . gapdh ( ab181602 ) was used as an internal control . after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature . ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals . comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) . in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) . it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy . we next performed experiments in the mouse model to analyze the function of baicalin in septic aki . mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) . thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) . bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) . protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) . it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy . we next performed experiments in the mouse model to analyze the function of baicalin in septic aki . mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) . thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) . bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) . protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . this study performed investigations in pediatric sepsis patients and in a clp - induced mouse sepsis model to reveal the role of baicalin in aki of pediatric sepsis . baicalin adjunctive therapy in the patients and treatment in the mouse model decreased bun and cr levels . further histological and expression analyses suggested the suppressive function of baicalin in renal cell apoptosis . the 50 pediatric patients participating in this study were chosen from pediatric sepsis patients diagnosed with aki , and the mouse model induced by clp was detected with elevated bun and cr levels , which suggested potential kidney injury . previous studies using mouse sepsis models used various baicalin treatment methods , including topical application on the mouse skin to analyze the effect of baicalin on epidermis , intraperitoneal injection to investigate the role of baicalin in mammary glands , and baicalin - containing diets to study liver injury , inflammation in the kidney , and lung carcinoma [ 22,2729 ] . in these studies , the dose of baicalin was usually 100200 mg/(kgd ) , and the duration ranged from 3 to 50 days . thus , in the present study we chose to perform baicalin treatment in the model mouse via intragastric administration of 200 mg/(kgd ) for 6 days , and the significantly elevated bun and cr levels in the clp group compared to the sham group indicated that this treatment method effectively induced kidney injury in the mouse sepsis model . clinical trials with baicalin adjunctive therapy have focused on its protective roles in ulcerative colitis , hepatic fibrosis , and diabetes mellitus [ 3032 ] , and several studies have investigated the potential of baicalin in treating patients with early diabetic nephropathy , in which baicalin inhibits aldose reductase activity and reduces urinary albumin excretion rate and the blood 2-microglobulin . the present study further discovered the protective role of baicalin against aki in pediatric sepsis patients , which was reflected in the reduced bun and cr levels . moreover , the experiments in the mouse model confirmed that baicalin treatment in a sepsis animal model could improve renal functions . these findings show the potential of using baicalin to attenuate aki in pediatric sepsis patients , but the proper dosage and possible complications need to be assessed in future research . tunel results showed that baicalin treatment in the mouse model significantly suppressed renal cell apoptosis , and western blot and qrt - pcr analyses indicated consistent changes in the expression of bax and bcl2 . a similar mechanism has been reported in mouse mammary glands , where baicalin inhibits cell apoptosis via regulating bax and bcl2 . in addition , baicalin inhibits hepatocyte apoptosis in mouse kidney injury induced by concanavalin a ; therefore , suppressing renal cell apoptosis is likely to be one of the mechanisms by which baicalin attenuates septic kidney injury . however , the anti - proliferative role of baicalin has also been reported in studies on lung cancer and mouse embryonic stem cells , and it is converted to baicalin by intestinal gut flora to act as a pro - apoptotic and anti - proliferative substance . baicalin is capable of promoting neuronal differentiation , and thus may benefit therapies for parkinson and alzheimer diseases . we speculate that there are multiple potential mechanisms behind the effects of baicalin in various diseases ; therefore , it is necessary to uncover the detailed mechanisms in order to optimize use of baicalin to control aki in pediatric sepsis patients . the investigations of baicalin in pediatric sepsis patients and the clp - induced mouse sepsis model reveal the protective role of baicalin against aki in pediatric sepsis . baicalin can inhibit renal cell apoptosis , which may be one of its functional mechanisms . this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and future research will concentrate on more detailed mechanisms of baicalin .

backgroundpediatric sepsis has high morbidity in children , may lead to acute kidney injury ( aki ) , and further aggravate the disease . baicalin is a kind of flavonoid in scutellaria baicalensis georgi and has been reported to protect against several diseases , but its roles in septic aki remain unclear . this study aimed to uncover the effects of baicalin in aki during pediatric sepsis.material/methodsblood urea nitrogen ( bun ) and serum creatinine ( cr ) levels were detected in 50 pediatric patients , who underwent basic therapy with or without baicalin adjunctive therapy . mouse sepsis models were constructed by cecal ligation and puncture ( clp ) and treated with baicalin intragastrically , after which bun and cr examination , tunel apoptosis assay , and expression analyses of bax and bcl2 were performed.resultsbaicalin adjunctive therapy significantly decreased bun and cr levels in pediatric sepsis patients ( p<0.05 ) . clp led to elevated bun and cr levels in the mouse model ( p<0.01 ) , indicating kidney injury accompanied by sepsis . baicalin decreased bun and cr levels ( p<0.05 ) , and reduced the apoptotic cell percent in the renal tissue ( p<0.05 ) of the clp model . it inhibited bax and promoted bcl2 in the renal tissue , which was consistent with cell apoptosis changes.conclusionsbaicalin is capable of suppressing renal cell apoptosis and protecting against aki in pediatric sepsis . this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and further research is necessary to reveal its deeper mechanisms .

fucosidosis is a rare lysosomal storage disorder , which is inherited in autosomal recessive pattern due to deficient activity of the enzyme alpha l fucosidase . type i presents in infancy with a very rapid progression of illness and death in early childhood , whereas type ii presents later with lesser severity although it is gradually progressive , and sometimes people survive into adulthood . so far around 100 cases have been reported worldwide with only one case from india . here , we present two siblings with fucosidosis with characteristic clinical , radiological and laboratory features with a brief review of the literature . an 8-year - old girl presented with features of neuroregression from around 3 years of age . her developmental milestones were delayed in all domains ( social smile - 3 months , head control - 5 months , rolling over - 8 months and walking unassisted and stranger awareness - 2 years ) . she had steady developmental gains till 3 years of age after which she developed insidious onset global regression of her acquired milestones . she presented to us at 8 years of age in bed bound state with spasticity and generalized dystonia . she had lost her language skills and currently indicates toilet needs and makes sounds without any meaningful words . she had elevated telangiectatic lesions on her palms and soles [ figure 1a ] , which was noticed from 7 years of age . her younger sibling , a 3-year - old girl , who was delivered preterm has mild developmental delay with autistic traits and mild coarse facies , but has been having good gains with developmental stimulation . ( a ) sole of the foot of a child showing multiple elevated telangiectatic lesions . widening of the medial end of clavicles ( b ) , deficiency of the medial end of radial epiphysis ( c ) , inferior beaking of thoraco lumbar vertebrae ( d ) , widening of the acetabulum ( e ) her blood counts , liver and renal functions were normal . x - rays [ figure 1b e ] showed widening of medial ends of clavicles , deficiency of medial part of radial epiphysis , inferior beaking of thoracolumbar vertebrae and widening of acetabulum . magnetic resonance imaging ( mri ) brain revealed features of hypomyelination with diffuse t2-weighted hyperintensity in subcortical and periventricular cerebral white matter . globus pallidus ( gp ) , substantia nigra and thalamus were hypointense in t2-weighted images , and t1 showed hyperintensity of the gp [ figure 2a d ] . there were two hyperintense curvilinear streaks within the lentiform nucleus on t2-weighted images corresponding to the lateral and medial medullary lamina of the gp [ figure 3 ] . magnetic resonance imaging brain t2-weighted axial image showing hypointensity of globus pallidus bilaterally and the subcortical hyperintensity ( a ) , t1-weighted axial image is showing the hyperintensity of globus pallidi ( b ) , t2 fluid attenuated inversion recovery images shows hypointensity of globus pallidi and hyperintensity of subcortical white matter ( c ) , t2 axial image showing the hyperintensity of subcortical u fibers and deep cerebral white matter suggestive of hypomyelination ( d ) t2 coronal sections showing the hyperintensity of medial and lateral medullary lamina of the globus pallidi the clinical picture of neuroregression , spasticity , dystonia , coarse facies , dysostosis multiplex , telangiectasia , mri feature of hypomyelination with t2 hypointense and t1 hyperintense gp and substantia nigra was suggestive of fucosidosis . fucosidase enzyme activity could not be detected in the leukocytes , which confirmed the diagnosis of fucosidosis . the defect leads to intracellular accumulation of fucose containing glycolipids and glycoproteins in various organs leading to the clinical manifestations . the clinical picture consists of progressive mental and motor deterioration , coarse facies , recurrent infections , dysostosis multiplex , angiokeratoma corporis diffusum , visceromegaly and seizures . our patient had coarse facies , dysostosis multiplex , telangiectatic skin lesions , ( which later evolve into angiokeratomas ) , progressive mental and motor deterioration . our patients probably had a milder form with slow progression , yet had developmental delay noticed from early infancy . classical mri finding described in fucosidosis include hypomyelination and demyelination with hyperintensity on t1 and marked hypointensity on t2-weighted images in the gp , diffuse symmetric t2 w hyperintensity of bilateral subcortical and deep white matter . the reason for signal alteration in gp is not exactly known , although several postulates like calcification , iron , manganese , cerebral glycolipid and triglyceride accumulation has been proposed . our patient had classical mri finding , in addition to that our case had t2-weighted hyperintensity of medial and lateral medullary lamina of gp , which passes between the hypointense gp . characteristic magnetic resonance spectroscopy findings reported are reduced n - acetyl aspartate / choline with elevated choline / creatine ratios and myoinositol , and a specific spectral peak at 3.8 - 3.9 ppm . various forms of dysostosis multiplex has been described , which was noted in our case also . our case had coarse facies with sociocognitive and motor regression , telangiectatic lesions in palms and soles , dysostosis multiplex and characteristic mri features of hypomyelination with gp and substantia nigra hypointensity in t2-weighted images and hyperintensity in t1 images with an undetectable fucosidase enzyme activity in leukocytes . the enzymes levels were not detected in her younger sibling also who has developmental delay with autistic traits . since she is early in the course of illness , she has been referred for consideration of hematopoietic stem cell transplantation . the treatment options at present are limited to hematopoietic stem cell transplant , which has shown to be curative in anecdotal studies . have described 22 disease causing mutations in fuca 1 gene , located on the short arm of chromosome 1 , which included point mutations , deletions , insertions , missense and inactivating mutations . early identification might lead to screening of presymptomatic siblings who might potentially be offered stem cell transplantation , which could be curative if done at an appropriate age .

fucosidosis is a rare lysosomal storage disorder due to deficiency of fucosidase enzyme , with around 100 cases reported worldwide . here , we describe the clinical and imaging features in two siblings with fucosidosis . an 8-year - old girl presented with global developmental delay , followed by regression of acquired milestones from 3 years of age with bipyramidal , extrapyramidal involvement , coarse facies , telangiectatic lesions , dysostosis multiplex , characteristic magnetic resonance imaging finding along with undetectable levels of the fucosidase activity , which confirmed the diagnosis . younger sibling has mild developmental delay with autistic traits with no neuroregression until now . he also has undetectable level of fucosidase enzyme activity and is being considered for stem cell transplantation . new case reports would expand the clinical spectrum , early diagnosis and help formulating appropriate therapy . early diagnosis is crucial and hence sibling screening can be done , and those in the presymptomatic stage can undergo hematopoietic stem cell transplantation , which is potentially curable .

in order to collect a set of images useful to test the algorithm , we run 10 plasma samples using 2d - ge . 2d - ge was performed according to maresca et al . , with each sample run in duplicate . for the first - dimension of electrophoresis , 200 g of plasma protein samples ( approximately 3 l ) were applied to 18-cm linear ipg strips 47 ( ge healthcare , uppsala , sweden ) and focused until 72,000 v / h were reached . prior to sds page , the ipg strips were equilibrated twice for 15min in equilibration buffer ( 50 mm tris hcl ph 8.8 , 6 m urea , 30% glycerol ( v / v ) , 2% sds ( w / v ) and traces of bromophenol blue ) containing 1% dtt ( w / v)for the first equilibration step and 2.5% iodoacetamide ( w / v ) for the second step , respectively . page was performed on 12.5% polyacrylamide gels at 60 ma / gel at 16 c and terminated when the dye front reached the lower end of the gel . after sds page , the gels were stained overnight with rubps in fixing solution ( 30% ethanol and 10% acetic acid ) according to the manufacturer s instructions . the gel images were acquired by gel - doc - it 310 imaging system ( uvp , upland , ca ) , with the following setting exposure 7 s , gain 3 and three different apertures : 6 , 7 and 8 . aperture is expressed as f - stop ( e.g. , f2.8 or f/2.8 ) : the smaller is the f - stop number ( or f / value ) , the larger is the aperture . using a larger exposure yields a larger number of spots and , at the same time , more saturated areas . in our case , only the image acquired at the lower exposure ( figure 1c ) could be properly analysed by commercial and academic software currently available , while the saturation in some areas in the other two images ( figure 1a and b ) prevented accurate evaluation of protein expression . using the algorithm described in this work , we have been able to approximate the intensity values of the non - saturated spots within the saturated area . after image scanning , the 2d - ge images are noisy and usually affected by several artefacts such as scratches , air bubbles and spikes . scratches and air bubbles are unintentionally introduced by the operator , while spikes are caused by the presence of precipitated staining particles or imperfect polyacrylamide gel matrix . in particular , spikes are a sort of impulsive noise because of the small area of high intensity . the aim of reducing speckles in a 2d - ge image ( despeckling ) is to remove the noise without introducing any significant distortion in quantitative spot volume data . noise suppression methods employed in commercial software packages for 2d - ge image analysis are based on spatial filtering , particularly the most widely used techniques are based on local median filter . although several other methods have been developed , the advantage of using the median filter is the easy application , since it normally requires the setting of only one main parameter , the filter kernel , which is generally related to the noise size . at the same time , the size of the filter matrix also accounts for the major impact exerted on the resulting image . we evaluated the performance of filter size on a set of test images from 3 3 to 7 7 and observed remarkable deterioration for larger filter size , due to the decreased the contrast of the image . therefore , a 3 3 filter was chosen , which was capable of removing most of the spikes , while preserving the shape of the spots . the result of a 3 3 median filter on a sample image is shown in figure 5 . a portion of gel with spikes in 2d and 3d view was shown in figure 5a and c , respectively . the results of a 3 3 median filter are shown in figure 5b and d accordingly . saturation effects in 2d gel images could occur for several reasons : ( i ) intense staining , causing complete absorption of light passing through the spot , ( ii ) long exposure time during the acquisition step , ( iii ) too high protein concentration , or ( iv ) accumulation of pigment on top of high intensity spots , leading to the loss of dynamic linear range . saturation effects might also result from manipulation of image data with image processing software that is not designed for quantitative image analysis ( e.g. , photoshop ) . for instance , visually - enhanced images often show plateau areas in the high and low intensity regions . the commercial software is able to detect a saturated area when the intensity reaches the maximum value of grey - scale . however , in most cases saturated areas are very similar to plateau zones , i.e. , where the pixels have similar intensity without reaching the maximum value of grey - scale . in these cases , the software might also fail to detect the plateau regions , causing the operator to underestimate the problem . in order to identify the plateau regions , we implemented a morphological filter , inspired by the rolling - ball algorithm described by sternberg , which allows segmentation of the plateau zones . this method is based on a structural element ( se ) defined by a circle of given radius ( rd ) and a grey - scale value tolerance ( gvt ) . in particular , for each pixel ( x , y ) of the image i , the se is defined as a circular neighbourhood of rd:(1a)se=(s , t)ini/(s - x)2+(t - y)2<rdwhere(1b)i=(0,image width)[0,image height]denotes the spatial domain of the image . the gvt represents the rate of grey values of the pixel in the centre of the ses ( figure 6b ) . for instance , setting the parameter to 10 , the rd is 10 pixels and the gvt in the 10% of grey values of the pixel at position ( x , y ) . the centre of se is moved along each pixel of the image and the maximum and minimum grey values of the pixels for each point ( x , y ) within the given rd are calculated . when the difference between maximum and minimum is less than gvt , the area defined by the local operator is considered as a plateau area . it is worth noting that , when the value of the parameter is low , the rd of the ball is small and only few pixels are included in se . we examined the performance with rd values from 10 to 25 and for this analysis we selected a value of 15 . after localizing the plateau regions , we segmented the image to identify the isolated protein spots containing plateau areas on each gel image . the segmentation procedure yields a set of image segments , consisting of connected neighbouring pixels enclosed by a spot boundary . the image is considered as a landscape , and the segmentation is to identify all the local minima in the landscape and then find the catchment basins associated with each local minima . the boundary between several catchment basins is called a watershed . for 2d - ge each separated catchment basin is considered as an isolated protein spot . unlike the usual watershed segmentation , only the plateau areas in our method have been assigned to a catchment basin . finally , the grey values of the pixels inside the spot , excluding the region identified as a plateau area , were used in the gaussian extrapolation step to recover the distribution of the unsaturated spot . the final step consists of reconstructing the saturated spots resulting from high exposure images and approximating the unknown grey values in the plateau region . this has been done considering the unsaturated spot to be described by an analytical function , depending on a restricted set of parameters . in particular , we assumed each cross section of the spot intensity along both vertical and horizontal axes to be approximated by a generalized gaussian distribution . namely , for each value of the y - coordinates we considered a function of the form:(2)f(x , m(y),(y),x0,b)=m(y)(y)exp-|x - x0(y)|bb(y)b for b = 2 , eq . ( 1b ) defines the kernel of a standard gaussian distribution centred in x0(y ) , where (y ) and m(y ) is the standard deviation and the maximum of intensity values , respectively . note that , unlike the other parameters , b does not depend on y , assuming that the approximating gaussian can have different maximum , center and variance in different sections . ,nyand the corresponding intensities { iij } , we determine the set of parameters ( m , , x0 , b ) for which the function defined in ( 2 ) fits at best the values of the intensity in the unsaturated region . in practice , we have to minimize an error function that defines how good a particular parameter set is . for example , a standard least - squared criterion can be used(4)em(yj),(yj),x0(yj),b=i=1nxfxi , m(yj),(yj),x0(yj),b - iij2 however , the error measure can be defined in different ways , e.g. , according to different norms or including different weighs for the different parameters and/or the different pixels . in particular , we modified ( 4 ) in order to control the variation of the parameters for different sections . in our case the error function ( 5 ) can be formulated as follows(5)em(yj),(yj),x0(yj),b = em(yj),(yj),x0(yj),b+m(yj)-m(yj-1)2+(yj)-(yj-1)2+x0x0(yi)-x0(yj-1)2 for positive values of the parameters ( m , , x0 ) , the problem is then reduced to finding the parameters yielding the minimum of the selected error function . one possibility is to perform an exhaustive search on all the values of a pre - defined parameters space . however , if the size of the parameter space is large , a more effective newton after image scanning , the 2d - ge images are noisy and usually affected by several artefacts such as scratches , air bubbles and spikes . scratches and air bubbles are unintentionally introduced by the operator , while spikes are caused by the presence of precipitated staining particles or imperfect polyacrylamide gel matrix . in particular , spikes are a sort of impulsive noise because of the small area of high intensity . the aim of reducing speckles in a 2d - ge image ( despeckling ) is to remove the noise without introducing any significant distortion in quantitative spot volume data . noise suppression methods employed in commercial software packages for 2d - ge image analysis are based on spatial filtering , particularly the most widely used techniques are based on local median filter . although several other methods have been developed , the advantage of using the median filter is the easy application , since it normally requires the setting of only one main parameter , the filter kernel , which is generally related to the noise size . at the same time , the size of the filter matrix also accounts for the major impact exerted on the resulting image . we evaluated the performance of filter size on a set of test images from 3 3 to 7 7 and observed remarkable deterioration for larger filter size , due to the decreased the contrast of the image . therefore , a 3 3 filter was chosen , which was capable of removing most of the spikes , while preserving the shape of the spots . the result of a 3 3 median filter on a sample image is shown in figure 5 . a portion of gel with spikes in 2d and 3d view was shown in figure 5a and c , respectively . the results of a 3 3 median filter are shown in figure 5b and d accordingly . saturation effects in 2d gel images could occur for several reasons : ( i ) intense staining , causing complete absorption of light passing through the spot , ( ii ) long exposure time during the acquisition step , ( iii ) too high protein concentration , or ( iv ) accumulation of pigment on top of high intensity spots , leading to the loss of dynamic linear range . saturation effects might also result from manipulation of image data with image processing software that is not designed for quantitative image analysis ( e.g. , photoshop ) . for instance , visually - enhanced images often show plateau areas in the high and low intensity regions . the commercial software is able to detect a saturated area when the intensity reaches the maximum value of grey - scale . however , in most cases saturated areas are very similar to plateau zones , i.e. , where the pixels have similar intensity without reaching the maximum value of grey - scale . in these cases , the software might also fail to detect the plateau regions , causing the operator to underestimate the problem . in order to identify the plateau regions , we implemented a morphological filter , inspired by the rolling - ball algorithm described by sternberg , which allows segmentation of the plateau zones . this method is based on a structural element ( se ) defined by a circle of given radius ( rd ) and a grey - scale value tolerance ( gvt ) . in particular , for each pixel ( x , y ) of the image i , the se is defined as a circular neighbourhood of rd:(1a)se=(s , t)ini/(s - x)2+(t - y)2<rdwhere(1b)i=(0,image width)[0,image height]denotes the spatial domain of the image . the se is depicted in figure 6a . the gvt represents the rate of grey values of the pixel in the centre of the ses ( figure 6b ) . for instance , setting the parameter to 10 , the rd is 10 pixels and the gvt in the 10% of grey values of the pixel at position ( x , y ) . the centre of se is moved along each pixel of the image and the maximum and minimum grey values of the pixels for each point ( x , y ) within the given rd are calculated . when the difference between maximum and minimum is less than gvt , the area defined by the local operator is considered as a plateau area . it is worth noting that , when the value of the parameter is low , the rd of the ball is small and only few pixels are included in se . we examined the performance with rd values from 10 to 25 and for this analysis we selected a value of 15 . after localizing the plateau regions , we segmented the image to identify the isolated protein spots containing plateau areas on each gel image . the segmentation procedure yields a set of image segments , consisting of connected neighbouring pixels enclosed by a spot boundary . the image is considered as a landscape , and the segmentation is to identify all the local minima in the landscape and then find the catchment basins associated with each local minima . the boundary between several catchment basins is called a watershed . for 2d - ge each separated catchment basin is considered as an isolated protein spot . unlike the usual watershed segmentation , only the plateau areas in our method have been assigned to a catchment basin . finally , the grey values of the pixels inside the spot , excluding the region identified as a plateau area , were used in the gaussian extrapolation step to recover the distribution of the unsaturated spot . the final step consists of reconstructing the saturated spots resulting from high exposure images and approximating the unknown grey values in the plateau region . this has been done considering the unsaturated spot to be described by an analytical function , depending on a restricted set of parameters . in particular , we assumed each cross section of the spot intensity along both vertical and horizontal axes to be approximated by a generalized gaussian distribution . namely , for each value of the y - coordinates we considered a function of the form:(2)f(x , m(y),(y),x0,b)=m(y)(y)exp-|x - x0(y)|bb(y)b for b = 2 , eq . ( 1b ) defines the kernel of a standard gaussian distribution centred in x0(y ) , where (y ) and m(y ) is the standard deviation and the maximum of intensity values , respectively . note that , unlike the other parameters , b does not depend on y , assuming that the approximating gaussian can have different maximum , center and variance in different sections . the reconstruction problem can be formulated as follows . ,nyand the corresponding intensities { iij } , we determine the set of parameters ( m , , x0 , b ) for which the function defined in ( 2 ) fits at best the values of the intensity in the unsaturated region . in practice , we have to minimize an error function that defines how good a particular parameter set is . for example , a standard least - squared criterion can be used(4)em(yj),(yj),x0(yj),b=i=1nxfxi , m(yj),(yj),x0(yj),b - iij2 however , the error measure can be defined in different ways , e.g. , according to different norms or including different weighs for the different parameters and/or the different pixels . in particular , we modified ( 4 ) in order to control the variation of the parameters for different sections . in our case the error function ( 5 ) can be formulated as follows(5)em(yj),(yj),x0(yj),b = em(yj),(yj),x0(yj),b+m(yj)-m(yj-1)2+(yj)-(yj-1)2+x0x0(yi)-x0(yj-1)2 for positive values of the parameters ( m , , x0 ) , the problem is then reduced to finding the parameters yielding the minimum of the selected error function . one possibility is to perform an exhaustive search on all the values of a pre - defined parameters space . however , if the size of the parameter space is large , a more effective newton mn conceived the study , developed the algorithm for the detection of overexposed areas and plateau regions . mn and ac drafted the manuscript with the help of ef . all authors read and approved the final manuscript .

analysis of images obtained from two - dimensional gel electrophoresis ( 2d - ge ) is a topic of utmost importance in bioinformatics research , since commercial and academic software available currently has proven to be neither completely effective nor fully automatic , often requiring manual revision and refinement of computer generated matches . in this work , we present an effective technique for the detection and the reconstruction of over - saturated protein spots . firstly , the algorithm reveals overexposed areas , where spots may be truncated , and plateau regions caused by smeared and overlapping spots . next , it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions , using a two - dimensional least - squares fitting based on a generalized gaussian distribution . pixel correction in saturated and smeared spots allows more accurate quantification , providing more reliable image analysis results . the method is validated for processing highly exposed 2d - ge images , comparing reconstructed spots with the corresponding non - saturated image , demonstrating that the algorithm enables correct spot quantification .

pulmonary arterial hypertension ( pah ) is a devastating disease with 50% mortality at 2.8 years in patients left untreated or unresponsive to treatment.1 diagnostic evaluation in patients with pulmonary hypertension ( ph ) includes an echocardiogram , which when performed as it routinely undertaken even in tertiary referral centers , will diagnose an elevated pulmonary artery pressure . patients are frequently misdiagnosed as having pah ( diagnostic group i classification [ ie , pah ] ) and started on pah - specific therapy based on an echocardiogram in the absence of a right heart catheterization ( rhc ) . when the diagnostic cardiac catheterization is subsequently undertaken , many patients referred for evaluation and treatment of pah have an elevated pulmonary capillary wedge pressure ( pcwp ) , suggesting left heart dysfunction ( lhd ) ( diagnostic group ii classification ) as a cause of ph.2,3 the distinction is important as the prognosis and therapy are different between these two disorders.4 the diagnosis of pah requires a normal pcwp , or a normal left ventricular end - diastolic pressure at the time of cardiac catheterization.5 elevated pcwp may be an indicator of lhd in patients with preserved ejection fraction . lhd is considered a frequent cause of ph.2,6 left atrial ( la ) size is considered a marker of lhd with preserved ejection fraction.710 we undertook this study to examine the role of computed tomography of the chest ( ct chest ) in evaluating la size as an indicator of pcwp in patients undergoing ph evaluation . our hypothesis was that a la appearing large on the ct chest would be an indicator of elevated pcwp during a rhc in patients with normal ejection fraction ( ef ) ( > 40% ) and normal aortic and mitral valves . after obtaining institutional review board approval , a retrospective study was undertaken of those patients evaluated for pah and underwent a right heart catheterization at baylor college of medicine pulmonary hypertension center from july 1 , 2005 to june 30 , 2006 . at this center , ct chest and echocardiography are routine evaluations undertaken for suspected ph . of those patients , 26 were excluded from analysis , 20 due to the absence of a recent ( defined as 60 days ) ct chest ; 3 due to the presence of congenital intra - cardiac shunts which frequently are associated with la enlargement ; 1 due to the presence of lung cancer and significant pleural effusion ; and 1 due to failure to obtain pcwp . ct chests from the remaining 37 patients were reviewed on the picture archiving and communications systems by 3 independent investigators ( adult pulmonologists ) , 2 of whom were blinded to the name , cardiac catheterization results , and diagnosis of the subjects ( mk and af ) . subjective impression of la size ( graded as small - normal or large ) was recorded ( figure 1 ) . approximate la area was measured as follows : la area was measured in the mediastinal window at the point where la appeared largest to the reader ( figure 1 ) ; horizontal length ( l ) and vertical width ( w ) of the la were measured ; la area was calculated ( la area = l w ) . la area was corrected for patient size by dividing the calculated area of the la by the horizontal chest wall length ( lcw ) , measured in the same plane and along the same line as the measurement of the horizontal length of the la ( corrected la = [ l w]/lcw ) ( figure 1 ) . the impression of the la by each investigator was recorded and grouped as either small - normal ( group i ) or large ( group ii ) ( figure 1 ) . when there was agreement between two investigators and disagreement with the third , the impression of the 2 agreeing investigators was accepted . rhc had been undertaken in patients with normal mitral and aortic valves on echocardiogram with preserved left ventricular ( lv ) systolic function ( ie , no evidence of lv systolic failure ) . the provided diagnosis was ph , therefore , all measurements were the standard evaluations undertaken for a diagnosis of ph . comparisons of all measurements between the two groups were made with unpaired t - test . pearson correlation analyses of clinical and hemodynamic parameters with la area and impression of investigators were performed with spss software . shrout - fleiss intra - class correlation coefficient was used to measure the inter - rater reliability among the 3 investigators . the inter - rater reliability calculated using the shrout - fleiss intraclass correlation for la area was 0.865 , indicating acceptable level of agreement among investigators . the estimated la area was 19.4 4.9 cm in group i and 39.9 7.6 cm in group ii ( mean sd ; p < 0.001 ) ( table 2a ) . the estimated la areas corrected for the chest wall length ( l w / lcw ) were 0.78 0.19 cm and 1.65 0.26 cm ( p < 0.001 ) in groups i and ii , respectively ( table 2b ) . there was no significant difference in the mean pulmonary artery pressures between the two groups ( p = 0.42 ) ( table 1 ) . the pcwp was 12 6 mmhg in group i and 21 7 mmhg in group ii ( p = 0.001 ) . in group i , 24 subjects had normal pcwp and 5 subjects had elevated pcwp on the rhc . of the 5 subjects with elevated pcwp , 3 had impaired lv relaxation with preserved lv ejection fraction ( lvef ) ( range 60%70% ) , 2 had elevated filling pressures on the echocardiogram suggestive of lhd , and the remaining 2 of the 5 subjects had normal lv filling pressures , lv relaxation , and la size ( figure 2 ) . in 1 subject with elevated pcwp and a diagnosis of pah , the rhc revealed marked elevation of both central venous pressure and right ventricular end - diastolic pressure and echocardiogram demonstrated a small lv with cavitary compromise due to markedly abnormal paradoxical interventricular systolic motion secondary to right ventricle systolic pressure overload . the second patient had portopulmonary hypertension and a high cardiac output that may have contributed to the high pcwp . in group ii , 7 of the 8 subjects had elevated pcwp at time of rhc ( figure 2 ) ; 1 subject with normal pcwp also had normal pa pressures during the rhc . this indicates that in all but 1 subject with a large appearing la on the ct chest , also had elevated pcwp . subjective observer impression of la size correlated with pcwp ( r = 0.51 , p = 0.001 ) . there was a positive correlation between the subjective observer impression of la size on the ct chest and la size on measured echocardiogram ( r = 0.61 , p < 0.001 ) . the positive predictive value ( ppv ) of the la size in determining elevated pcwp in ph subjects was 87.5% ; negative predictive value ( npv ) was 83% . ppv of la size on ct chest in determining la size on echocardiogram was 90% ; npv was 67% . these results suggest that looking at the la on the ct chest may be an indicator of elevated pcwp . there was a significant correlation between pcwp and estimated la area , both uncorrected ( r = 0.45 , p = 0.005 ) and corrected ( r = 0.47 , p = 0.003 ) ( figure 3 ) . this result indicates that the la area increases as the pcwp became elevated and may be an important indicator of elevated pcwp . it also suggests that there is no need to correct a la size measurement for parameters of patient size . though no patient had a lvef < 45% , uncorrected la area negatively correlated with lvef ( r = 0.34 , p = 0.03 ) ( table 3 ) suggesting that as lvef decreased not unexpectedly la area increased . this provides some internal consistency and clinical validation of the observation . to determine if la area on a ct chest could be used to discriminate between subjects with pah ( diagnostic group i ) from those with elevated pcwp ( diagnostic group ii ) , we calculated area under the receiver operating characteristic ( roc ) curves for both uncorrected and corrected la area . the largest area under the roc for uncorrected la area of 31 cm was 0.996 ( figure 4 ) . therefore , uncorrected la area of 31 cm may be a good diagnostic tool to discriminate between the two groups . value greater than 1.3 for the corrected la area yielded a sensitivity and specificity for prediction of left heart failure with preserved ef of 100% . these promising results indicate that both uncorrected and corrected la area measurements were reasonable values to distinguish between subjects with elevated pcwp ( diagnostic group ii ) and pulmonary arterial hypertension ( diagnostic group i ) . the inter - rater reliability calculated using the shrout - fleiss intraclass correlation for la area was 0.865 , indicating acceptable level of agreement among investigators . the estimated la area was 19.4 4.9 cm in group i and 39.9 7.6 cm in group ii ( mean sd ; p < 0.001 ) ( table 2a ) . the estimated la areas corrected for the chest wall length ( l w / lcw ) were 0.78 0.19 cm and 1.65 0.26 cm ( p < 0.001 ) in groups i and ii , respectively ( table 2b ) . there was no significant difference in the mean pulmonary artery pressures between the two groups ( p = 0.42 ) ( table 1 ) . the pcwp was 12 6 mmhg in group i and 21 7 mmhg in group ii ( p = 0.001 ) . in group i , 24 subjects had normal pcwp and 5 subjects had elevated pcwp on the rhc . of the 5 subjects with elevated pcwp , 3 had impaired lv relaxation with preserved lv ejection fraction ( lvef ) ( range 60%70% ) , 2 had elevated filling pressures on the echocardiogram suggestive of lhd , and the remaining 2 of the 5 subjects had normal lv filling pressures , lv relaxation , and la size ( figure 2 ) . in 1 subject with elevated pcwp and a diagnosis of pah , the rhc revealed marked elevation of both central venous pressure and right ventricular end - diastolic pressure and echocardiogram demonstrated a small lv with cavitary compromise due to markedly abnormal paradoxical interventricular systolic motion secondary to right ventricle systolic pressure overload . the second patient had portopulmonary hypertension and a high cardiac output that may have contributed to the high pcwp . in group ii , 7 of the 8 subjects had elevated pcwp at time of rhc ( figure 2 ) ; 1 subject with normal pcwp also had normal pa pressures during the rhc . this indicates that in all but 1 subject with a large appearing la on the ct chest , also had elevated pcwp . subjective observer impression of la size correlated with pcwp ( r = 0.51 , p = 0.001 ) . there was a positive correlation between the subjective observer impression of la size on the ct chest and la size on measured echocardiogram ( r = 0.61 , p < 0.001 ) . the positive predictive value ( ppv ) of the la size in determining elevated pcwp in ph subjects was 87.5% ; negative predictive value ( npv ) was 83% . ppv of la size on ct chest in determining la size on echocardiogram was 90% ; npv was 67% . these results suggest that looking at the la on the ct chest may be an indicator of elevated pcwp . there was a significant correlation between pcwp and estimated la area , both uncorrected ( r = 0.45 , p = 0.005 ) and corrected ( r = 0.47 , p = 0.003 ) ( figure 3 ) . this result indicates that the la area increases as the pcwp became elevated and may be an important indicator of elevated pcwp . it also suggests that there is no need to correct a la size measurement for parameters of patient size . though no patient had a lvef < 45% , uncorrected la area negatively correlated with lvef ( r = 0.34 , p = 0.03 ) ( table 3 ) suggesting that as lvef decreased not unexpectedly la area increased . this provides some internal consistency and clinical validation of the observation . to determine if la area on a ct chest could be used to discriminate between subjects with pah ( diagnostic group i ) from those with elevated pcwp ( diagnostic group ii ) , we calculated area under the receiver operating characteristic ( roc ) curves for both uncorrected and corrected la area . the largest area under the roc for uncorrected la area of 31 cm was 0.996 ( figure 4 ) . therefore , uncorrected la area of 31 cm may be a good diagnostic tool to discriminate between the two groups . value greater than 1.3 for the corrected la area yielded a sensitivity and specificity for prediction of left heart failure with preserved ef of 100% . these promising results indicate that both uncorrected and corrected la area measurements were reasonable values to distinguish between subjects with elevated pcwp ( diagnostic group ii ) and pulmonary arterial hypertension ( diagnostic group i ) . clinical suspicion of ph is usually conf irmed by an echocardiogram,11 which is typically reliable for discriminating systolic dysfunction , valvular lesions , and congenital defects . ct chest is frequently undertaken as part of the routine evaluation of ph patients to exclude parenchymal lung disease . estimation or measurement of la size by ct chest is simple and remarkably reliable , akin to the routine evaluation of heart size from chest x - ray . this study demonstrates the utility of routinely assessing la size / area in the evaluation of patients with ph . the data presented here indicate that most patients with a large la on ct chest , even without formal measurement , had elevated pcwp suggestive of lhd . these findings may allow patients to be stratified for risk of pah enabling prioritization for right heart catheterization and evaluation of the need for initiation of ph specific therapy . in addition inappropriate use of pah specific therapy could be avoided.4 pah is an orphan disease with a prevalence of 15 cases per million.12 in this single center study we present data from 37 patients who underwent both rhc and ct chest within 1 month of each other . there were 26 patients with pah in group i , 7 patients had lhd with preserved ejection fraction in group ii . 1 patient in group three patients in group i were diagnosed with lhd with preserved ejection fraction based on the findings of impaired lv relaxation on the echocardiogram in all 3 and elevated filling pressures in 2 of the 3 subjects . one of the patients in group i with elevated pcwp and small appearing la on ct chest had markedly enlarged right atrium and right ventricle , and small lv with shifting of the interventricular septum towards lv on the echocardiogram . this may reflect ventricular interdependence with elevated right ventricular pressure and volume shifting the interventricular septum to the left leading to obliteration of the lv cavity and lv systolic / diastolic dysfunction . , is increasingly recognized in pah populations.13,14 the elevated pcwp in left heart failure with preserved ef , on the other hand , is due to the stiffened lv with decrease compliance and associated with enlargement of the la.15 in such cases , lhd results from noncompliance of the lv , which in the presence of normal lv systolic function , produces elevated left ventricular filling pressures ( elevated pcwp or lv end - diastolic pressures),16 further aggravated by increases in heart rate in response to effort or stress . lhd with preserved ef is the commonest confounding diagnosis in the evaluation and management of patients with ph and an increasingly frequent diagnostic entity in the aging , diabetic , and hypertensive population . evaluation of echographic parameters of lhd is beyond the scope of this paper and will be undertaken in subsequent studies . la size is considered a marker of lhd with preserved ejection fraction.710 advancing age alone does not contribute to la enlargement and the impact of gender on la volume may be due to differences in body surface area.8 however , in our study there was no difference in the body surface area or body mass index between the two groups ( table 1 ) . factors influencing la size include valvular abnormalities and atrial arrhythmias.17 none had atrial fibrillation at the time of rhc . prevalence of lhd with preserved ef is high and treatment remains poorly defined , risk factors include : essential hypertension , obesity , obstructive sleep apnea , renal failure , thyroid dysfunction , female gender , and older age.16,18,19 patients may be presumptively started on pah specific therapy without undergoing a diagnostic rhc . a ct chest , however , can be easily and non - invasively undertaken and our data indicates la size could give an indication of the presumed diagnosis pending rhc . the impression of la enlargement on ct in patients undergoing evaluation for ph may be an important predictor that ph specific therapy was not indicated . this data should not be construed as a recommendation to base diagnosis of ph on ct chest without proceeding to rhc but rather to identify patients at risk of lhd with enlarged la . different imaging techniques such as 2- or 3-dimensional echocardiography , magnetic resonance imaging , and cine computerized tomography , have been used to estimate la size and/or volume.2023 these techniques are limited by availability , technical difficulties , time requirements , or need for special software.20,24 though tertiary care centers have the ability to undertake an echocardiography evaluation of patients with suspected ph , such studies have not been validated in large trials nor are they routinely applied to all echocardiograms evaluations.2527 our data demonstrate that a routine ct chest may be used to reliably predict elevated pcwp that may suggest the diagnosis of lhd in patients with preserved lv systolic function and echocardiograhic evidence of ph . estimation of la size by ct chest may avoid initiation of unnecessary and inappropriate ph - specific therapy and delays in diagnostic rhc in patients undergoing ph evaluation . the limitations of this study were the retrospective study design , small sample size , and one center review . la area and impression may have been influenced by the cardiac cycle at the time of the ct chest and intravascular volume status of the patient . , future studies that prospectively and simultaneously evaluate the echocardiographic and hemodynamic parameters in patients undergoing ph evaluation will be needed . the results of this single - center study will need to be validated in a large prospective multi - center study .

background : one of the commonest causes of pulmonary hypertension ( ph ) is left heart dysfunction associated with elevated pulmonary capillary wedge pressure ( pcwp ) . in contrast , the pathology of pulmonary arterial hypertension ( pah ) originates in the pulmonary vascular bed . accurate diagnosis of pah requires right heart catheterization ( rhc ) with normal pcwp . this study examines the role of computed tomography of the chest ( ct chest ) in evaluating left atrial ( la ) size as an indicator of elevated pcwp in patients undergoing ph evaluation.methods:ct chest and rhc data were reviewed in 37 subjects at the baylor ph center . both subjective estimates and objective measurements of left atrial size from the ct chest were recorded separately by 3 investigators . patients were categorized as group i ( small - normal la ) and group ii ( large la ) and rhc results compared.the objective and subjective measurements were compared by receiver operator characteristic ( roc).results : the mean pcwp was 12 6 mmhg in group i and 21 7 mmhg in group ii ( p = 0.001 ) . the estimated la area was 19.4 4.9 cm2 in group i and 39.9 7.6 cm2 in group ii ( mean sd ; p < 0.001 ) . the estimated la area , corrected for the chest wall length , was 0.78 0.19 cm2 and 1.65 0.26 cm2 in groups i and ii , respectively ( p < 0.001 ) . significant correlations were found between uncorrected pcwp and la area ( r = 0.45 , p = 0.005 ) , corrected pcwp and la area ( r = 0.47 , p = 0.003 ) , and the subjective observer impression of la enlargement and measured pcwp ( r = 0.51 , p = 0.001).conclusion : in this pilot study , enlarged la area on the ct chest was associated with an elevated pcwp on rhc . for patients undergoing ph evaluation , increased la area on ct chest could suggest left heart dysfunction in patients as a possible cause of ph .

dual antiplatelet therapy consisting of aspirin and an adenine diphosphate ( adp ) p2y12 receptor antagonist is recommended as the standard antiplatelet regimen for patients with coronary artery disease.1 clopidogrel , an adp p2y12 receptor antagonist , strongly inhibits formation of thrombosis by impeding platelet binding regardless of the degree of degranulation or blood flow status.2,3 aspirin has antiplatelet effects by irreversible inhibition of the formation of thromboxane a2 by acetylation of platelet cyclooxygenase , thereby contributing to decreasing the mortality caused by myocardial infarction , stroke , and cardiovascular diseases.4 in several studies , combined clopidogrel and aspirin treatment increased platelet aggregation , and this effect was superior to administration of aspirin alone in reducing the incidence of cardiovascular death and nonfatal myocardial infarction or stroke.5 a recent meta - analysis concluded that short - term aspirin in combination with clopidogrel is more effective than monotherapy as a secondary preventive treatment for stroke or transient ischemic attack without increasing the risk of hemorrhagic stroke and major bleeding events.6 in addition , approximately 50% of asians , including koreans , have a gene variant that leads to resistance to clopidogrel ; therefore , there is a safety issue of thrombus formation.1 however , it is well recognized that low - dose aspirin strongly enhances cyp2c19 enzyme activity and increases drug efficacy in patients having clopidogrel resistance.6 fixed - dose combination ( fdc ) formulations have an important role in the management of coronary artery disease . the fdc of aspirin and clopidogrel has increased therapeutic compliance , which is a major factor in the poor outcome of stenting patients for acute coronary syndrome , by simplification of the medical regimen.7 in addition , overall costs can be lowered by decreasing the number of prescriptions , bottles , labels , etc.8 clopidogrel is rapidly absorbed from the gastrointestinal tract , with a maximum concentration ( cmax ) of 2 ng / ml at 1.4 hours following administration of clopidogrel 75 mg , and is eliminated with a half - life ( t1/2 ) of 1.7 hours.9 in contrast , enteric - coated formulations of aspirin 100 mg show continuous absorption from the intestine with a time - to - reach cmax approximately 4 hours , and then the drug is rapidly eliminated with a t1/2 less than 1 hour . based on these pharmacokinetic properties of the two drugs , there should be little possibility of interaction in the absorption profiles , and their short t1/2 indicates that they are suitable for fdc because both drugs are almost completely eliminated within 12 hours after dosing . in this study , we compared the pharmacokinetics of clopidogrel , aspirin , and its active metabolite , salicylic acid , in healthy men after administration of an fdc formulation and coadministration of clopidogrel and aspirin . the study protocol and written informed consent form were reviewed and approved by the institutional review board of inje university busan paik hospital ( irb number 11 - 100 ) . the study was carried out in compliance with the declaration of helsinki ( 2010 ) , the international conference on harmonization of good clinical practice , and the current korean good clinical practice guidelines.10 healthy men aged between 20 and 55 years within 20% of the ideal body weight according to broca s formula were enrolled in this study . all subjects were judged to be healthy based on the results of a detailed physical examination , 12-lead electrocardiogram , clinical laboratory tests , and vital signs at the screening . the following exclusion criteria were used : excessive consumption of caffeine ( > 5 cups / d ) , cigarettes ( > 10 cigarettes / d ) , or alcohol ( > 30 g of alcohol / d ) , administration of inducers or inhibitors of drug - metabolizing enzymes including proton pump inhibitors ( ppis ) ( lansoprazole , omeprazole , pantoprazole , and rabeprazole ) , rifampicin , carbamazepine , and barbitals within 28 days ; history of disease influence on drug absorption , distribution , metabolism , and excretion ( eg , gastrointestinal ulcer , ulcerative colitis , and history of abdominal operation ) ; history of hemorrhagic symptoms and disease ; aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) exceeding 1.25 times the upper limit of the normal range ; total bilirubin exceeding 1.5 times the upper limit of the normal range ; prothrombin time , activated partial thromboplastin time , and bleeding time extended beyond the normal range ; platelet level < 15010/l or > 35010/l ; and participation in another clinical study within 90 days prior to the start of the study . a randomized , open , two - period , two - treatment , two - sequence crossover study was conducted at the clinical trial center of inje university busan paik hospital , busan , republic of korea ( clinicaltrials.gov identifier : nct01496261 ) . the 60 enrolled subjects were divided into two groups and randomized to one of two sequences ( rt , tr ) at the beginning of the study at a ratio of 1:1 using a randomization schedule generated using the sas software ( v 9.3 ; sas institute inc . , each group of 30 subjects was administered the study drugs in consecutive periods with a gap of 3 days between them . the assigned study drugs were administered orally with 240 ml of water after a 10-hour overnight fast , and subjects continued fasting until 4 hours postdose . subjects were administered two capsules of clopidogrel / aspirin 75 mg/100 mg fdc ( chong kun dang pharm . , subjects were coadministered two tablets of clopidogrel 75 mg ( plavix ; sanofi - aventis , schiltigheim , france ) and two capsules of aspirin 100 mg ( astrix ; boryung pharm . , seoul , republic of korea ) in the other period with a 14-day washout period . the length of the washout period was estimated to be greater than seven times the reported t1/2 of clopidogrel , aspirin , and salicylic acid ( 7.27.6 , 0.4 , and 2.1 hours , respectively).9,11 blood samples for clopidogrel measurement were collected at preset time intervals of predose and 0.33 , 0.67 , 1 , 1.5 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 24 hours after dosing . samples for aspirin measurement were taken predose and 1 , 2 , 2.5 , 3 , 3.5 , 4 , 4.5 , 5 , 6 , 8 , 10 , 12 , and 24 hours after dosing . whole blood samples ( 5 ml ) collected in edta ( k2 ) tubes were centrifuged at 1,800 g for 10 minutes , and plasma samples were transferred to microtubes and stored at 70c . subjects were asked about the occurrence of any adverse events ( aes ) between consent and the last poststudy visit . physical examinations and clinical laboratory tests were performed before and 24 hours after dosing in each period . vital signs were monitored before and at 12 and 24 hours after dosing in each period . subjects who took the study drugs at least once during the study were included in the safety assessment . concentrations of clopidogrel , aspirin , and the active aspirin metabolite salicylic acid were analyzed using validated ultraperformance liquid chromatography tandem mass spectrometry ( uplc ms / ms ) , acquity uplc system ( waters , milford , ma , usa ) . clopidogrel bisulfate and its internal standard ( rac clopidogrel - d4 hydrogen sulfate ) and aspirin and salicylic acid and their internal standards ( aspirin - d4 and salicylic acid - d4 , respectively ) were prepared using a 96-well solid - phase extraction plate , bond elut , plexa 10 mg ( varian , palo alto , ca , usa ) . detection and quantification were performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization interface in positive mode and multiple reaction monitoring mode . chromatographic separation of the compounds was accomplished using an acquity uplc beh c18 column ( 1.7 m , 2.150 mm ; waters ) with acetonitrile in 1,000 ml of water containing 0.1% formic acid as the mobile phase . a full validation of the assay was carried out with respect to selectivity , accuracy , precision , recovery , calibration curve , and stability . the calibration curves for clopidogrel , aspirin , and salicylic acid were linear over ranges of 0.0120.0 ng / ml , 10.02,000 ng / ml , and 10020,000 ng / ml , respectively , with coefficients of determination ( r ) greater than 0.995 for all . the pharmacokinetic parameters of clopidogrel , aspirin , and salicylic acid were estimated using a noncompartmental method using the winnonlin software ( v 6.1 ; pharsight corp , mountain view , ca , usa ) . the below the limit of quantitation values occurring prior to the first measurable concentration were set to zero , and those occurring after the first measureable concentration were not included in the pharmacokinetic analysis . the cmax and time to cmax ( tmax ) were calculated directly from the concentration the area under the plasma concentration time curve ( auc ) from time 0 to 24 hours ( auclast ) was calculated according to the linear trapezoidal rule . auc0 was calculated using the following equation : auc0 = auclast + ct/z , where ct is the last observed concentration and z is the elimination rate constant . the log - transformed cmax and auclast for clopidogrel , aspirin , and salicylic acid were compared using the average bioequivalence approach . this comparison was performed using analysis of variance ( anova ) with effects for sequence , subject within sequence , treatment , and period . because , for logistical reasons , the two groups were administered the study drugs at different times , group effect was also included as a factor.12 using anova , the 90% confidence intervals ( cis ) of the least - squares geometric means were calculated for cmax and auclast . the fdc formulation was considered bioequivalent if the 90% cis of cmax and auclast for clopidogrel , aspirin , and salicylic acid fell within the range of 0.801.25.12 to evaluate the difference of tmax between coadministration and fdc , wilcoxon signed - rank test was used . all statistical calculations were performed using the sas software ( v 9.3 ; sas institute inc . ) . healthy men aged between 20 and 55 years within 20% of the ideal body weight according to broca s formula were enrolled in this study . all subjects were judged to be healthy based on the results of a detailed physical examination , 12-lead electrocardiogram , clinical laboratory tests , and vital signs at the screening . the following exclusion criteria were used : excessive consumption of caffeine ( > 5 cups / d ) , cigarettes ( > 10 cigarettes / d ) , or alcohol ( > 30 g of alcohol / d ) , administration of inducers or inhibitors of drug - metabolizing enzymes including proton pump inhibitors ( ppis ) ( lansoprazole , omeprazole , pantoprazole , and rabeprazole ) , rifampicin , carbamazepine , and barbitals within 28 days ; history of disease influence on drug absorption , distribution , metabolism , and excretion ( eg , gastrointestinal ulcer , ulcerative colitis , and history of abdominal operation ) ; history of hemorrhagic symptoms and disease ; aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) exceeding 1.25 times the upper limit of the normal range ; total bilirubin exceeding 1.5 times the upper limit of the normal range ; prothrombin time , activated partial thromboplastin time , and bleeding time extended beyond the normal range ; platelet level < 15010/l or > 35010/l ; and participation in another clinical study within 90 days prior to the start of the study . a randomized , open , two - period , two - treatment , two - sequence crossover study was conducted at the clinical trial center of inje university busan paik hospital , busan , republic of korea ( clinicaltrials.gov identifier : nct01496261 ) . the 60 enrolled subjects were divided into two groups and randomized to one of two sequences ( rt , tr ) at the beginning of the study at a ratio of 1:1 using a randomization schedule generated using the sas software ( v 9.3 ; sas institute inc . , each group of 30 subjects was administered the study drugs in consecutive periods with a gap of 3 days between them . the assigned study drugs were administered orally with 240 ml of water after a 10-hour overnight fast , and subjects continued fasting until 4 hours postdose . subjects were administered two capsules of clopidogrel / aspirin 75 mg/100 mg fdc ( chong kun dang pharm . , subjects were coadministered two tablets of clopidogrel 75 mg ( plavix ; sanofi - aventis , schiltigheim , france ) and two capsules of aspirin 100 mg ( astrix ; boryung pharm . , seoul , republic of korea ) in the other period with a 14-day washout period . the length of the washout period was estimated to be greater than seven times the reported t1/2 of clopidogrel , aspirin , and salicylic acid ( 7.27.6 , 0.4 , and 2.1 hours , respectively).9,11 blood samples for clopidogrel measurement were collected at preset time intervals of predose and 0.33 , 0.67 , 1 , 1.5 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 24 hours after dosing . samples for aspirin measurement were taken predose and 1 , 2 , 2.5 , 3 , 3.5 , 4 , 4.5 , 5 , 6 , 8 , 10 , 12 , and 24 hours after dosing . whole blood samples ( 5 ml ) collected in edta ( k2 ) tubes were centrifuged at 1,800 g for 10 minutes , and plasma samples were transferred to microtubes and stored at 70c . subjects were asked about the occurrence of any adverse events ( aes ) between consent and the last poststudy visit . physical examinations and clinical laboratory tests were performed before and 24 hours after dosing in each period . vital signs were monitored before and at 12 and 24 hours after dosing in each period . subjects who took the study drugs at least once during the study were included in the safety assessment . concentrations of clopidogrel , aspirin , and the active aspirin metabolite salicylic acid were analyzed using validated ultraperformance liquid chromatography tandem mass spectrometry ( uplc ms / ms ) , acquity uplc system ( waters , milford , ma , usa ) . clopidogrel bisulfate and its internal standard ( rac clopidogrel - d4 hydrogen sulfate ) and aspirin and salicylic acid and their internal standards ( aspirin - d4 and salicylic acid - d4 , respectively ) were prepared using a 96-well solid - phase extraction plate , bond elut , plexa 10 mg ( varian , palo alto , ca , usa ) . detection and quantification were performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization interface in positive mode and multiple reaction monitoring mode . chromatographic separation of the compounds was accomplished using an acquity uplc beh c18 column ( 1.7 m , 2.150 mm ; waters ) with acetonitrile in 1,000 ml of water containing 0.1% formic acid as the mobile phase . a full validation of the assay was carried out with respect to selectivity , accuracy , precision , recovery , calibration curve , and stability . the calibration curves for clopidogrel , aspirin , and salicylic acid were linear over ranges of 0.0120.0 ng / ml , 10.02,000 ng / ml , and 10020,000 ng / ml , respectively , with coefficients of determination ( r ) greater than 0.995 for all . the pharmacokinetic parameters of clopidogrel , aspirin , and salicylic acid were estimated using a noncompartmental method using the winnonlin software ( v 6.1 ; pharsight corp , mountain view , ca , usa ) . the below the limit of quantitation values occurring prior to the first measurable concentration were set to zero , and those occurring after the first measureable concentration were not included in the pharmacokinetic analysis . the cmax and time to cmax ( tmax ) were calculated directly from the concentration the area under the plasma concentration time curve ( auc ) from time 0 to 24 hours ( auclast ) was calculated according to the linear trapezoidal rule . auc0 was calculated using the following equation : auc0 = auclast + ct/z , where ct is the last observed concentration and z is the elimination rate constant . the log - transformed cmax and auclast for clopidogrel , aspirin , and salicylic acid were compared using the average bioequivalence approach . this comparison was performed using analysis of variance ( anova ) with effects for sequence , subject within sequence , treatment , and period . because , for logistical reasons , the two groups were administered the study drugs at different times , group effect was also included as a factor.12 using anova , the 90% confidence intervals ( cis ) of the least - squares geometric means were calculated for cmax and auclast . the fdc formulation was considered bioequivalent if the 90% cis of cmax and auclast for clopidogrel , aspirin , and salicylic acid fell within the range of 0.801.25.12 to evaluate the difference of tmax between coadministration and fdc , wilcoxon signed - rank test was used . all statistical calculations were performed using the sas software ( v 9.3 ; sas institute inc . ) . sixty healthy men ( 30 subjects per group ) were enrolled in the study and randomized the mean age of the subjects was 25.53 years ( range , 2138 years ) , and the mean body weight was 69.95 kg ( range , 50.387.5 kg ) . three subjects withdrew consent during the study and four subjects withdrew because of laboratory abnormalities before dosing . time profiles of clopidogrel , aspirin , and salicylic acid are shown in figure 1 . descriptive statistics for the pharmacokinetic parameters of clopidogrel , aspirin , and salicylic acid are provided in table 2 . similar to the coadministration of clopidogrel and aspirin , the tmax of clopidogrel and aspirin administered as fdc were 0.67 ( p=0.85 ) and 4.00 hours ( p=0.88 ) , respectively . anova indicated a lack of group , period , sequence , and treatment effects for both cmax and auclast . the mean t1/2 of coadministered clopidogrel was 6.31 hours , which was comparable with that of fdc clopidogrel at 5.21 hours . the mean t1/2 of aspirin was also similar for coadministration ( 0.65 hour ) and fdc administration ( 0.63 hour ) . the 90% cis for the ratio ( fdc / coadministration ) of the geometric means for cmax , auclast , and aucinf are presented in table 3 . the mean log - transformed ratios of the primary parameters and their 90% cis were all within the predefined equivalence limit of 0.801.25 . no serious or significant aes occurred during the study . two aes were reported for one subject ( 1.67% ) after administration of fdc capsules : increases in alt and ast . four aes were reported for three subjects ( 5% of study population ) after coadministration of clopidogrel 75 mg and aspirin 100 mg : an increase in alt and ast ( in the same subject ) , an increase in blood bilirubin , and catheter site pain . of these aes , increases in alt and ast were considered possibly related to the study drugs and the others were assessed as unlikely to be or definitely not related to the study drugs . sixty healthy men ( 30 subjects per group ) were enrolled in the study and randomized the mean age of the subjects was 25.53 years ( range , 2138 years ) , and the mean body weight was 69.95 kg ( range , 50.387.5 kg ) . three subjects withdrew consent during the study and four subjects withdrew because of laboratory abnormalities before dosing . mean plasma concentration time profiles of clopidogrel , aspirin , and salicylic acid are shown in figure 1 . descriptive statistics for the pharmacokinetic parameters of clopidogrel , aspirin , and salicylic acid are provided in table 2 . similar to the coadministration of clopidogrel and aspirin , the tmax of clopidogrel and aspirin administered as fdc were 0.67 ( p=0.85 ) and 4.00 hours ( p=0.88 ) , respectively . anova indicated a lack of group , period , sequence , and treatment effects for both cmax and auclast . the mean t1/2 of coadministered clopidogrel was 6.31 hours , which was comparable with that of fdc clopidogrel at 5.21 hours . the mean t1/2 of aspirin was also similar for coadministration ( 0.65 hour ) and fdc administration ( 0.63 hour ) . the 90% cis for the ratio ( fdc / coadministration ) of the geometric means for cmax , auclast , and aucinf are presented in table 3 . the mean log - transformed ratios of the primary parameters and their 90% cis were all within the predefined equivalence limit of 0.801.25 . two aes were reported for one subject ( 1.67% ) after administration of fdc capsules : increases in alt and ast . four aes were reported for three subjects ( 5% of study population ) after coadministration of clopidogrel 75 mg and aspirin 100 mg : an increase in alt and ast ( in the same subject ) , an increase in blood bilirubin , and catheter site pain . of these aes , increases in alt and ast were considered possibly related to the study drugs and the others were assessed as unlikely to be or definitely not related to the study drugs . the objective of the current study was to compare the pharmacokinetics of a newly developed fdc of clopidogrel 75 mg / aspirin 100 mg with that of coadministration of corresponding doses of clopidogrel and aspirin . although exposure to clopidogrel can be highly variable with a coefficient of variance over 30% , we applied conventional bioequivalence criteria using a nonreplicative single - dose crossover design because of the narrow therapeutic window and safety issues with clopidogrel.13 pharmacokinetic analysis showed that the fdc capsule was bioequivalent to the coadministration of the individual drugs . in practice , clopidogrel is prescribed as a loading dose of 300600 mg , with 75 mg given subsequently as a maintenance dose . in this study , a single oral dose of 150 mg , half the loading dose , was administered because of the high intraindividual variability in the degree of absorption . although the aspirin dose of 200 mg is twice the usual maintenance dose ( corresponding to 75100 mg ) , it was considered acceptable because this was a single - dose study and aspirin shows linear pharmacokinetic characteristics.14 at the time this study was planned , there had been no studies investigating the intraindividual variability of the primary pharmacokinetic parameters of enteric - coated aspirin . therefore , we calculated the sample size using the ratio of intra- and interindividual variability for the general formulation of aspirin.15 the sample size , based on an estimated intraindividual variability of cmax for aspirin of 38% , was calculated to be 24 subjects per group to detect a 20% difference between the test and reference treatments with a power of 80% at a significance level of 5% . thus , 60 subjects were considered sufficient to compare the relative pharmacokinetics of fdc and the coadministration of individual formulations . our extrapolated intraindividual variability is in agreement with the 36.68% for an enteric - coated formulation of aspirin estimated by jung et al.16 when we recruited subjects for this study , strict selection criteria were applied for alcohol uptake , smoking status , and other medications that have an effect on the clopidogrel - metabolizing enzyme cyp2c19 , because variability in clopidogrel pharmacokinetics has been attributed to alcohol , smoking , cyp2c19 genotype , and genotype - related drug drug interactions . clopidogrel is probably converted to ethyl clopidogrel in the presence of ethyl alcohol , the major ingredient of alcoholic beverages , causing a decrease in the total metabolism of clopidogrel . in addition , ethyl alcohol increases the cytotoxic effect of clopidogrel and the pharmacological activity by prolonged presence of clopidogrel by decreasing the total metabolism of clopidogrel ( hydrolysis and transesterification ) in the presence of ethyl alcohol.17 a potential role of smoking in the metabolism of clopidogrel and high on - treatment platelet reactivity were suggested . pharmacodynamic studies and post hoc analyses of large clinical trials support a link between smoking status and the efficacy of clopidogrel therapy.18 in addition , clopidogrel is mainly metabolized to its active metabolite by cyp2c19,19 so it is important to consider comedication with drugs having the same metabolic route . the present study has several limitations . in clinical situations , dual therapy with clopidogrel and aspirin however , all participants of this study were healthy men , who may not represent the target patients , and only a single dose of treatment was given . because the antiplatelet effect of clopidogrel decreases with patient age,20 further multiple dosing studies , preferably in patients with cardiovascular disease , are needed to apply the fdc to clinical practice . the results from the present study indicate that the clopidogrel 75 mg / aspirin 100 mg fdc capsule was bioequivalent to individual formulations of corresponding doses of the two drugs .

backgroundsimultaneous prescription of clopidogrel and low - dose aspirin is recommended for the treatment of acute coronary syndrome because of improvements in efficacy and patient compliance . in this study , the pharmacokinetics of a fixed - dose combination ( fdc ) of clopidogrel and aspirin was compared with coadministration of individual formulations to clarify the equivalence of the fdc.methodsthis was a randomized , open - label , two - period , two - treatment , crossover study in healthy korean men aged 2055 years . subjects received two fdc capsules of clopidogrel / aspirin 75/100 mg ( test ) or two tablets of clopidogrel 75 mg and two capsules of aspirin 100 mg ( reference ) with a 14-day washout period . plasma concentrations of clopidogrel , aspirin , and salicylic acid were measured using validated ultraperformance liquid chromatography tandem mass spectrometry . bioequivalence was assessed by analysis of variance and calculation of the 90% confidence intervals ( cis ) of the ratios of the geometric means ( gmrs ) for auclast and cmax for clopidogrel and aspirin.resultssixty healthy subjects were enrolled , and 53 completed the study . clopidogrel , aspirin , and salicylic acid showed similar absorption profiles and no significant differences in cmax , auclast , and tmax between fdc administration and coadministration of individual formulations . the gmrs ( 90% ci ) for the cmax and auclast of clopidogrel were 1.08 ( 0.95 , 1.23 ) and 0.93 ( 0.84 , 1.03 ) , respectively . the gmrs ( 90% ci ) for the cmax and auclast of aspirin were 0.98 ( 0.84 , 1.13 ) and 0.98 ( 0.93 , 1.04 ) , respectively . both treatments were well tolerated in the study subjects.conclusionthe fdc of clopidogrel and aspirin was bioequivalent to coadministration of each individual formulation . the fdc capsule exhibited similar safety and tolerability profiles to the individual formulations . therefore , clopidogrel / aspirin 75 mg/100 mg fdc capsules can be prescribed to improve patient compliance .

fulvestrant ( faslodex ; astrazeneca ) is a novel steroidal estrogen receptor ( er ) antagonist lacking agonist effects . by covalent binding to the er , receptors are rapidly downregulated , resulting in a decrease of cellular er levels and complete abrogation of estrogen - sensitive gene transcription . the efficacy of fulvestrant in patients with tamoxifen - resistant disease [ 2 , 3 ] and in patients progressing on aromatase inhibitors has been confirmed , thus giving the opportunity to extend the overall period during which hormonal agents may be used in women with endocrine - responsive advanced breast cancer . two recent clinical trials are focusing on fulvestrant high - dose regimens ( 500 mg monthly ) to optimize fulvestrant treatment . the neoadjuvant newest trial showed significantly greater biological activity benefit with high - dose fulvestrant compared to the approved dose . the randomized , double - blind confirm ( comparison of faslodex in recurrent or metastatic breast cancer ) trial presented recently has shown a small but statistically significant increase in time to progression for fulvestrant 500 mg compared with fulvestrant 250 mg , without increase of toxicity . we report a case achieving long - term complete remission under fulvestrant introduced after no or poor response to prior endocrine therapies , in whom the most efficacious dosing regimen was below the approved dose . in january 1993 , a 47-year - old , premenopausal woman was diagnosed with a left breast cancer ( pt1c(m)pn0m0 ) after bilateral subcutaneous mastectomy for relapsing fibroadenomas . the tumor was er - positive ( 90% ) , pgr - positive ( 70% ) as evaluated by immunohistochemistry and had a low - level her2-gene amplification ratio of 2.22 ( positive > 2.20 ) as assessed in 2007 . axillary dissection was carried out followed by radiotherapy ( 50 gy total dose ) , but no adjuvant endocrine therapy was given . in january 1998 , distant recurrence was diagnosed in soft tissue ( er - negative , pgr - positive by histological assessment ) , in bone , pleura and lung ( lymphangiosis carcinomatosa ) . anastrozole was started as 1st - line endocrine treatment in a clinical trial , but discontinued after 3 months due to progression in pleura / lung and in the contralateral breast . 1 ) . six cycles of chemotherapy with paclitaxel ( 175 mg / m ) and doxorubicin ( 60 mg / m ) , every 21 days ( eortc 10961 ) , were given , followed by monthly pamidronate for 8 months . partial remission for 10 months was achieved with normalized chest x - ray and ca 15 - 3 . in june 1999 she received 9 months of tamoxifen as 2nd - line endocrine treatment , which resulted in stable disease for 6 months prior to progression ( ca 15 - 3 re - rising ) . exemestane was started as 3rd - line endocrine treatment , but discontinued 14 days later due to severe allergic exanthema . in march 2001 , cytological assessment of a further progression in the contralateral breast revealed an er - positive , pgr - negative tumor and fulvestrant ( 250 mg intramuscular , monthly ) was initiated as 4th - line endocrine treatment . as the drug had not yet been licensed in switzerland at that time , fulvestrant was initially provided free of charge through a named patient program ( supported by astrazeneca ) . twelve months later complete remission was achieved ( normal cervical / thoracic / abdominal computed tomography scan , disappearance of previous tracer uptake in bone scan ) . however , the patient experienced painful oral and vaginal dryness with loss of libido , ulcerative stomato - pharyngitis and an extensive thrombophlebitis of the left arm requiring temporary treatment interruptions . symptomatic treatment was given in different combinations , but mucosal dryness and inflammation became an ongoing problem diminishing her quality of life . therefore , in february 2004 , we decided to continue fulvestrant at half dose ( 125 mg monthly ) . mucosal symptoms improved ; however , when fulvestrant was once administered in full dose by error , painful oral and vaginal mucositis reoccurred . in june 2006 , complete remission was reconfirmed and fulvestrant was tentatively stopped 63 months after the 1st injection . however , complete remission was lost shortly thereafter ( ca 15 - 3 re - rising , small suspicious pleura effusion ) , but re - achieved 10 months after renewal of fulvestrant ( 125 mg monthly ) ( fig . , we finally decided to determine the patient 's fulvestrant plasma concentrations under the 125-mg dose by the high - performance liquid chromatography ( hplc ) method in the standard lab for fulvestrant plasma sample analyses . the maximum concentration detected was even below the predicted level ( table 1 ) . at follow - up in september 2008 thereafter , ca 15 - 3 ranged between upper normal limit ( unl ) and 1.3 unl , but so far progression of disease has not been confirmed on restaging . at the last follow - up in march 2010 , the patient was doing well . as observed in several clinical trials and case studies , some patients experience prolonged duration of response with fulvestrant treatment for endocrine - responsive advanced breast cancer [ 3 , 8 , 9 ] . to our knowledge , a 9-years response to 4th - line endocrine treatment with fulvestrant has not been reported in the literature so far . this case is remarkable in several aspects : first , achievement of long - lasting complete remission ( last reconfirmed 90 months after treatment start ) is very rare , especially after no or poor response to prior anastrozole / tamoxifen , obviously both not predictive for the efficacy of fulvestrant in this patient . second , reducing the dose of fulvestrant due to unanticipated intensity of mucosal toxicity maintained complete remission in this case . we think it is important to alert oncologists to the fact that the prior response to an aromatase inhibitor does not appear to be predictive for the benefit of fulvestrant , an observation also made in a phase ii trial investigating fulvestrant in patients with primary or acquired aromatase inhibitor - resistance . abram et al . presented a case with endocrine - responsive advanced breast cancer , which also appeared to respond better to fulvestrant than to prior endocrine therapies . they assumed that the intramuscular administration by a health care professional may have resulted in improved compliance . a belgian study found a greater activity of fulvestrant in tumors expressing both er and pgr . other investigators found fulvestrant activity not being reduced in pgr - negative tumors . in our case , hormone receptor status was er - positive / pgr - negative when repeated prior to start of fulvestrant treatment , though the primary tumor was er - and pgr - positive . pgr expression within the tumor is known to be patchy and a sampling error might have led to the er - positive , pgr - negative result in the immunocytochemical analysis of the fine needle aspiration material . tumors for which fulvestrant therapy might be particularly appropriate are those er - positive tumors with her2 overexpression assuming ligand - independent er activation seems to be a key feature . however , available data are conflicting . in our case , her2 amplification with a ratio of 2.22 was low and unlikely to contribute to the superiority of fulvestrant compared to anastrozole and tamoxifen by the above - mentioned mechanism . several large trials have shown that fulvestrant is well tolerated [ 2 , 3 , 4 , 14 ] . our patient experienced unusually severe mucosal toxicity together with complete remission under the approved 250-mg dose , but clearly less toxicity while maintaining complete remission under a 125-mg dose . the pharmacokinetic profile excluded faster absorption or a variation in drug metabolism which theoretically could have contributed to the observed toxicity and efficacy ( table 1 ) . her fulvestrant plasma concentrations were below the observed mean for a 250-mg dose and , importantly , even below the predicted mean for a 125-mg dose . we therefore hypothesize an extraordinary sensitivity , but not a metabolic phenomenon as a key factor in the observations made in this case . this observation is of particular interest in the ongoing fulvestrant dosing debate : our case might serve as a basis for future strategies investigating individual dosing as a promising approach for optimizing fulvestrant treatment .

we report a case of long - term ( 9 years ) response to 4th - line endocrine treatment with fulvestrant given for advanced breast cancer after no or poor response to prior endocrine therapies . complete remission was achieved with full dose and maintained even after dose reduction due to unanticipated intensity of mucosal toxicity . complete remission was temporarily lost after fulvestrant was tentatively withdrawn ( 63 months after treatment start ) , but was re - achieved after renewal of half - dose treatment and last reconfirmed 90 months after treatment start . the pharmacokinetic profile provides evidence to hypothesize a unique sensitivity to fulvestrant in this patient which might explain both : toxicity and extraordinary efficacy .

infestation with the liver flukes fasciola hepatica and fasciola gigantica , has traditionally been associated with substantial production and economic losses in livestock , particularly sheep and cattle ( 1 ) . several studies have shown that f. hepatica occurs in temperate areas , while f. gigantica occurs mainly in tropical zones , and both species overlap in subtropical areas ( 24 ) . fasciola hepatica and f. gigantica were previously reported from a range of domestic animals ( buffalo , cattle , sheep , and goat ) and humans based on morphological and some molecular features in iran ( 5 , 6 ) . the differential diagnosis between f. hepatica and f. gigantica infection is very important because of their different transmission and epidemiological characteristics . fasciola hepatica and f. gigantica can generally be distinguished based on their morphology ( 3 ) . however , it is usually difficult to discriminate accurately between f. hepatica and f. gigantica because of the high variations in their morphological characteristics . the two species can be discriminated by dna sequences of nuclear ribosomal internal transcribed spacer 1 ( its1 ) , its2 , and 28s rdna regions and of mitochondrial nadh dehydrogenase i ( nd1 ) and cyto - chrome c oxidase i ( co1 ) genes ( 7 , 8) . as intraspecific genetic variations among liver flukes may reflect differences in virulence , host specificity and drug susceptibility or resistance ( 9 ) , the population genetics of fasciola parasites in iran needs to be studied . the genetic variation within f. hepatica sequences of the mitochondrial dna and ribosomal regions has been reported previously from iran and several other countries ( 5 , 6 , 912 ) . however , limited information is available about intraspecific genetic diversity among iranian f. gigantica isolates . its1 , its2 , 18s rdna and co1 regions have been used as markers for genetic characterization of fasciola flukes from iran ( 9 , 13 , 14 ) . in previous studies , there was little information available on the characterization of iranian f. gigantica existing genotypes based on variations in nadh dehydrogenase subunit 1 ( nd1 ) dna sequences . the present study was designed to molecularly differentiate fasciola species and characterize genetic diversity of f. gigantica using nd1gene in the endemic areas of iran . fasciola specimens ( n=40 ) were collected at local abattoirs during post - slaughter inspection from livers of naturally infected sheep ( n=15 ) , cattle ( n=15 ) , buffalos ( n=5 ) and camels ( n=5 ) from four distinct areas in iran including fars ( 29 37n , 5231e ) , khorasan razavi ( 3617n , 5936e ) , gilan ( 3716n , 4934e ) and khuzestan ( 32 22 n figure 1 shows the iran s map and the geographic locations where the parasitic flukes were collected . fresh adult worms were washed thoroughly in physiological saline , fixed in 70% ethanol and stored at 20 until further use . the geographic locations where the parasitic flukes ( fasciola spp . ) were collected from different hosts in iran individual worms were pressed slightly between two slides and were subjected to computerized morphometric examination using computer image analysis system ( image analyzer version 1.34 ) . body length ( bl ) , body width ( bw ) , body area ( ba ) and body perimeter were measured and bl / bw ratio was calculated . the initial differentiation between f. hepatica and f. gigantica species was made based on the morphometric criteria according to standard taxonomic keys ( 15 ) . samples were transferred from fixative solution to sterile microtubes and left until complete evaporation of ethanol . dna was extracted from a small part of the parasite apical zone , to avoid inclusion of female genitalia likely to include foreign sperm . dna extraction was carried out using a commercial extraction kit ( dneasy blood & tissue kit , qiagen , germany ) in according to the manufacturer s instructions . the purity of the extracted dna samples was evaluated using spectrophotometric method ( eppendorf , germany ) . the mitochondrial dna was amplified using specific primers for nadh dehydrogenase 1(nd1 ) ( jb11 : 5-aga ttc gta agg ggc cta ata-3 and jb12 : 5-acc act aac taa cta attcac ttt c-3 ) ( 16 , 17 ) . the primers for amplification of the its2 region ( 5-tct tga acg cat att gcg gc-3 and 5-agt tca gcg ggt aat cac gt-3 ) were used as described by ghavami and others ( 11 ) . the expected pcr amplicons are fragments of 534 bp from nd1 gene and fragments of 456 bp from its2 region . reactions were carried out in a final volume of 50l containing 5l and 4l of genomic dna for ndi and its2 assays , respectively . each its2 pcr reaction contained 50 mm kcl , 10 mm tris - hcl ( ph=9.0 ) , 1 mm mgcl2 , 200 m dntps , 0.4 m of each primer , and 2.5 u taq dna polymerase ( fermentas , usa . each nd1 pcr reaction contained 50 mm kcl , 10 mm tris - hcl ( ph=9.0 ) , 1.5 mm mgcl2 , 100 m dntps , 0.48 m of each primer , and 2.6 u taq dna polymerase ( fermentas , usa ) . pcr amplification was performed under the following conditions : initial denaturation at 94c for 5 min , followed by 35 cycles of denaturation at 94c for 60 s , annealing at 55c for 45 s ( nd1 ) and at 60c for 45 s ( its2 ) , extension at 72c for 45 s , and a final synthesis at 72c for 7 min , in the mastercycler gradient eppendorf thermal cycler ( germany ) . 5l of each reaction product was subjected to electrophoresis in tae buffer ( 40 mm tris base , 20 mm acetic acid,1 mm edta ) using 1% agarose gels , stained with ethidium bromide , ( 0.5g / ml ) and visualized on a uv transilluminator . briefly , ten microliters of pcr products were incubated with 2 l of pagi restriction endonuclease ( er1282 , 00022771 , fermentas , usa ) and 2 l of supplied buffer at 37c for 16 hours . the final reaction volume was adjusted to 20 l by adding enough sterile distilled water . the digested dna was analyzed by electrophoresis in 2% agarose gels in tae buffer , visualized by ethidium bromide staining , and photographed . lengths of resulting restriction fragments from f. hepatica and f. gigantica were predicted by means of the gene runner software v. 3.05 . based on computerized cutting , this enzyme has no cutting site on its2 pcr amplicons for fasciola hepatica and one cutting site for f. gigantica , producing fragments of 165 and 291bp . the pcr products of ndi for f. gigantica parasites ( characterized by morphological data and rflp analysis of its2 ) were directly sequenced using capillary dna analyzer ( abi 3730 , applied biosystems , foster city , ca , usa ) after sequencing reactions with a big dye terminator v3.1 cycle sequencing kit ( applied biosystems ) . forward and reverse nucleic acid sequence data were used to construct a continuous sequence of inserted dna . further comparison of the continuous ndi sequences was made with previously available sequences in national center for biotechnology information ( ncbi ) using basic local alignment search tool ( blast ) and compared with other homologues by means of neighbor joining and maximum parsimony trees reconstructed from kimura 2-parameter distances from 1,000 bootstrap replicates of multiple sequence alignments created using clustalw , as implemented by mega version 4.0 ( 18 ) . this experiment was performed under the approval of the state committee on animal ethics , shiraz university , shiraz , iran . in addition , the recommendations of european council directive ( 86/609/ec ) of november 24 , 1986 , regarding the protection of animals used for experimental purposes , were considered . fasciola specimens ( n=40 ) were collected at local abattoirs during post - slaughter inspection from livers of naturally infected sheep ( n=15 ) , cattle ( n=15 ) , buffalos ( n=5 ) and camels ( n=5 ) from four distinct areas in iran including fars ( 29 37n , 5231e ) , khorasan razavi ( 3617n , 5936e ) , gilan ( 3716n , 4934e ) and khuzestan ( 32 22 n figure 1 shows the iran s map and the geographic locations where the parasitic flukes were collected . fresh adult worms were washed thoroughly in physiological saline , fixed in 70% ethanol and stored at 20 until further use . the geographic locations where the parasitic flukes ( fasciola spp . ) were collected from different hosts in iran individual worms were pressed slightly between two slides and were subjected to computerized morphometric examination using computer image analysis system ( image analyzer version 1.34 ) . body length ( bl ) , body width ( bw ) , body area ( ba ) and body perimeter were measured and bl / bw ratio was calculated . the initial differentiation between f. hepatica and f. gigantica species was made based on the morphometric criteria according to standard taxonomic keys ( 15 ) . samples were transferred from fixative solution to sterile microtubes and left until complete evaporation of ethanol . dna was extracted from a small part of the parasite apical zone , to avoid inclusion of female genitalia likely to include foreign sperm . dna extraction was carried out using a commercial extraction kit ( dneasy blood & tissue kit , qiagen , germany ) in according to the manufacturer s instructions . the purity of the extracted dna samples was evaluated using spectrophotometric method ( eppendorf , germany ) . the mitochondrial dna was amplified using specific primers for nadh dehydrogenase 1(nd1 ) ( jb11 : 5-aga ttc gta agg ggc cta ata-3 and jb12 : 5-acc act aac taa cta attcac ttt c-3 ) ( 16 , 17 ) . the primers for amplification of the its2 region ( 5-tct tga acg cat att gcg gc-3 and 5-agt tca gcg ggt aat cac gt-3 ) were used as described by ghavami and others ( 11 ) . the expected pcr amplicons are fragments of 534 bp from nd1 gene and fragments of 456 bp from its2 region . reactions were carried out in a final volume of 50l containing 5l and 4l of genomic dna for ndi and its2 assays , respectively . each its2 pcr reaction contained 50 mm kcl , 10 mm tris - hcl ( ph=9.0 ) , 1 mm mgcl2 , 200 m dntps , 0.4 m of each primer , and 2.5 u taq dna polymerase ( fermentas , usa . each nd1 pcr reaction contained 50 mm kcl , 10 mm tris - hcl ( ph=9.0 ) , 1.5 mm mgcl2 , 100 m dntps , 0.48 m of each primer , and 2.6 u taq dna polymerase ( fermentas , usa ) . pcr amplification was performed under the following conditions : initial denaturation at 94c for 5 min , followed by 35 cycles of denaturation at 94c for 60 s , annealing at 55c for 45 s ( nd1 ) and at 60c for 45 s ( its2 ) , extension at 72c for 45 s , and a final synthesis at 72c for 7 min , in the mastercycler gradient eppendorf thermal cycler ( germany ) . 5l of each reaction product was subjected to electrophoresis in tae buffer ( 40 mm tris base , 20 mm acetic acid,1 mm edta ) using 1% agarose gels , stained with ethidium bromide , ( 0.5g / ml ) and visualized on a uv transilluminator . briefly , ten microliters of pcr products were incubated with 2 l of pagi restriction endonuclease ( er1282 , 00022771 , fermentas , usa ) and 2 l of supplied buffer at 37c for 16 hours . the final reaction volume was adjusted to 20 l by adding enough sterile distilled water . the digested dna was analyzed by electrophoresis in 2% agarose gels in tae buffer , visualized by ethidium bromide staining , and photographed . lengths of resulting restriction fragments from f. hepatica and f. gigantica were predicted by means of the gene runner software v. 3.05 . based on computerized cutting , this enzyme has no cutting site on its2 pcr amplicons for fasciola hepatica and one cutting site for f. gigantica , producing fragments of 165 and 291bp . the pcr products of ndi for f. gigantica parasites ( characterized by morphological data and rflp analysis of its2 ) were directly sequenced using capillary dna analyzer ( abi 3730 , applied biosystems , foster city , ca , usa ) after sequencing reactions with a big dye terminator v3.1 cycle sequencing kit ( applied biosystems ) . forward and reverse nucleic acid sequence data were used to construct a continuous sequence of inserted dna . further comparison of the continuous ndi sequences was made with previously available sequences in national center for biotechnology information ( ncbi ) using basic local alignment search tool ( blast ) and compared with other homologues by means of neighbor joining and maximum parsimony trees reconstructed from kimura 2-parameter distances from 1,000 bootstrap replicates of multiple sequence alignments created using clustalw , as implemented by mega version 4.0 ( 18 ) . this experiment was performed under the approval of the state committee on animal ethics , shiraz university , shiraz , iran . in addition , the recommendations of european council directive ( 86/609/ec ) of november 24 , 1986 , regarding the protection of animals used for experimental purposes , were considered . based on the morphometric criteria , 14 parasitic samples were initially identified to be f. gigantica while 26 samples were diagnosed to be f. hepatica . morphometric data of the f. gigantica samples from different geographical locations and host origins captured in iran are present in table 1 . morphometric data of adult liver flukes of fasciola gigantica form different geographical locations and host origins used for genotyping in iran the expected pcr products of 456bp were produced in all morphologically like f. gigantica samples using its2 gene primers independently from the different geographical origins and the different host species . restriction fragment length polymorphism ( rflp ) patterns of parasitic flukes were obtained after digestion of the pcr products with pagi enzyme , in order to differentiate two fluke species ( fig . 2b ) . based on sequence differences of amplified portions of the its2 gene between f. gigantica and f. hepatica , the restriction enzyme pagi was expected to have one cutting site on the its2 pcr amplicons from f. gigantica . accordingly , all of the its2 gene pcr amplicons of 456 bp from f. gigantica flukes were cut by the pagi endonuclease to fragments of 165 and 291bp . none of its2 pcr products from f. hepatica like samples was digested by the restriction enzyme treatment ( fig . all samples confirmed to be f. gigantica ( n=14 ) were subjected to pcr amplification of nd1 gene . the expected pcr products of 534 bp were produced in all reactions from f. gigantica samples ( fig . sequencing results of the nd1 pcr products were used to characterize the genotypic diversity of the f. gigantica flukes obtained from endemic areas of iran . comparison of partial sequencing results of nd1 gene ( 410 bp ) with previously available sequences in ncbi using blast showed that 13 out of 14 studied fasciola samples belonged to f. gigantica . interestingly , one fluke sample ( 12 m : kf356181 ) that had been classified as f. gigantica according to morphometric criteria and its2 pcrrflp pattern , belonged to f. hepatica based on nd1 nucleotide sequence . agarose gel electrophoresis ; ( a ) ndi pcr products from fasciola samples compared with the molecular 100 bp weight marker . ( b ) pcr - rflp for its2 products using pagi ( f.hep = f. hepatica , lane 4 and f. gig = f. gigantica , lanes 2 , 5 and 6 ) , lane 3=blank control the phylogenetic analysis of the nd1 sequence data revealed no host and geographic specificity among genotypes ( fig . twenty - six sites out of 410 base pairs of analyzed nd1 gene in 10 isolates of f. gigantica were diagnosed to be polymorphic denoting noticeable genetic diversity between different isolates of the parasite in iran . nucleotide sequence variation among the iranian f. gigantica isolates for nd1 analyzed in this study ranged from 0% to 4.63% . from 26 polymorphism sites , only eight ones were leaded to the post - translational amino acid changes in nd1 gene product ( fig . the nucleotide sequence data obtained in this study have been deposited in the genbank under accession numbers kf356168 to kf356181 . the ndi phylogenetic analysis , constructed according to maximum parsimony ( mp ) method to show the position of fasciola spp . isolates in sheep , camel , cow and water buffalo in various locations in iran partial alignment of the nd1 amino acid sequences for fasciola gigantica ( n=13 isolates ) obtained from different geographical locations and host origins in iran . the species of genus fasciola ( platyhelminthes : digenea ) are the etiological agents of hepatic damage in a wide range of vertebrate hosts worldwide ( 4 ) , resulting in profound economic losses ( 19 ) and public health challenge ( 9 , 20 ) . the present study characterized the genetic diversity of f. gigantica obtained from different hosts and different geographic locations in iran . intraspecific genetic variations among liver flukes may reflect differences in virulence , host specificity and drug susceptibility or resistance ( 9 ) . the differential diagnosis between f. hepatica and f. gigantica infection is very important because of their different transmission and epidemiological characteristics . due to the limitations of morphological methods , various molecular approaches have been developed and used successfully for the identification and differentiation of fasciola species ( 21 ) . pcr analyses of ribosomal dna genes have been widely used for molecular identification of fasciolides . previous studies have confirmed that the first and second internal transcribed spacers ( its1 and its2 ) of rdna located between the nuclear small and large subunit rdna genes can provide genetic markers for species - level identification of fasciola . several studies on species identification of fasciola flukes based on its2 sequence as molecular marker have been conducted ( 10 , 12 , 2225 ) . in the current study , the application of pcr - rflp analysis of the its2 sequence was successful to differentiate fasciola species and results were in agreement with morphological classification of two fluke species . however , it seems that the accurate characterization of some types of intermediate form of fasciola needs both mitochondrial ndi and ribosomal its sequence data . the mitochondrial markers have been extensively used for phylogenetic studies and population differentiation because of its relativity rapid rate of evolution , importance in differentiation and discrimination of closely related organism , maternally inherited and does not undergo any recombination ( 26).for example , nadh dehydrogenase subunit 1 gene ( nd1 ) have been shown to be well suited for investigating the population genetics of trematodes , i.e. , f. hepatica ( 27 ) , clonorchis sinensis ( 28 ) and opisthorchis viverrini ( 29 ) . in the present study , mitochondrial nd1 gene was amplified using pcr from all f. gigantica samples and partial sequencing results were compared with previously available sequences in ncbi using blast and compared with other homologues by means of maximum parsimony trees . nd1 gene sequence showed a considerable genetic variability among samples obtained from the different geographical origins and the different host species in iran . all of the isolates were classified as f. gigantica based on both its2 and nd1 gene sequences except for one sample , which categorized as f. hepatica based on nd1 gene sequence . the presence of intermediate forms of f. gigantica and f. hepatica has been reported from several countries . previous studies has confirmed the existence of mixed infections of the both fluke species in the same animal , which imply that hybridization and/or introgression phenomena are possible cause of formation of intermediate forms ( 3 , 6 ) . phylogenetic analysis revealed that it was impossible to identify accurately genotype using a host , suggesting that host associations are not likely to be useful characters for fasciola genotype classification . for example , geno - types of a and j were reported from different hosts . similarly , no associations were found between geographic location and phylogenetic lineage , indicating that these characters were not useful for classification of f. gigantica . in addition , the results of this study showed heterogeneity might also exist in one location . the existence of the heterogeneity in one area may be due to traditional shepherding in iran , which includes regional movements between different pastures as well as transportation of sheep during slaughtering process . the amino acid substitutions identified in the nd1 peptide sequence of f. gigantica suggests that this polymorphism may exhibit considerable functional heterogeneity in nd1 , which may be of importance to the parasite biological activities . some authors believe that the capacity of fasciolids to adapt rapidly to new definitive hosts species and environments is most probably related to the high genetic variability of this parasite ( 30 ) . the mitochondrial genetic variations among the iranian f. gigantica isolates ( 0% to 4.63% ) were higher than those previously reported for f. hepatica in iran were . formerly , moazeni et al . ( 2101 ) showed a limited genetic variation using mitochondrial co1 sequence ( 0% to 0.98% ) among 19 different isolates of f. hepatica from cattle and sheep in different areas of iran ( 5 ) . these authors only defined four different co1 haplotypes - based on five nucleotide substitutions - among this trematode isolates . the number of different nd1 genotypes obtained in this study demonstrates that , the mitochondrial gene is a suitable marker to show variability within f. gigantica isolates . additional studies involving more isolates and other mitochondrial polymorphic genes , such as that for cytochrome c oxidase gene ( co1 ) , may help us to clarify genetic divergence of f. gigantica worldwide .

background : fasciola hepatica and f. gigantica are the causative agents of fasciolosis in domestic animals and humans . based on the morphometric criteria , differential diagnosis between them is problematic . in addition , intermediate forms of fasciola have been found in iran , which makes the differentiation more difficult . the aim of the present study was to provide molecular evidence for the existence of f. gigantica in iran using sequencing analysis of nd1 and pcr - rflp analysis of its2 regions and to study the intraspecies variations of f. gigantica based on mitochondrial nd1 gene polymorphism.methods:forty fasciola spp . samples collected from four distinct provinces ( fars , khuzestan , gilan , khorasan razavi ) in iran were collected for morphological and molecular characterization . in molecular method , pcr - rflp analysis of its2 using pagi restriction enzyme was used as a screening approach for f. gigantica differentiation . then mitochondrial dna sequence variations in the nd1 gene were used for phylogenetic analysis.results:based on the morphometric criteria and rflp analysis , 14 parasitic samples were initially identified to be f. gigantica . phylogenetic results showed that there are at least 10 different genotypes of f. gigantica in iran , which are different from those existing in the genbank . twenty - six points out of 410 base pairs of sequenced nd1 gene in 10 varieties of f. gigantica were diagnosed to be polymorphic . from 26 points of polymorphism , only eight resulted in the post - translational amino acid changes in nd1 gene product structure.conclusion:data revealed noticeable genetic diversity ( up to 4.63% ) between different varieties of f. gigantica in iran .

the recent development of whole slide imaging ( wsi ) is rapidly making many advances in pathology and enabling the creation of novel tools and applications for the pathology community , including virtual microscopy , and computer - aided quantification and diagnosis . in contrast to conventional light microscopy that allows only a view of a fraction of a specimen at a time , wsi offers a digital replica of an entire histopathology slide . the data can be processed with a pattern recognition algorithm to identify features of interest . this technique , called whole slide pattern recognition , can provide more information than traditional pathology slide analysis , minimizes tedious visual inspection by trained pathologists and has the potential to improve clinical diagnosis . first of all , a typical wsi dataset can contain a number of pixels at tera - scale ( 10 ) and previous computational methods used to identify targets on traditional histopathology images , including texture - analysis , nuclei pattern classification , and edge detection , are impractical for wsi due to computational time requirements . parallel processing strategies have been developed to handle wsi ; however , boundary artifacts can jeopardize the integration of the analysis results and introduce a substantial amount of re - processing time . in addition , the abundant information provided by the whole slide analysis also requires a method to provide a quantitative assessment and to present meaningful results in a manner that is easy to interpret . in this paper , we report a wsi analysis method that conducts automatic stain recognition to estimate the density distribution of the recognized stains across an entire pathology slide . the whole slide images were tessellated into smaller image blocks with overlapping regions at the image boundary to enable parallel processing . pixel - based stain recognition with morphological smoothing was applied to identify stains in the tessellated images . the density distribution of the identified stains was calculated by the kernel density estimator , yielding a distribution image of a feature or a particular cell type labeled by the stains . to validate our method , we applied it to specimen slides from an ongoing study using a rat cardiac allograft rejection model and also another study using a rat heart ischemia / reperfusion injury model . in both studies , the macrophage infiltration was revealed by immunohistochemistry ( ihc ) with anti - rat ed1 antibody . the accuracy of the stain recognition algorithm was first validated by comparing its results with manual counting without involving the wsi technique . the pathology images were obtained with a standard light microscope and the results of our stain recognition algorithm were compared with those obtained from manual counting . then , our method was further validated / correlated with the results obtained from in - vivo cellular magnetic resonance imaging ( mri ) , an imaging technique that can detect macrophage infiltration when labeled with ultrasmall superparamagnetic iron - oxide ( uspio ) or micron - sized iron - oxide ( mpio ) particles . our method was also validated / correlated with the results obtained from ex - vivo cellular mri , which were conducted using magnetic resonance microscopy ( mrm ) to achieve a much higher resolution for our final examination . to demonstrate the potential utilities of our method , we trained the algorithm to recognize different targets under hematoxylin and eosin ( h and e ) stain and to present their distribution across the tissue sections . we further calculated the diameter of the recognized targets and estimated the spatial distribution of cell nuclear diameter , which may have potential application in characterizing tissue in histopathology slides . the rat cardiac allograft rejection model , as described in a study , was established using dark agouti and brown norway rats ( harlan , indianapolis , in ) as the donor - recipient transplantation pairs . the superior vena cava of the graft heart was anastomosed to the recipient inferior vena cava ( ivc ) and the aorta of the graft heart was anastomosed to the recipient abdominal aorta , with the recipient proximal ivc partially obstructed to increase the pre - load . the graft , with intact pulmonary circulation , received proper pressure and volume loading without detectable atrophy over time . on day 7 after the transplantation , the graft hearts were scanned using in - vivo cellular mri and then harvested for pathology inspections . a suture was tied around the left anterior descending artery for 45 min and then was cut to allow reperfusion . after 2 days , the heart was harvested and scanned by ex - vivo mrm . cellular mri experiments were conducted by using dextran - coated uspio particles or polystyrene - coated mpio particles ( bangs laboratories , fishers , in ) as an mri contrast agent to image macrophage distribution . the iron - oxide particles are taken up by macrophages in circulation and create signal - voids in t2 * -weighted images to reveal the macrophage infiltration in - vivo , offering a unique way to correlated the ihc stain distribution obtained from our method . in our study , the iron - oxide particles were given to the animals before surgery . the rats , undergoing heterotopic heart transplantation , received uspio particles and were scanned by in - vivo cellular mri . the in - vivo mri scan was conducted on a bruker 7-t scanner ( bruker , billerica ma ) . the rats were intubated and ventilated during the mri scan and electrocardiogram ( ecg ) leads were placed on the abdomen using a gating and monitoring system ( sa instruments , stony brook , ny ) . t2 * -weighted magnetic resonance ( mr ) images , an imaging modality known to be sensitive to the signal void artifact created by iron - oxide particles , were acquired using a flash sequence with ecg and respiratory gating . tr = the respiration cycle ( ~1 s ) , te = 5 ms , field of view ( fov ) = 4 cm , slice thickness = 1.5 mm , in - plane resolution = 156 m . the ex - vivo mri scans were conducted using a bruker 11.7-tesla scanner ( bruker , billerica ma ) . t2 * -weighted mr images were also acquired using a flash sequence with te = 5.8 ms , fov = 1.2 cm , and isotropic resolution = 23 m . the hearts were fixed in 4% paraformaldehyde for 1 - 2 weeks and embedded in paraffin . # dc2002 ) , which streamed slides in preheated high ph target retrieval solution ( dako # s3308 , carpinteria , ca , usa ) for 15 min . this was followed by cooling of the slides at room temperature for 20 min and rinsing the slides in distilled water before commencing the staining steps . to stain the slides , the tissue sections were incubated with 10% horse serum for 10 min at room temperature to block non - specific binding . the slides were then incubated with 1:200 anti - rat ed1 monoclonal purified immunoglobulin g ( abd serotec , oxford , uk ) for 2 h. after pbs wash , the slides were incubated with biotinylated secondary antibodies ( lsab kit , dako , carpinteria , ca , usa ) for 30 min and labeled with the streptavidin - horseradish peroxidase ( dako , carpinteria , ca , usa ) for 30 min . then the slides were stained with aminoethyl carbazole chromogen ( skytek laboratories , inc . , a rat spleen tissue section was used as a positive control section , whereas a normal rat cardiac tissue was used as a negative control section . the stained slides were scanned on a whole slide scanner ( nanozoomer 2.0-ht , hamamatsu , japan ) to acquire wsi at 20 magnification . the resulting pathology images were then processed by our whole slide pattern recognition method , as detailed in the following section . the whole slide image was tessellated to enable the whole slide pattern recognition , as shown in figure 1 . the tessellation lines divide the whole slide image into several n - by - n sized blocks and the choice of n depends on the application scenario and the applied recognition algorithm . a higher value offers a larger field of view , but it may exceed the maximum allowable image size for the algorithm . after tessellation , each block was padded with an additional margin , m - pixel wide , to the neighboring block . the padding setting was devised for accurate accounting of stains near the block boundary , since a strict boundary could bisect a stain , incorrectly assigning it to both neighboring blocks . the choice of m considers the span of individual stain and requires prior information about their morphology . an m - pixel - wide margin allows stains with a maximum span of m to be fully recognized within the tessellated image . in our study , we used n = 1000 and the value of m was set to 100 . the tessellation scheme for the whole slide pattern recognition . the whole slide image is tessellated into n - by - n sized blocks to enable parallel recognition . each block is patched with additional m - width margin to consider stains bisected by the tessellation boundary the center of each recognized stain can be used to determine whether it should be counted or ignored to avoid double counting of stains , as illustrated by the example scenario in figure 2 . one should note that the tessellation borders are present at all sides of the tessellated image and figure 2 shows only the border between two adjacent images blocks to simplify the illustration . as shown in figure 2a , our stain recognition algorithm is applied to the image block to the left and the recognized stains with center points inside the tessellation line ( annotated by yellow ) are counted , whereas those outside the line ( annotated by red ) are ignored . similarly , in figure 2b , our stain recognition algorithm is applied to the image block to the right . the recognized stains with center points inside the tessellation line ( annotated by yellow ) are counted , whereas the rest are ignored ( annotated by red ) . ( a ) the stain recognition is applied to the image block to the left to recognize the stains . the center locations of the stains ( marked by dot points ) are used to determine whether a recognized stain should be counted ( annotated by yellow ) or ignored ( annotated by red ) . ( b ) the stain recognition is applied to the image block to the right , showing how double counting is avoided . the counted stains ( annotated by yellow ) have center points located inside the tessellation line the flow chart of our stain recognition algorithm is shown in figure 3 . the method is composed of three modules , including a classification module that performs pixel - wise color classification , a morphology smoothing module that eliminates the fragments considered to be false positives and an analysis module that identifies each isolated stain region . it is noteworthy that a variety of stain recognition algorithms can be applied to this problem , and in this study , we have used the simplest pixel - wise color recognition approach for its low computation time and stable performance . the red - green - blue color channels were used as the classification features and the output ( classification result ) was either 0 ( background ) or 1 ( foreground ) . the stain classification was conducted using nearest neighbor method , a popular method in data mining . nearest neighbor method required a training dataset of background and foreground stain image . in this study , we manually cropped the background and foreground stain from the wsi data as the training dataset . alternatively , one may also get training data from the positive and negative controls . the nearest neighbor method classified each image pixel based on its color difference with the pixels in the training images . if the minimum difference was from the foreground stain , then the pixel was classified as foreground ; otherwise , it was classified as background . this classification was applied to each pixel in the input image [ figure 3b ] to obtain the binary recognition output [ figure 3c ] . this output image might have small fragments due to error classification or the partial slice coverage of the stained cells . to eliminate fragments in the binary images and to facilitate analysis , morphological opening and closing were applied 3 times using a 3-by-3 square structuring element to smooth the contours of the recognized stains , as shown in figure 3d . finally , the connected component analysis identified each isolated stain region in the image and the center location of each stain was calculated for further density estimation . the pixel - wise classification is trained by a foreground stain image and also a background stain image ( a ) . each pixel of the input image ( b ) is then classified using nearest neighbor classification to obtain a binary image ( c ) . the small fragments in the binary image are then eliminated by morphological closing and opening , resulting in a smoother output ( d ) . this image is then analyzed by connected component analysis to obtain the center locations of the recognized stains since the locations of the recognized stains were discrete points scattered across the wsi , a regression analysis method was used to calculate their density distribution . this was carried out by the kernel density estimator as shown in figure 4 , where figure 4a shows an exemplary result of the discrete stain locations and figure 4b shows their density distribution estimated by the kernel density estimator . we had a total of k stains obtained from our whole slide pattern recognition and each of them was located at coordinate xi . the kernel density estimator estimated the density distribution function by using the following formulae : the exemplary analysis result of the kernel density estimator . ( b ) their density distribution can be estimated by the kernel density estimator where h is the kernel bandwidth and is the kernel function . the scalar feature ( e.g. diameter or area ) of the k recognized stains can also be estimated using kernel regression . where yi is a scalar feature of the i stain . equation 2 can be used to estimate the spatial distribution of the feature across the entire pathology slide . the bandwidth of the kernel determines the variability of the density estimation . a higher value of the bandwidth generates a smoothing effect , leading to reduced variability . on the contrary , a lower value creates a sharper estimation , but may cause over - fitting and larger variability . in this study , we used a data - driven method to automatically determine the bandwidth . we used equation 1 to estimate the ed1 stain distribution images and equation 2 to estimate the spatial distribution of the stain feature . the source codes of our whole slide recognition program ( ws recognizer ) are publicly available at http://ws-recognizer.labsolver.org . the validation was conducted using a total of 4 rats from the acute cardiac allograft rejection study in our laboratory . for each rat , one tissue section was obtained from the mid portion of the excised heart . for each slide , 16 pathology images were acquired on a transmitted light microscope ( olympus proves ax 70 ) at 40 objective lens . the field of view covered different areas in the slide to acquire images with different macrophage density . in each pathology image , manual counting and automatic stain recognition were conducted separately to avoid a spurious correlation between the manual counting and the automated recognition . manual counting was conducted without knowing the stain recognition results and the training data for automatic recognition were selected without knowledge of the manual - counting results . the final comparison was conducted by performing regression analysis to calculate the correlation coefficient . to validate our method on wsi data , we compared the ed1 stain distribution image with both in - vivo and ex - vivo cellular mr images . to facilitate a visual comparison , the orientations of the mr images were manually rotated to match those of the pathology images . the hypointensity patches shown in the t2 * -weighted images were due to the accumulation of the iron - oxide labeled macrophages , a feature that allowed us to examine whether our ed1 stain distribution image was in good agreement with the distribution shown on cellular mri . the rat cardiac allograft rejection model , as described in a study , was established using dark agouti and brown norway rats ( harlan , indianapolis , in ) as the donor - recipient transplantation pairs . the superior vena cava of the graft heart was anastomosed to the recipient inferior vena cava ( ivc ) and the aorta of the graft heart was anastomosed to the recipient abdominal aorta , with the recipient proximal ivc partially obstructed to increase the pre - load . the graft , with intact pulmonary circulation , received proper pressure and volume loading without detectable atrophy over time . on day 7 after the transplantation , the graft hearts were scanned using in - vivo cellular mri and then harvested for pathology inspections . a suture was tied around the left anterior descending artery for 45 min and then was cut to allow reperfusion . after 2 days , the heart was harvested and scanned by ex - vivo mrm . cellular mri experiments were conducted by using dextran - coated uspio particles or polystyrene - coated mpio particles ( bangs laboratories , fishers , in ) as an mri contrast agent to image macrophage distribution . the iron - oxide particles are taken up by macrophages in circulation and create signal - voids in t2 * -weighted images to reveal the macrophage infiltration in - vivo , offering a unique way to correlated the ihc stain distribution obtained from our method . in our study , the iron - oxide particles were given to the animals before surgery . the rats , undergoing heterotopic heart transplantation , received uspio particles and were scanned by in - vivo cellular mri . the in - vivo mri scan was conducted on a bruker 7-t scanner ( bruker , billerica ma ) . the rats were intubated and ventilated during the mri scan and electrocardiogram ( ecg ) leads were placed on the abdomen using a gating and monitoring system ( sa instruments , stony brook , ny ) . t2 * -weighted magnetic resonance ( mr ) images , an imaging modality known to be sensitive to the signal void artifact created by iron - oxide particles , were acquired using a flash sequence with ecg and respiratory gating . tr = the respiration cycle ( ~1 s ) , te = 5 ms , field of view ( fov ) = 4 cm , slice thickness = 1.5 mm , in - plane resolution = 156 m . the ex - vivo mri scans were conducted using a bruker 11.7-tesla scanner ( bruker , billerica ma ) . t2 * -weighted mr images were also acquired using a flash sequence with te = 5.8 ms , fov = 1.2 cm , and isotropic resolution = 23 m . the hearts were fixed in 4% paraformaldehyde for 1 - 2 weeks and embedded in paraffin . # dc2002 ) , which streamed slides in preheated high ph target retrieval solution ( dako # s3308 , carpinteria , ca , usa ) for 15 min . this was followed by cooling of the slides at room temperature for 20 min and rinsing the slides in distilled water before commencing the staining steps . to stain the slides , the tissue sections were incubated with 10% horse serum for 10 min at room temperature to block non - specific binding . the slides were then incubated with 1:200 anti - rat ed1 monoclonal purified immunoglobulin g ( abd serotec , oxford , uk ) for 2 h. after pbs wash , the slides were incubated with biotinylated secondary antibodies ( lsab kit , dako , carpinteria , ca , usa ) for 30 min and labeled with the streptavidin - horseradish peroxidase ( dako , carpinteria , ca , usa ) for 30 min . then the slides were stained with aminoethyl carbazole chromogen ( skytek laboratories , inc . , a rat spleen tissue section was used as a positive control section , whereas a normal rat cardiac tissue was used as a negative control section . the stained slides were scanned on a whole slide scanner ( nanozoomer 2.0-ht , hamamatsu , japan ) to acquire wsi at 20 magnification . the resulting pathology images were then processed by our whole slide pattern recognition method , as detailed in the following section . the whole slide image was tessellated to enable the whole slide pattern recognition , as shown in figure 1 . the tessellation lines divide the whole slide image into several n - by - n sized blocks and the choice of n depends on the application scenario and the applied recognition algorithm . a higher value offers a larger field of view , but it may exceed the maximum allowable image size for the algorithm . after tessellation , each block was padded with an additional margin , m - pixel wide , to the neighboring block . the padding setting was devised for accurate accounting of stains near the block boundary , since a strict boundary could bisect a stain , incorrectly assigning it to both neighboring blocks . the choice of m considers the span of individual stain and requires prior information about their morphology . an m - pixel - wide margin allows stains with a maximum span of m to be fully recognized within the tessellated image . in our study , we used n = 1000 and the value of m was set to 100 . the tessellation scheme for the whole slide pattern recognition . the whole slide image is tessellated into n - by - n sized blocks to enable parallel recognition . each block is patched with additional m - width margin to consider stains bisected by the tessellation boundary the center of each recognized stain can be used to determine whether it should be counted or ignored to avoid double counting of stains , as illustrated by the example scenario in figure 2 . one should note that the tessellation borders are present at all sides of the tessellated image and figure 2 shows only the border between two adjacent images blocks to simplify the illustration . as shown in figure 2a , our stain recognition algorithm is applied to the image block to the left and the recognized stains with center points inside the tessellation line ( annotated by yellow ) are counted , whereas those outside the line ( annotated by red ) are ignored . similarly , in figure 2b , our stain recognition algorithm is applied to the image block to the right . the recognized stains with center points inside the tessellation line ( annotated by yellow ) are counted , whereas the rest are ignored ( annotated by red ) . ( a ) the stain recognition is applied to the image block to the left to recognize the stains . the center locations of the stains ( marked by dot points ) are used to determine whether a recognized stain should be counted ( annotated by yellow ) or ignored ( annotated by red ) . ( b ) the stain recognition is applied to the image block to the right , showing how double counting is avoided . the counted stains ( annotated by yellow ) have center points located inside the tessellation line the flow chart of our stain recognition algorithm is shown in figure 3 . the method is composed of three modules , including a classification module that performs pixel - wise color classification , a morphology smoothing module that eliminates the fragments considered to be false positives and an analysis module that identifies each isolated stain region . it is noteworthy that a variety of stain recognition algorithms can be applied to this problem , and in this study , we have used the simplest pixel - wise color recognition approach for its low computation time and stable performance . the red - green - blue color channels were used as the classification features and the output ( classification result ) was either 0 ( background ) or 1 ( foreground ) . the stain classification was conducted using nearest neighbor method , a popular method in data mining . nearest neighbor method required a training dataset of background and foreground stain image . in this study , we manually cropped the background and foreground stain from the wsi data as the training dataset . the nearest neighbor method classified each image pixel based on its color difference with the pixels in the training images . if the minimum difference was from the foreground stain , then the pixel was classified as foreground ; otherwise , it was classified as background . this classification was applied to each pixel in the input image [ figure 3b ] to obtain the binary recognition output [ figure 3c ] . this output image might have small fragments due to error classification or the partial slice coverage of the stained cells . to eliminate fragments in the binary images and to facilitate analysis , morphological opening and closing were applied 3 times using a 3-by-3 square structuring element to smooth the contours of the recognized stains , as shown in figure 3d . finally , the connected component analysis identified each isolated stain region in the image and the center location of each stain was calculated for further density estimation . the pixel - wise classification is trained by a foreground stain image and also a background stain image ( a ) . each pixel of the input image ( b ) is then classified using nearest neighbor classification to obtain a binary image ( c ) . the small fragments in the binary image are then eliminated by morphological closing and opening , resulting in a smoother output ( d ) . this image is then analyzed by connected component analysis to obtain the center locations of the recognized stains since the locations of the recognized stains were discrete points scattered across the wsi , a regression analysis method was used to calculate their density distribution . this was carried out by the kernel density estimator as shown in figure 4 , where figure 4a shows an exemplary result of the discrete stain locations and figure 4b shows their density distribution estimated by the kernel density estimator . we had a total of k stains obtained from our whole slide pattern recognition and each of them was located at coordinate xi . the kernel density estimator estimated the density distribution function by using the following formulae : the exemplary analysis result of the kernel density estimator . ( b ) their density distribution can be estimated by the kernel density estimator where h is the kernel bandwidth and is the kernel function . the scalar feature ( e.g. diameter or area ) of the k recognized stains can also be estimated using kernel regression . where yi is a scalar feature of the i stain . equation 2 can be used to estimate the spatial distribution of the feature across the entire pathology slide . a higher value of the bandwidth generates a smoothing effect , leading to reduced variability . on the contrary , a lower value creates a sharper estimation , but may cause over - fitting and larger variability . in this study , we used a data - driven method to automatically determine the bandwidth . we used equation 1 to estimate the ed1 stain distribution images and equation 2 to estimate the spatial distribution of the stain feature . the source codes of our whole slide recognition program ( ws recognizer ) are publicly available at http://ws-recognizer.labsolver.org . the validation was conducted using a total of 4 rats from the acute cardiac allograft rejection study in our laboratory . for each rat , one tissue section was obtained from the mid portion of the excised heart . for each slide , 16 pathology images were acquired on a transmitted light microscope ( olympus proves ax 70 ) at 40 objective lens . the field of view covered different areas in the slide to acquire images with different macrophage density . in each pathology image , manual counting and automatic stain recognition were conducted separately to avoid a spurious correlation between the manual counting and the automated recognition . manual counting was conducted without knowing the stain recognition results and the training data for automatic recognition were selected without knowledge of the manual - counting results . to validate our method on wsi data , we compared the ed1 stain distribution image with both in - vivo and ex - vivo cellular mr images . to facilitate a visual comparison , the orientations of the mr images were manually rotated to match those of the pathology images . the hypointensity patches shown in the t2 * -weighted images were due to the accumulation of the iron - oxide labeled macrophages , a feature that allowed us to examine whether our ed1 stain distribution image was in good agreement with the distribution shown on cellular mri . the sample result of the stain recognition algorithm is shown in figure 5a , where the recognized stains are annotated by the yellow contour . the training images are presented in figure 5b and c , which are the background and foreground ihc stain images , respectively . the image shows the infiltration of ed1 macrophages due to acute cardiac allograft transplant rejection and the brownish ihc stains were identified by our recognition method . the total numbers of macrophages were counted by summing up the number of annotated regions in the image and the center point of each annotated region can be calculated to facilitate further stain density estimation . the result of our stain recognition and the training images used in the stain classification . ( a ) the stain recognition is applied to a pathology image of myocardium under acute cardiac allograft rejection , where macrophage accumulation revealed by immunohistochemistry ( ihc ) stains can be recognized by our algorithm . the stain recognition algorithm is trained by using one background image ( b ) and one foreground ihc stain image ( c ) and the recognized stains are annotated by yellow to illustrate the performance of our algorithm the result of the manual validation is shown in figure 6 , where the numbers of ihc stains counted by our automated stain recognition algorithm were correlated with manual counting . the analysis shows an r = 0.8029 and a correlation coefficient of 0.8961 between the number of cells counted manually and that recognized by our algorithm , suggesting that the results obtained from our automatic recognition algorithm was in good agreement with the manual counting results . the narrow bank of the confidence interval further suggests that the high correlation result is highly reproducible . one may note that the coefficient of the regression line was 0.445 , meaning that the automatic recognition algorithm tends to count twice as many as that by the manual counting . the systematic overestimation of the algorithm could be due to the fact that the algorithm counted the ihc stains regardless of their size , whereas manual counting may have ignored small stains . a feasible approach to improve consistency is adding a size filter to the stain recognition algorithm . nonetheless , this discrepancy does not invalidate our stain distribution images , since the high correlation coefficient ensured that our recognition algorithm revealed the same distribution pattern and only differed by a scaling factor . the regression analysis shows the correlation between the number of stain recognized by our stain recognition algorithm and that by the manual counting . the analysis has included 64 observations and the regression line is presented between the 95% confidence band . the regression analysis shows an r = 0.8029 and a correlation coefficient of 0.8961 , suggesting a high correlation between manual counting and our stain recognition algorithm the in - vivo cellular mri is shown in figure 7 , where the t2 * -weighted image ( left ) was compared with the ed1 stain distribution image ( right ) . the animal from our rat heart transplantation model developed acute cardiac rejection and uspio was used as a macrophage - labeling agent in the cellular mri . as shown in figure 7a , the signal - void artifact in the t2 * -weighted mr image , as illustrated by the arrow , suggested the accumulation of uspio - labeled macrophages in the endocardium of the left ventricle . in comparison , our ed1 stain distribution image shown in figure 7b illustrated a similar pattern of accumulation of ed1 macrophages in the endocardium . one may note that the t2 * -weighted image does not match perfectly with the ed1 stain distribution image . this may be due to the fact that there could be tissue distortion during the fixation procedure and that the images may not align perfectly with the pathology slides . despite this difference , the highly similar pattern suggested that our method can be used to reveal ed1 macrophages within pathology slides . the in - vivo cellular magnetic resonance ( mr ) images correlates with the ed1 stain distribution image . ( a ) the in - vivo t2 * -weighted cellular mr image , where the macrophages labeled with ultrasmall superparamagnetic iron - oxide particles create hypo - intensity patches ( annotated by arrows ) in the myocardium that develops acute allograft rejection . ( b ) the corresponding ed1 stain distribution image shows a similar pattern of the macrophages accumulation in the endocardium . the hypo - intensity patches in ( a ) correspond to the hyper - intensity locations in ( b ) the ex - vivo mrm is shown in figure 8 , where the t2 * -weighted image of the ex - vivo tissue ( left ) was compared with the ed1 stain distribution image ( right ) . the rat was from our model of ischemia / reperfusion injury , with mpio particles used as the macrophage labeling agent in the cellular mrm . the tissue section is outlined ( black ) in the ed1 stain distribution image to facilitate comparison . as shown in figure 8a , the mpio - labeled macrophages generate dark patches and spots in the mrm images , suggesting massive macrophage infiltration in the myocardium with ischemia / reperfusion injury . similarly , our ed1 stain distribution image shown in figure 8b showed the same pattern of ed1 macrophage infiltration in the left ventricle . the high similarity of both imaging approaches confirmed that our method can reliably identify ed1 macrophages within pathology slides . the ex - vivo cellular magnetic resonance microscopy ( mrm ) correlates with the ed1 stain distribution image . ( a ) the ex - vivo t2 * -weighted cellular mrm , where the macrophages labeled with micron - sized iron - oxide particles create hypo - intensity patches in the myocardium that has ischemia / reperfusion injury ( b ) the corresponding ed1 stain distribution image shows highly similar pattern of macrophages accumulation in the myocardium , suggesting that the ed1 stain distribution image can reveal the macrophage infiltration across an entire pathology slide figure 9a shows the spatial distribution of the cell nucleus , lipofusin and red blood cell in 3 rats with heart ischemia / reperfusion injury . the stain recognition algorithm was trained to recognize cell nuclei ( n ) , lipofusin ( l ) and red blood cells ( r ) under h and e stain , as shown in figure 9b . the distribution of cell nuclei can be used to investigate the increase of immune cells during the inflammation process , whereas the distribution of lipofusin may be used to investigate oxidative stress in the myocardium . both three distribution images offer paranomic pathology information of the ischemia / reperfusion injury across tissue sections . the spatial distribution of cell nucleus , lipofusin and red blood cell in rat hearts with ischemia / reperfusion injury . ( a ) the spatial distribution of cell nucleus , lipofusin and red blood cell in 3 rats . ( b ) the stain recognition algorithm was trained to cell nuclei ( n ) , lipofusin ( l ) and red blood cells ( r ) under h and e stain . these three distribution images offer paranomic pathology information that facilitates quantitative characterization in histopathology figure 10 is an example of feature distribution image showing the spatial distribution of the cell nuclear diameter . the diameter of the recognized cell nuclei was estimated using the kernel regression formulated in equation 2 . the feature distribution image can be used to provide a quantitative evaluation of a particular feature and to assist tissue characterization in histopathology . a feature distribution image showing the spatial distribution of the cell nuclear diameter . the cell nuclei were recognized under h and e stain and the spatial distribution of their diameter can be estimated using kernel regression . in this paper , we report on an automated method that conducts whole slide pattern recognition to obtain the stain distribution . regression analysis showed a correlation coefficient of 0.8961 between manual counting and our stain recognition algorithm , suggesting that the labor intensive manual counting may be replaced by our automated recognition algorithm , which is critical for further analysis of the image data from wsi . moreover , the ed1 stain distribution image was in good agreement with both in - vivo cellular mri and ex - vivo cellular mrm . the high density areas revealed in the ed1 stain distribution images correlated well with the hypo - intensity areas generated by iron - oxide - labeled macrophages , suggesting that our method can serve as a surrogate to reveal the distribution of macrophages across tissue sections . the novelty of our approach includes the following : first , we have implemented a parallel processing approach that makes whole slide pattern recognition possible . it is possible that the same processing approach can be combined with more advanced pattern recognition algorithms to extract meaningful features from pathology / ihc digital slides . second , the stain distribution images obtained from our method offered a panorama of pathology information instead of a fraction of a view under the light microscope . this panoramic view is less susceptible to errors caused by subjective selection of the data . in contrast , traditional pathology validation often requires expert selection of the lesion site , making the process subjective and potentially biased . whole slide pattern recognition leaves no bias and presents all the information across the pathology slide to offer more objective results . third , the stain distribution images are quantitative information that can be further analyzed statistically . this unique feature is provided by the kernel density estimator , which is a widely - used regression approach in the field of statistics and has been used in a variety of real world applications . lastly , our method can be fully automated and no human intervention is needed to obtain the stain distribution mapping . this offers a highly objective and efficient method that may enable a high throughput processing of data and massive screening . despite the many advantages mentioned above , there are several possible pitfalls in our method that are noteworthy of discussion . the accuracy of our method depends on the performance of the recognition algorithm and it is possible that an algorithm may give false positive results secondary to poor slide preparation or artifacts in the preparation . similarly , the stain variability may alter the recognition results and may create a false perception of the changes in the stain distribution . both pitfalls may lead to misleading results that are hard to identify and thus , a post - processing inspection may be needed for quality control . first , we did only a qualitative comparison and did not conduct rigorous registration to compare our stain distribution imaging with mri . this limitation is due to the fact that the tissue specimen deforms substantially in the fixation process and the spatial information between mri and wsi can not be perfectly aligned . furthermore , the ex - vivo mri of the tissue - embedded paraffin block can not be acquired because the mr spectrum is dominated by the paraffin resonance . another limitation of our experiment is that we did not examine the positive and negative predicting values of our stain recognition algorithm . although the accuracy of the morphology - based stain recognition has already been examined , it is unclear whether the same performance can be achieved in counting macrophages due to their highly pleomorphic shapes . the current setting is optimized to identify stains with an oval shape , but there can be other morphological presentations in the pathology slides . to overcome this limitation , the algorithm can be customized to accommodate different morphology of the stains . moreover , although there is a positive correlation between the number of the recognized stains and manual cell counts , our stain recognition algorithm still tends to count more than that of manual counting . this may be due to the fact that macrophages have cytoplasmic projections that can lead to separated regions and result in duplicated counts . in addition , the nuclei of macrophages are often lobulated , further adding the complexity of the cell recognition in our use case scenario . counting last , the stain distribution value can be affected by several factors , including the thickness of the sections , the z - depth and the bandwidth used in the kernel density estimator . therefore , a comparison based on the stain - distribution value should be conducted in a carefully controlled manner . the obtained stain distribution image represents a panorama of the pathology information and can be used in both qualitative and quantitative analyses . this imaging approach may also assist pathologists in clinical diagnosis by offering them a toolkit for multiple applications in diagnostic pathology as well as yield newer methods to use image analysis to quantitate and analyze biomarkers , which are both diagnostic and prognostic . in addition , the algorithm described in this study may serve as a new and systematic approach to validate other imaging modalities . the ed1 stain distribution image confirms that the cellular mri technique is able to detect the macrophage infiltration of cardiac transplantation rejection or cardiac ischemic injury in our rat models .

background : whole slide imaging ( wsi ) offers a novel approach to digitize and review pathology slides , but the voluminous data generated by this technology demand new computational methods for image analysis.materials and methods : in this study , we report a method that recognizes stains in wsi data and uses kernel density estimator to calculate the stain density across the digitized pathology slides . the validation study was conducted using a rat model of acute cardiac allograft rejection and another rat model of heart ischemia / reperfusion injury . immunohistochemistry ( ihc ) was conducted to label ed1 + macrophages in the tissue sections and the stained slides were digitized by a whole slide scanner . the whole slide images were tessellated to enable parallel processing . pixel - wise stain classification was conducted to classify the ihc stains from those of the background and the density distribution of the identified ihc stains was then calculated by the kernel density estimator.results:the regression analysis showed a correlation coefficient of 0.8961 between the number of ihc stains counted by our stain recognition algorithm and that by the manual counting , suggesting that our stain recognition algorithm was in good agreement with the manual counting . the density distribution of the ihc stains showed a consistent pattern with those of the cellular magnetic resonance ( mr ) images that detected macrophages labeled by ultrasmall superparamagnetic iron - oxide or micron - sized iron - oxide particles.conclusions:our method provides a new imaging modality to facilitate clinical diagnosis . it also provides a way to validate / correlate cellular mri data used for tracking immune - cell infiltration in cardiac transplant rejection and cardiac ischemic injury .

survival rates continue to improve with the advent of more refined treatments and better supportive care . today over 80% of children with cancer are alive for at least five years , and the majority of these are cured . as a consequence , many sufferers of childhood cancer are now living into adulthood and having families of their own . it has been estimated that about 1 in 900 adults aged 18 to 44 years is a cancer survivor . among this group , those with an inherited susceptibility to cancer will transmit their genetic fault to a proportion of their children . they are also at risk of developing a second cancer during their adult life . while the causes of the majority of childhood cancers are largely unknown , there are a number of clinical syndromes for which the evidence for an excess cancer risk in children is most persuasive . these include li - fraumeni syndrome , neurofibromatosis type i , inherited retinoblastoma mutations , familial wilms tumor , and some disorders of dna repair . these syndromes collectively account for less than 10% of all childhood cancers , however . beyond this , it is not clear if cancer in childhood increases the risk of cancer in relatives . a number of recent studies have described an association between childhood cancer and cancer in first - degree relatives [ 57 ] , particularly siblings [ 5 , 7 ] and mothers . in this study , we describe the distribution of cancers in australian children and estimate the risks of cancer in their relatives . it was established from the clinical services at the royal children 's hospital ( rch ) and the monash medical centre ( mmc ) , between which over 95% of children under 10 years of age and 83% of children aged 1015 years with cancer within the state of victoria , australia , are treated . children were eligible for inclusion if they had a diagnosis of any cancer before age 15 and when initial treatment was given at one of the participating centres between october 1998 and october 2002 . the human research ethics committee at each participating institution reviewed and approved the protocol before enrolment commenced . a total of 486 ( 422 at rch and 64 at mmc ) incident cases of childhood cancer were diagnosed during the study period . of these , 21 families were found to be ineligible ( seven non - english speaking parents , seven where the proband was aged over 15 years , six where the proband did not have their initial treatment at either service , and one where the proband was adopted ) . of the remaining 465 families , 21 were not contactable , 53 refused to participate , 6 were lost to followup , and 6 later withdrew consent . this left 379 ( 82% ) families participating ( 340 at rch and 39 at mmc ) , from which we interviewed 351 ( 92.6% ) fathers and 371 ( 97.9% ) mothers . in 343 ( 90.5% ) families , both the mother and father were interviewed , while in 28 ( 7.4% ) families only the mother was interviewed and in 8 ( 2.1% ) families only the father was interviewed . at the time of enrolment , both parents of the affected child were asked a baseline questionnaire that included information on lifestyle and environmental risk factors and a detailed family history of cancer . where half siblings were identified , the appropriate , biologically relevant family history was obtained . the family history of cancer questionnaire asked about all of the affected child 's siblings , parents , aunts , uncles , and grandparents ( i.e. , all first - degree and second - degree relatives ) . parents were asked to identify each first - degree and second - degree relative , any known cancer diagnoses with the age ( in years ) at diagnosis , and type of malignancy , together with their current age or age at the time of death . the information collected was reviewed and classified by the authors with expertise in cancer ( jah ; es ) . the world health organization 's international classification of diseases for oncology , 3rd edition ( id - o-3 ) scheme , was used for classification of site of malignancy . nonmelanoma skin cancers , nonmalignant tumours , and in situ cancerswere not included in the analyses . to address the potential contribution of known familial cancer syndromes , we examined the medical records of the 26 affected children who had a first - degree relative with cancer . in particular , we sought clinical evidence for li - fraumeni syndrome , neurofibromatosis type 1 , familial retinoblastoma , and familial wilm 's tumour syndrome . person - years at risk for the cohort of relatives were calculated as the time to date of diagnosis of cancer , date of death , or date of interview completion , whichever occurred earliest . australian population - based cancer incidence data , specific for gender , age , and year of birth , were obtained from the australian institute of health and welfare . standardised incidence ratios were used to compare the number of observed cancers in relatives with the number expected in the australian population . robust estimates for confidence intervals were calculated to account for potential clustering within a family . all statistical tests were two - sided and a p value < 0.05 was considered statistically significant . the distribution of childhood cancers diagnosed in the cohort of 379 children is summarized in table 1 . a family history for cancer was identified in 4,736 ( 1,337 first - degree and 3,399 second - degree ) relatives with a total of 211,394 person - years of followup . fourteen of the 379 ( 3.7% ) families reported a positive history for childhood cancer in any relative , with none having more than one case identified . twenty - six children with cancer ( 6.9% ) had a first - degree relative ( parent or sibling ) with a history of cancer . the results for the family history of cancer in all childhood cancer patients are summarized in table 2 . there was a higher than expected , though not statistically significant , incidence of childhood cancer among first - degree relatives ( sir 1.43 ; 95% ci 0.545.08 ) . there was also a higher than expected , though not statistically significant , incidence of cancer among first - degree relatives ( sir 1.45 ; 95% ci 0.932.1 ) . there was a statistically significant increase in cancer among female first - degree relatives ( sir 1.82 ; 95% ci 1.263.39 ) . the increased family cancer history in first - degree females was largely attributable to an effect in mothers ( sir 1.78 ; 95% ci 1.273.33 ) but was also observed in sisters ( sir 2.15 ; n = 1 ) . elevated cancer incidence rates were also observed in aunts ( sir 1.48 ; 95% ci 1.112.00 ) . the gender - specific association was reflected in higher than expected incidence rates of breast cancer in mothers ( sir 1.92 ; 95% ci 0.726.83 ) , aunts ( sir 1.64 ; 95% ci 0.982.94 ) and to a lesser extent grandmothers ( sir 1.04 ; 95% ci 0.781.40 ) . although numbers were small , an increased cancer incidence in relatives appeared to be most apparent in the children with sarcomas ( sir 2.58 ; 95% ci 0.998.52 ) , embryonal tumors ( sir 2.12 ; 95% ci 0.719.34 ) , and brain tumors ( sir 1.52 ; 95% ci 0.703.92 ) . of the 26 families with a history of cancer within a first - degree relative , only one met the criteria for a clinically recognizable familial cancer syndrome ( li fraumeni syndrome ) . the increased rates of cancer in female first - degree relatives ( sir 1.68 ; 95% ci 1.032.93 ) , including mothers ( sir 1.78 ; 95% ci 1.093.09 ) and aunts ( sir 1.49 ; 95% ci 1.122.02 ) , remained when this child and family were removed from the analyses . our small , population - based study suggests an increased risk of childhood cancer and some adult cancers among relatives of childhood cancer patients that are not accounted for by clinically identifiable familial cancer syndromes . these findings were seen in both first- and second - degree relatives . although some of our findings did not reach statistical significance , they are largely consistent with previously published unselected childhood cancer cohorts [ 57 , 12 ] and add to a growing body of evidence that unidentified genetic risk exists in some families . in addition to the small size of the study , other limitations include the use of family history in isolation and the absence of some parental input to family history of cancer . while we were not able to cross - reference reported history of cancer with cancer registry data , a similar study in sweden confirmed underreporting of cancer in relatives by history . furthermore , in the small minority of cases where both parents were not available to provide the relevant family history of cancer , underreporting of the family history of cancer is also likely . allowing for these two study biases towards the null hypothesis is therefore likely to strengthen the positive findings we have reported . this underreporting may also explain why a large number of apparent protective effects occurred in second - degree relatives , where direct , personal access to a family history of cancer is not always possible . overall , first - degree relatives ( siblings and parents ) had an increased incidence of childhood tumors . this is consistent with the higher occurrence of childhood cancers among siblings demonstrated in a study of 51,000 children who developed cancer under the age of 15 in the uk , even after those with a genetic etiology were excluded . it was also reported in a cohort of 13,703 childhood cancer survivors in north america ( sir 1.5 ; 95% ci 1.351.7 ) . the apparent association of childhood cancer with adult cancers in female relatives is fascinating and supports a recently published population - based study from utah , usa , where a higher risk of adult cancer was restricted to mothers and siblings ( sir 1.31 ; 95% ci 1.111.56 ) but was not observed in fathers . it is interesting to note that the effect was greatest for children younger than 5 years at diagnosis ( sir 1.48 ; 95% ci 1.131.95 ) , suggesting a strong ante- or perinatal effect . factors which may be related to maternal effects on childhood cancer risk include maternal age of pregnancies , altered in utero exposures , and birth weight . our finding of an increased prevalence of breast cancer in female relatives also supports recently published swedish data . a number of related studies have also observed higher rates of breast cancer in mothers and sisters [ 1416 ] . the potential link between breast cancer and childhood cancer is of great interest , given past reports of increased rates of childhood cancer in families carrying a brca1 or brca2 mutation [ 17 , 18 ] . our study was not designed nor powered to examine the relationship between specific childhood cancer types and family history of cancer , so it must be acknowledged that potential associations may have gone undetected . certainly , others have postulated a link between family history of cancer and the more common subtypes , childhood leukaemia , childhood lymphomas , and childhood brain tumours . the epidemiology of childhood cancer described here is the starting point for further explorations into identifying previously unrecognized genetic predisposition to childhood cancer . we are currently undertaking whole genome sequencing of these children with cancer and their affected first - degree relatives ' germline dna in order to identify the role of previously described and novel genetic mutations to account for our findings .

we determined the extent and distribution of cancers in relatives of 379 children newly diagnosed with cancer . family history was collected from 1,337 first - degree and 3,399 second - degree relatives and incidence compared with national age- and gender - specific rates . overall , 14 children ( 3.7% ) had a relative with a history of childhood cancer and 26 children ( 6.9% ) had a first - degree relative with a history of cancer , with only one of these having an identifiable familial cancer syndrome . there was a higher than expected incidence of childhood cancer among first - degree relatives ( parents and siblings ) ( standardized incidence ratio ( sir ) 1.43 ; 95% ci 0.545.08 ) . there was also a higher than expected incidence of adult cancers among first - degree relatives ( sir 1.45 ; 95% ci 0.932.21 ) , particularly in females ( sir 1.82 ; 95% ci 1.263.39 ) . the increased family cancer history in first - degree females was largely attributable to an effect in mothers ( sir 1.78 ; 95% ci 1.273.33 ) . the gender - specific association was reflected in higher than expected incidence rates of breast cancer in both mothers ( sir 1.92 ; 95% ci 0.726.83 ) and aunts ( sir 1.64 ; 95% ci 0.982.94 ) . these findings support the hypothesis that previously undetected familial cancer syndromes contribute to childhood cancer .

this special issue of fungal genetics and biology describes a comprehensive effort to develop methods and expertise to tackle one of the world s most serious fungal diseases of wheat . the series of papers within this issue are designed to provide a resource for a new generation of plant pathologists who we hope will be inspired to investigate the biology of septoria blotch of wheat and develop new and durable disease control strategies . wheat is the world s most widely cultivated crop and is responsible for providing about a quarter of the calories to humankind . global wheat production was more than 700 million tons in 2013 , with the european union and china producing the largest harvests , and wheat being cultivated across most of the temperate regions of the planet ( gurr and fones , 2015 ; rudd et al . , 2015 ) . as well as being the most widely grown crop , wheat is also the most traded food on the international markets indeed more wheat is traded than all other crops combined . along with rice and maize , there are many threats to wheat productivity , including the availability of high quality land for cultivation , which is threatened by urbanisation , the sustainable use of fertilisers , which are used in enormous quantities , especially in europe , and the prevalence of diseases . wheat diseases , such as septoria blotch , provide an ever - present threat to wheat production and if they could be controlled effectively , would provide a much - needed boost to productivity that will be required to satisfy increasing global demand for food in the next two decades ( courbot et al . , 2015 ; what then are the essential pre - requisites to allow us rapidly to understand the biology of zymoseptoria tritici ? what requirements are necessary to allow a fungal pathogen to be regarded as a model system ( perez - nadales et al . , 2014 ) and for new insights into the underlying mechanisms of infection and virulence to be understood ? the last 20 years have seen pathogens , such as the corn smut fungus ustilago maydis and the rice blast fungus magnaporthe oryzae , for example , emerge as model systems ( dean et al . , 2012 ) . in both cases , this required the development of many tools , which took many years . it is by considering these issues that this collection of reviews and primary publications has been put together . arguably , the first pre - requisite to being able to investigate any disease is a comprehensive description of the life cycle of the organism and the spatial and temporal dynamics of the infection process . for z. tritici this means a cell biological investigation of what happens from the moment a spore germinates on the leaf surface , to its location of a stoma and invasion of underlying leaf tissue . using live cell imaging approaches , facilitated by the tools generated and described elsewhere in this special issue , the cell biology of infection is addressed , with an emphasis on the developmental biology of the fungus , its dimorphic growth habit , its ability to perceive and respond to the leaf surface , and its ability to undertake developmental transitions during its establishment of a wheat infection ( steinberg , 2015 ) . without a road - map of how an infection proceeds , it is difficult to identify points of disease intervention or to understand how any single gene product , or family of proteins ( from the pathogen or its host ) , might be important in disease . the next essential pre - requisite for the model pathogen tool - kit is the ability to test the function of any gene . without an ability to construct mutants , it is hard to carry out any reverse genetic approach to test the importance of any selected biological process . therefore , a high frequency transformation system is essential , with a ready supply of selectable marker genes to allow multiple constructs to be expressed , and the ability to carry out targeted mutations ( kilaru and steinberg , in 2015 ; kilaru et al . , 2015a ; schuster et al . , the latter can be dramatically improved by development of a strain of z. tritici in which the non - homologous dna end - joining pathway is impaired ( sidhu et al . coupled to this , it would be advantageous to have a means by which the function of a gene could be attenuated but without complete loss of function , such as virus - induced gene silencing , and the ability to regulate genes driven by a set of highly controllable promoters by which gene expression can be predictably controlled ( kilaru et al . the latter will also provide the means to over - express , or mis - time the expression of a given gene to tests its function , or requirement ( cairns et al . , 2015 ) . next , a means by which gene products can be localised using expression of fluorescently - labelled fusion proteins is essential , preferably with optimised fluorescent markers , calibrated and tested for the pathogen and in amenable vectors , with easy cloning strategies for ready construction ( kilaru et al . , 2015c ; mehrabi et al . , 2015 ; schuster et al . , 2015a ; sidhu et al . this provides the means to carry out live cell imaging of a pathogen undergoing infection and directly observing the position , fate and turnover of a gene product , which is absolutely pivotal to understanding its function . but this will make no sense without some context , so having the ability to unequivocally identify organelles , such as nuclei , er , golgi bodies , peroxisomes , and mitochondria is essential ( schuster et al . , 2015b ; kilaru et al . , 2015b ; guo et al . , 2015a , b ) . also , to understand intracellular trafficking it is necessary to be able to identify and track the movement of endosomes , secretory vesicles and the associated components of the actin and microtubule cytoskeleton and their corresponding motor proteins , so that the transport of proteins and the regulation of such processes can be studied ( guo et al . , 2015a , b ; kilaru et al . , 2015c ; schuster et al . when considered together , these tools will therefore allow rapid elucidation of gene function in z. tritici , as rapidly as in any fungal pathogen studied today . so far , of course , we have only considered addressing the functions of individual genes as opposed to gene families and the function of the whole genome and the corresponding , context - dependent proteomes that are expressed during pathogenesis . to address these requires first of all a detailed understanding of the genome ( goodwin et al . , 2011 ; testa et al . , 2015 ) and its inherent , strain - to - strain variation , and how this correlated with pathotype . understanding the role , for example , of accessory , or supernumerary chromosomes , as opposed to the core , invariant gene repertoire of the genome will be key ( mcdonald et al . , 2015 ) . an ability to analyse global patterns of gene expression , proteomic and metabolomics data sets during infection , will also be necessary so that the total expressed proteome can be defined and sub - sets associated with the effector repertoire of the pathogen , proteins required for symptom development and those necessary for fungal proliferation can be readily classified , define and then functionally analysed ( ben mbarek et al . methods to define transcriptional networks by chromatin immuno - precipitation coupled to next generation sequencing to define targets genes downstream of transcription factors , will also allow networks of gene expression to be distinguished readily ( soyer et al . , protein interactions between fungal proteins , but also between fungal proteins that act upon host plant proteins , is also essential , with the ready availability of yeast two - hybrid libraries and associated bespoke protocols to z. tritici ( ma et al . , 2015 ) finally , of course we need to consider the plant host and within this context , transgenic wheat lines expressing organelle - specific markers are already available and being constructed to augment host - pathogen cell biological analysis , while detailed analysis of sources of resistance can guide plant breeding strategies such that durable combinations of resistance genes can be identified and introduced into elite , high yielding commercial wheat cultivars ( brown et al . , 2015 ) . with wider knowledge of the fungal pathogen population and prevailing virulence specificities , should some the ability to breed for exclusion of the most prevalent , prevailing pathotypes of the fungus ( vallet et al . , 2015 ) , providing a direct route to disease control at least in the medium term . the new methods and resources presented in this special issue mean that the z. tritici research community now has , arguably , the same level of research tools as any fungal model system . this is remarkable , because it has happened in a very short period of time in response to an acute need , articulated by growers and the agricultural biotechnology industry . an integrated programme has been funded and undertaken to carry out this method development and to begin to apply the newly acquired expertise to understanding this disease . many research questions immediately present themselves . what is the purpose of the morphogenetic plasticity exhibited by z. tritici and how are these dimorphic transitions regulated how is long - distance control of pathogenesis - associated gene expression achieved and how does the fungus perceive and respond to the internal plant tissue environment during infection ? how is sporulation triggered and correctly regulated ? all of these questions can now be addressed and the answers provided will result not only in many of the fungicide targets of the future , but also perhaps to some longer - term strategies for broad spectrum disease control . so , to those of you reading this commentary who have never previously considered studying z. tritici , or perhaps not even considered studying a fungal pathogen before , think of the obstacles that have just been removed .

septoria blotch of wheat is one of the world s most serious plant diseases , which is difficult to control due to the absence of durable host resistance and the increasing frequency of fungicide - resistance . the ascomycete fungus that causes the disease , zymoseptoria tritici , has been very challenging to study . this special issue of fungal genetics and biology showcases an integrated approach to method development and the innovation of new molecular tools to study the biology of z. tritici . when considered together , these new methods will have a rapid and dramatic effect on our ability to combat this significant disease .

endometrial carcinoma is the most common gynecologic malignancy , with an estimated 40 100 incident cases and 7470 deaths in 2009 . the current standard of care for patients with endometrial cancer consists of surgical staging , including total hysterectomy , bilateral salpingo - oophorectomy , and pelvic and para - aortic lymph node dissection . depending on the surgical stage and histological grade , the adjuvant treatment may include radiation therapy and/or chemotherapy . in approximately 10% of patients , however , there is a relative lack of consensus regarding the most appropriate management strategy for these patients . patients with clinically occult stage ii disease usually have undergone simple hysterectomy rather than radical hysterectomy , which is then followed by adjuvant radiation therapy . patients with clinical stage ii disease may be treated with preoperative ( neoadjuvant ) radiation therapy followed by simple hysterectomy , or alternatively , primary radical hysterectomy and possible adjuvant tumor - directed radiation therapy . it is especially advocated for patients having co - morbidities that limit their candidacy for radical surgery , including obesity . in all patients with clinical stage ii disease , neoadjuvant radiotherapy can decrease the size and extent of bulky cervical disease , making it easier to obtain clear surgical margins . further , it avoids the increased morbidity associated with lymphadenectomy followed by radiation therapy , as is seen in cervical cancer . conversely , advocates of primary treatment by radical hysterectomy cite the ability to debulk large tumor prior to attempting radiation therapy , to clarify the origin of the primary tumor as cervical or endometrial , and to clarify the stage and grade of disease prior to initiating adjuvant therapy . most of the published literature on preoperative treatment is with low dose rate ( ldr ) brachytherapy [ 912 ] . due to the relative rarity of this clinical scenario , there is no published data on dose , fractionation or pathological response to high dose rate ( hdr ) intracavitary brachytherapy in the neoadjuvant setting . the goal of this study was to evaluate pathological response , tolerance , and outcome with this latter approach . twelve women clinically diagnosed with clinical stage ii endometrial carcinoma from 1999 - 2010 were treated with preoperative radiation therapy . all patients had biopsy - confirmed endometrial cancer and had pretreatment ct scan and/or mri for staging . the dose of external beam ( ebrt ) was 45 - 50.4 gy in 25 to 28 fractions followed by hdr brachytherapy . the hdr brachytherapy was done with placement of a smit sleeve and tandem and ring applicator . prior to 2004 , three patients received a dose of 4 gy in four fractions , but since that time all patients received doses between 5 - 5.5 gy in three fractions . initially , for the first six patients , the dose was prescribed to point a with orthogonal film based dosimetry . the last six patients were treated with mri or ct based planning with the clinical target volume being the entire uterus and cervix ( fig . 1 ) . demographics , tumor histology , and pathological response to neoadjuvant hdr brachytherapy prior to simple hysterectomy in twelve patients with clinical stage ii endometrial carcinoma histological and figo grading from endometrial biopsy , not surgical specimen eqd2 equivalent dose in 2-gray increments , pr partial response , cr complete response microscopic residual disease , ned no evidence of disease , awd alive with disease sagittal ( left ) and coronal ( right ) view showing clinical target volume ( red ) covered by prescription isodose line of 100% ( red ) simple hysterectomy with lymph node sampling was performed four to eight weeks after the last brachytherapy treatment . the total doses from ebrt and brachytherapy were summated and normalized to a biologically equivalent dose of two gy per fraction ( eqd2 ) using the linear quadratic model and 10 gy . all patients were followed every three months for the first two years , and every six months thereafter , with vaginal cytology at each surveillance visit . the median age was 57 ( range 41 - 76 ) with median body mass index ( bmi ) of 35 ( range 29 - 45 ) . all patients completed the planned course of preoperative radiation therapy with a median eqd2 dose of 65.0 gy ( range 56.3 - 75.4 gy ) . all patients had simple hysterectomy with lymph node sampling with uneventful recovery from the operation . complete pathological response ( pcr ) was seen in two patients ( 17% ) while five patients ( 42% ) had only microscopic residual disease confined to either the endometrium or cervical glands . two patients were found to have para - aortic disease and received adjuvant para - aortic radiation . two patients with high grade pathology ( one each with serous and clear cell histology ) also received adjuvant chemotherapy after surgery . at a median follow up of 37 months ( 1 - 91 months ) , one patient has developed recurrence at the vaginal apex six months after completing initial therapy and is alive with the disease at 91 months , while another developed a lung recurrence at 28 months . one patient has died of comorbidities at 86 months with no evidence of the disease at last follow up . two - year actuarial disease - free and cause - specific survivals were 88% and 100% , respectively ; these were calculated for only those patients with at least two years of follow - up . the goals of this neoadjuvant therapy are to shrink the primary disease , to treat pelvic nodes and parametria , and to make the patient more suitable for simple hysterectomy . there is no published experience with hdr intracavitary brachytherapy as preoperative treatment for endometrial cancer . the american brachytherapy society in its published guidelines for hdr brachytherapy for endometrial cancer have not recommended a dose or fractionation schedule for preoperative hdr brachytherapy for stage ii endometrial cancer , likely due to the lack of published data . multiple studies have shown equivalency of hdr and ldr brachytherapy for cervical cancer [ 14 , 15 ] . the advantages of hdr include short treatment time , ability for optimization , and a lack of radiation exposure to healthcare personnel . since 2004 , our institution has followed a fractionation schedule of three fractions of 5 - 5.5 gy to deliver a dose equivalent to 65 - 70 gy . the schedule has been well tolerated , has shown a good response rate , and has not revealed significant morbidity . more recently , we have integrated three - dimensional planning for hdr brachytherapy , which helps to conform better the dose to the tumor while reducing the dose to critical organs ( fig . 1 ) . the results of this small series are comparable to published studies using ldr brachytherapy . in a series of 74 patients with stage ii endometrial cancer treated at the university of kentucky ( uk ) with a combined dose of 65 gy , the complete pathological response rate was seen in 31% and the residual disease confined to endometrium in 29% [ 10 , 11 ] . in our study , which had a median eqd2 dose of 66.31 gy , pcr was seen in 22% , with superficial residual disease confined to the endometrium in 33% . in the uk series , para - aortic nodal disease was noted at the time of surgery in five patients , compared to two patients in our series . patients with cervical involvement are at increased risk of both pelvic and para - aortic nodal involvement ; since only the pelvic nodes are treated during the whole pelvic radiation therapy given preoperatively , para - aortic nodal sampling at the time of surgery should be considered . the optimal management for clinical stage ii disease that provides a better outcome , in terms of disease control and morbidity , it is difficult to study prospectively because of the comparative rarity of this clinical scenario . in a retrospective analysis of 184 consecutive patients with clinical or pathologic stage ii endometrial cancer treated with definitive intent with either preoperative or postoperative radiation therapy , only the grade and histology were found to be independent predictors of disease free survival . since a subset of these patients are not ideal candidates for radical surgery because of medical co - morbidities , advanced age , and obesity , the preoperative radiation therapy followed by simple hysterectomy may be a better option . ours is the first published series using hdr and shows the hdr fractionation schedule , as done in our series , is well tolerated and would be an option for patients treated with neoadjuvant radiation therapy .

purposewe sought to evaluate pathological response , tolerance , and outcome after preoperative ( neoadjuvant ) high dose rate brachytherapy in a small series of patients with clinical stage ii endometrial carcinoma , and to evaluate a dose and fractionation protocol for this treatment.material and methodstwelve women diagnosed with clinical stage ii endometrial carcinoma from 1999 - 2010 were treated with preoperative radiation therapy . their medical charts were retrospectively analyzed for hdr treatment regimen , pathological response , and longitudinal outcomes . radiation doses were normalized to a biologically equivalent dose of 2 gy per fraction ( eqd2).resultstwo patients had complete pathological response to neoadjuvant therapy ; five more had only microscopic residual disease at the time of surgery . at a median follow up of 37 months ( 1 - 91 months ) , one patient has developed recurrence at the vaginal apex six months after completing initial therapy , while another developed a lung recurrence at 28 months . two - year disease - free and cause - specific survivals were 88% and 100% , respectively.conclusionsour small study shows that the hdr fractionation schedule , as done in our series for preoperative radiation therapy for clinical stage ii endometrial cancer , is well tolerated and would be an option for patients treated with neoadjuvant radiation therapy .

the availability or utilization of health services does not always guarantee better health outcomes and it requires safe delivery of quality health services . unsafe care is a global challenge , which often leads to adverse events and poor treatment outcomes . the limited data from low- and middle - income countries reports wrong identification of patients , medication errors , misdiagnosis , etc . patient safety is an important element of an effective and efficient healthcare and is one of the most referred components in quality of care . though broad approaches to safer patient care are applicable for the majority of the settings , there is always need to understand the local issues to tailor - made strategies to increase patient safety . this study was conducted to understand the key challenges and to propose solutions to improve overall patient care in the hospitals in delhi . the population of delhi state was 16.8 million in 2011 with 97.5% urban population , 1483 km geographical area and population density of 11,297 ( range : 3800 to 37,346/km ) . the health services in the state are provided by a number of agencies , and the city has good health infrastructure including bed population ratio , both in public and private sectors . the study was planned as qualitative research design with some quantitative data from desk review . the desk review of the available published and unpublished information on patient safety , in general , and in gynecology and obstetrics set - ups in specific , in india since the year 2000 was conducted . the desk review was followed by in - depth interviews ( idis ) and focused group discussions ( fgds ) to triangulate the information . the head of the departments or the nodal officers from the departments / units of obstetrics and gynecology in the identified hospitals participated in the discussion . these were restricted to secondary and tertiary care facilities as gynecology and obstetrics specialty usually exist at these levels only . the participants were grouped into two sets ( hospitals with more than 500 bed capacity and those 500 or less bed capacity ) . this grouping was done to keep the fgd participants homogenous and to ensure that the representatives from the larger health facilities not trying to over - emphasize their experiences . therefore , nodal persons responsible for patient safety at gynecology and obstetrics departments from 20 of 39 hospitals run by government of delhi were included . for the literature review , it was agreed that studies prior to the year 2000 may not be representative of the ground scenario as a lot has changed in the last few years . the participants for in - depth interview were purposively selected to supplement and triangulate the information generated . the desk review and in - depth discussions were conducted by all authors during the months of january the fgds were conducted at maulana azad medical college , new delhi and were facilitated by resource persons from govind ballabh pant hospital and world health organization country office for india . the discussions were moderated using a few probe topics to ensure that relevant aspects are addressed . one of the authors ( cl ) has extensive experience in conducting idis and fgds for almost a decade . the fgds were conducted in english , were recorded and a sociogram for each fgd was prepared to check the participant interactions . the facilitators role was to ensure that discussion were free flowing , not dominated by any one individual and also to keep it on track . the findings from desk review , idis , and fgds were summarized and analyzed using health system framework . the process helped in generating list of codes , which were grouped as per health system functions and analyzed . the population of delhi state was 16.8 million in 2011 with 97.5% urban population , 1483 km geographical area and population density of 11,297 ( range : 3800 to 37,346/km ) . the health services in the state are provided by a number of agencies , and the city has good health infrastructure including bed population ratio , both in public and private sectors . the study was planned as qualitative research design with some quantitative data from desk review . the desk review of the available published and unpublished information on patient safety , in general , and in gynecology and obstetrics set - ups in specific , in india since the year 2000 was conducted . the desk review was followed by in - depth interviews ( idis ) and focused group discussions ( fgds ) to triangulate the information . the head of the departments or the nodal officers from the departments / units of obstetrics and gynecology in the identified hospitals participated in the discussion . these were restricted to secondary and tertiary care facilities as gynecology and obstetrics specialty usually exist at these levels only . the participants were grouped into two sets ( hospitals with more than 500 bed capacity and those 500 or less bed capacity ) . this grouping was done to keep the fgd participants homogenous and to ensure that the representatives from the larger health facilities not trying to over - emphasize their experiences . therefore , nodal persons responsible for patient safety at gynecology and obstetrics departments from 20 of 39 hospitals run by government of delhi were included . for the literature review , it was agreed that studies prior to the year 2000 may not be representative of the ground scenario as a lot has changed in the last few years . the participants for in - depth interview were purposively selected to supplement and triangulate the information generated . the desk review and in - depth discussions were conducted by all authors during the months of january the fgds were conducted at maulana azad medical college , new delhi and were facilitated by resource persons from govind ballabh pant hospital and world health organization country office for india . the discussions were moderated using a few probe topics to ensure that relevant aspects are addressed . one of the authors ( cl ) has extensive experience in conducting idis and fgds for almost a decade . the fgds were conducted in english , were recorded and a sociogram for each fgd was prepared to check the participant interactions . the facilitators role was to ensure that discussion were free flowing , not dominated by any one individual and also to keep it on track . the findings from desk review , idis , and fgds were summarized and analyzed using health system framework . the process helped in generating list of codes , which were grouped as per health system functions and analyzed . the available published literature on patient safety in india was found to be limited and mainly focuses on reuse of syringes , accidental needle stick injuries , and on biomedical waste disposal practices . the authors did not find any published study on patient safety from obstetrics and gynecology facilities in india since the year 2000 . a brief summary of the findings from the literature review on different aspects on patient safety in india is provided in table 1 . identified challenges and proposed solutions in patient safety in maternal healthcare idis were conducted with five senior level government officials working in the area of patient safety . in total , two fgds were also conducted , which were attended by 20 participants ( 12 participants in first and 8 participants in second fgd ) . each fgd session lasted for approximately 90 min . in both the fgds , in the beginning the participants highlighted challenges and issues , and possible solutions , which have been summarized by health system functions [ table 1 ] . in addition , the appropriate management of emergency cases , the issue of near miss cases and interchange of the newborn babies were flagged as patient safety issues . the participants felt that any consideration for improvement in the quality of health services should be linked to strengthening of health services at all levels and as part of the continuity of care . it was emphasized by a number of participants that the patient safety is a very appropriate entry point for overall improvement in quality of healthcare services . intra - hospital prescription audits and medical audits , use of checklists to monitor and maintain the equipment , standardized patient consent forms ( possibly in vernacular ) , implementation and compliance with the safe surgery checklist , and use of monitoring indicators i.e. , surgical site infections and reporting of errors / monitoring ( nonmedical incident reporting ) , development of protocols appropriate for specific health facilities , provision of benchmarks for the hospital workforce were suggested as approaches or solutions to improve patient safety . the quality in simple to understand terms is meeting the expectations of the users . quality has been defined as the degree to which health services for individuals and population increase the likelihood of desired health outcomes and are consistent with professional knowledge . in operational terms , quality in health services has six dimensions of effective , efficient , accessible , acceptable and patient - centered , equitable , and safe care . the review of the literature conducted for this study indicates that there has been insufficient research in the field of patient safety ( excluding injection safety that has relatively more data ) in india . the lack of research evidence could probably be a reason for limited attention on patient safety and quality of health services and the related issues in health facilities in india , both in public and private sector . this highlights the need for more research for evidence - informed decision making , to initiate efforts to improve the quality of healthcare and to measure the trends in patient safety in india . the frequent reports in the print and online media often highlight the gap between the needs and what is being done . these media reports have at times highlighted the unsafe care at all levels , beginning from primary to tertiary levels . these findings have been corroborated by the experience of the participants in this study as well . other corroborative evidence of low quality of health services have been analyzed and noted that in the 5 years period ( 200610 ) ; there was nearly 100% increase in institutional deliveries in india but maternal mortality ratio has come down by 50% only . the rate of reduction in maternal mortality during this period was not commensurate to the rate of increase in the institutional deliveries in india . this reflects that while the institutional deliveries or access to services might have increased quantitatively , the quality of services offered during these deliveries may not have been up to the mark to reduce maternal deaths . a proportion of these deliveries was being conducted at primary healthcare set - up level which are often reported to have limited facilities to handle complicated cases and provision for appropriate and timely referrals . though , this study had participants from secondary and tertiary care facilities primary care facilities which are often less equipped are likely to have similar challenges and should be systematically studied . a common theme across all discussions was the need for adoption of approaches such as prescription audits , random checking of case sheets , cases record audits , the audits at pharmacy levels and the formulation , adoption of standard treatment guidelines ( stgs ) , and drug dosages charts . there are emerging evidence from existing literature in different parts of the world that audits in hospital and community set - ups and use of stgs improve patient safety and treatment outcomes . the findings that the delayed referrals often are more prone to unsafe practices are consistent with the observations by other researchers . this is very well - linked to the often - recognized need and demand for effective patient transport and emergency transport system . the improved patient transport systems are closely linked to the patient safety and quality of care . the referrals and transportations are important not only between facilities but the patient handovers and transportation within the different units and the departments within the same facilities and were reported to be closely linked to patient safety issues and participants highlighted the need for strengthening at all levels . the studies on needle stick injuries noted that the reasons for such injuries included professional hierarchy where orders have to be strictly followed without questioning , poor communication regarding guidelines among staff members and heavy patient load . the limited attention on incident reporting and administrative sanctions have also been recognized as barrier in patient safety . a consultation on regulation and accreditation of health service delivery institutions in india recommended that the quality of health services has to be a mainstream and explicit discourse in india . it suggested that the stakeholders need to innovate and accelerate efforts to improve the quality of health services and that there is a need for establishing the multi - stakeholder forum on regulation and accreditation as a linkage platform . there were recommendations related to the capacity building of policy makers , other stakeholders , and institutions , in improving quality of health service , need to provide technical assistance for implementation of the clinical establishment ( registration and regulation ) act , 2010 and that of to document best practices for licensing , regulation , and accreditation of health service delivery institutions in india . though there is an increasing dialogue among stakeholders and more attention on accreditation , the fact is that only a limited numbers of hospitals in india are accredited by any system . many of the participants in this study commented that there is a culture of not recognizing the patient safety issues . unless the mindset is going to change , the quality of care is unlikely to improve . in this context , it is suggested that for improved patient safety , the initial step could be the identification of problems and agreeing on a pragmatic roadmap and implementation plan to improve patient safety at facility levels . this study highlights the need for increased community participation , awareness of patient 's rights , community awareness , and the advocacy for patient communication in the field of patient safety . this is likely to help by on one hand , the caregivers demanding for a better quality of health care and on the other hands , setting up realistic expectations for community and the family members of patients . analysis of the findings from health system framework indicated that to improve patient safety ( and quality of services ) , there is need to look at various functions of health system by making all components of services available , making referrals timely and which are respected at the next levels of health facilities ( service provision ) ; having different categories of people available and trained in the safety issues ( creating resources ) ; providing sufficient finances for meeting the additional requirement , and conducting trainings ( financing ) and this has to be done with a clearly laid out strategies and plans , well - supported by management and leadership of the facilities ( stewardship and governance ) [ table 1 ] . though the primary healthcare facilities were not represented in the sample selected for this study , the participants highlighted the role of primary healthcare facilities and provider could play in the improved patient safety by decision on timely referral , ensuring all precautions are taken while providing health care at lower levels . it was discussed that the patient safety is across the continuity of care , which start from primary care level . moreover , there are more providers ( both in public and private sector combined ) at the primary care and changing their practices in patient safety would have a major contribution to the overall outcome . it is proposed that in future while conducting such studies , inclusion of primary care facilities and providers could be given due consideration . this is an area where even less data are available in india and more research is required . one of the impacts of this study had been that in the following months , the information generated from this study was used for designing of a training program on patient safety ; inclusion of patient safety agenda in two conferences / meetings organized in delhi ; and the government of delhi conducting 2-day capacity building workshops in patient safety for hospital and health facility focal points . these are not the ultimate solutions ; however , they are the right steps for generating awareness and preparing the system for better patient care . a limitation of this work is that it is based on interactions with medical doctors from one specialty in government owned secondary and tertiary care facilities in one state only . it is possible that inclusion of private sector hospitals , other categories of staff ( nurses and orderly , etc . ) ; care providers from other departments , and inclusion of primary care facility could have provided additional insight . however , it is expected that the findings from this study are still valid , though more studies are needed for additional set - ups and types of providers . the qualitative narrative of this study was by design and the authors supplemented quantitative component by conducting literature review . however , these limitations should not affect the conclusions as the key objectives of this study was to understand the local context , to improve the patient safety through selection of appropriate interventions and to implement the best practices . the quality of health services in india , with specific case of maternal health services as in this study , needs major improvement . the providers are often aware of the challenges and are keen to implement the solutions . available local information could be used for selection , designing , and implementation of measures to improve patient safety . a systematic and sustained approach with attention on all functions of health systems patient safety could be used as an entry point for bringing attention on overall quality of healthcare services in india . the momentum and opportunity to bring improvement in quality of health services has to be sustained by more funding , capacity building , and strong leadership with clearly outlined intentions through policy proposals . the quality has to be a mainstream discourse in public policy and programmatic discourses , as india intends to advance toward universal health coverage . the opinions expressed in this article are solely those of the individual authors and should not be attributed to the institutions / organizations they have been affiliated to in the past or at present . the opinions expressed in this article are solely those of the individual authors and should not be attributed to the institutions / organizations they have been affiliated to in the past or at present .

background : there is insufficient information on causes of unsafe care at facility levels in india . this study was conducted to understand the challenges in government hospitals in ensuring patient safety and to propose solutions to improve patient care.materials and methods : desk review , in - depth interviews , and focused group discussions were conducted between january and march 2014 . healthcare providers and nodal persons for patient safety in gynecology and obstetrics departments of government health facilities from delhi state of india were included . data were analyzed using qualitative research methods and presented adopting the health system approach.results:the patient safety was a major concern among healthcare providers . the key challenges identified were scarcity of resources , overcrowding at health facilities , poor communications , patient handovers , delay in referrals , and the limited continuity of care . systematic attention on the training of care providers involved in service delivery , prescription audits , peer reviews , facility level capacity building plan , additional financial resources , leadership by institutional heads and policy makers were suggested as possible solutions.conclusions:there is increasing awareness and understanding about challenges in patient safety . the available local information could be used for selection , designing , and implementation of measures to improve patient safety at facility levels . a systematic and sustained approach with attention on all functions of health systems could be beneficial . patient safety could be used as an entry point to improve the quality of health care services in india .

5xfad tg mouse model ( b6sjl - tg(appswfllon , psen1*m146l*l286v)6799vas ) ( jackson laboratories ) is bred on a non - transgenic background ( b6sjlf1 ) . hela is a human cervical cancer cell line ; nih/3t3s is a mouse embryonic fibroblast line ; hepg3 is from hepatocellular carcinoma ; raw264.7 is a mouse leukemic monocyte macrophage cell line . 16 nm magnetite nanoparticles were synthesized by decomposition of iron - oleate at 320c as described in an earlier report . 10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex . 18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute . resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation . to make the organic phase synthesized mns suitable for biological application we stabilized the mns using carboxylate terminated ligand with ndopa as an anchor . synthesis of carboxylate terminated ndopa ligand and functionalization of the mns was carried out according to the following protocol . tetraethylene diacide , n - hydroxysuccinimide ( nhs ) , n , n-dicyclohexylcarbodiimide ( dcc ) , ndopa hydrochloride and anhydrous sodium bicarbonate was dissolved in chloroform under argon atmosphere and stirred for 4 hr . hexane stabilized mns were added and stirred for another 24 hr . the precipitate formed was separated by magnet , dispersed in water and purified by dialysis . the conjugation of buffer stabilized mns with antibody was done using a conventional carboxyl - amine crosslinking method . we first activated the carboxyl terminated mns by sulfo - n - hydroxy succinimide ( snhs ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc ) followed by incubation with corresponding antibody ( nu4 or igg1 , with or without fluorescent label ) overnight . conjugated mns were separated by magnet to remove excess reagent and antibody then re - dispersed in working media . conjugation efficiency was estimated using uv spectroscopy ( absorbance at 280 nm ) of the magnetically separated supernatant . of note , because both fluorophore and mns conjugation use carboxyl - amine crosslinking , fluorescent nu4 that has more than 2.5 mol fluorophore / mol antibody does not conjugate well to the mns . optimal concentration of fluorophore / nu4 is ~1.02.5 mol fluorophore / mol antibody for fluorescent - nu4mns preparation . mice were anesthetized with isoflurane and then placed on their backs with their heads positioned to maximize the residency time for the delivered material to remain on the olfactory surface . each naris was administered with 568-nu4 , nu4mns or non - immune iggmns ( 5 l / naris ) , using a sterile micropipette , slowly over a period of 1 min , keeping the opposite naris and mouth closed to allow complete aspiration of delivered material . a falcon tube containing the mns treated tissue samples was placed in a bruker pharmascan 7 t animal mri scanner . sample were imaged using a t2 * -weighted gradient echo ( gre ) flash pulse sequence 3-dimensional acquisition . the image resolution was 0.07 mm isotropic , with field - of - view ( fov ) 4.62cm/2.52cm/0.65 cm and image matrix ( mtx ) 700 pixels/385 pixels/100 pixels . the number of averages ( nex ) was 20 to improve the signal - to - noise ratio . following intranasal inoculation , the probe was allowed to distribute for 4 hours before mr imaging was performed according to imaging methodology described in mundt et al . t1 , t2 , and t2 * weighted mr images were acquired on a bruker biospec 7 t magnet , using a 25 mm rf quadrature coil . t1- and t2-weighted images provide anatomical guidance as well as some localization of the nu4mns and were acquired with a fat suppressed spin echo sequence ( rapid acquisition with relaxation enhancement , rare ) with the following parameters for t1-weighted ( tr=1000 ms , teeff=13.2 ms , rare factor 2 , number of excitations , nex=4 ) and for t2-weighted ( tr=3500 ms , teeff=58.5 ms , rare factor 4 , nex=4 ) . t2 * -weighted imaging provides more of the localization of the nu4mns as the iron causes local changes in magnetic susceptibility which t2 * weighted images can be sensitive to . a gradient echo sequence was used with the following parameters ( gradient echo fast imaging , gefi ; tr=1200 ms , te=5.6 ms , flip angle 35 and nex=4 ) . 5xfad tg mouse model ( b6sjl - tg(appswfllon , psen1*m146l*l286v)6799vas ) ( jackson laboratories ) is bred on a non - transgenic background ( b6sjlf1 ) . hela is a human cervical cancer cell line ; nih/3t3s is a mouse embryonic fibroblast line ; hepg3 is from hepatocellular carcinoma ; raw264.7 is a mouse leukemic monocyte macrophage cell line . 16 nm magnetite nanoparticles were synthesized by decomposition of iron - oleate at 320c as described in an earlier report . 10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex . 18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute . resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation . 10.8 g of iron ( iii ) chloride hexahydrate and 36.5 g sodium oleate were dissolved in a mixture of 60 ml distilled water , 80 ml ethanol and 140 ml hexane and heated at 60c for 4 hr . the organic layer of the biphasic mixture becomes dark , indicating phase transfer of iron ( iii ) ions and formation of iron oleate complex . 18 g of iron oleate complex and 2.58 g of oleic acid were dissolved in 100 g of octadecene at room temperature and heated to 320c at a rate of 3.3c per minute . resulting nanoparticles are separated from the solution by addition of ethanol and ethyl acetate followed by centrifugation . to make the organic phase synthesized mns suitable for biological application we stabilized the mns using carboxylate terminated ligand with ndopa as an anchor . synthesis of carboxylate terminated ndopa ligand and functionalization of the mns was carried out according to the following protocol . tetraethylene diacide , n - hydroxysuccinimide ( nhs ) , n , n-dicyclohexylcarbodiimide ( dcc ) , ndopa hydrochloride and anhydrous sodium bicarbonate was dissolved in chloroform under argon atmosphere and stirred for 4 hr . hexane stabilized mns were added and stirred for another 24 hr . the precipitate formed was separated by magnet , dispersed in water and purified by dialysis . the conjugation of buffer stabilized mns with antibody was done using a conventional carboxyl - amine crosslinking method . we first activated the carboxyl terminated mns by sulfo - n - hydroxy succinimide ( snhs ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc ) followed by incubation with corresponding antibody ( nu4 or igg1 , with or without fluorescent label ) overnight . conjugated mns were separated by magnet to remove excess reagent and antibody then re - dispersed in working media . conjugation efficiency was estimated using uv spectroscopy ( absorbance at 280 nm ) of the magnetically separated supernatant . of note , because both fluorophore and mns conjugation use carboxyl - amine crosslinking , fluorescent nu4 that has more than 2.5 mol fluorophore / mol antibody does not conjugate well to the mns . optimal concentration of fluorophore / nu4 is ~1.02.5 mol fluorophore / mol antibody for fluorescent - nu4mns preparation . mice were anesthetized with isoflurane and then placed on their backs with their heads positioned to maximize the residency time for the delivered material to remain on the olfactory surface . each naris was administered with 568-nu4 , nu4mns or non - immune iggmns ( 5 l / naris ) , using a sterile micropipette , slowly over a period of 1 min , keeping the opposite naris and mouth closed to allow complete aspiration of delivered material . a falcon tube containing the mns treated tissue samples was placed in a bruker pharmascan 7 t animal mri scanner . sample were imaged using a t2 * -weighted gradient echo ( gre ) flash pulse sequence 3-dimensional acquisition . the image resolution was 0.07 mm isotropic , with field - of - view ( fov ) 4.62cm/2.52cm/0.65 cm and image matrix ( mtx ) 700 pixels/385 pixels/100 pixels . the number of averages ( nex ) was 20 to improve the signal - to - noise ratio . following intranasal inoculation , the probe was allowed to distribute for 4 hours before mr imaging was performed according to imaging methodology described in mundt et al . t1 , t2 , and t2 * weighted mr images were acquired on a bruker biospec 7 t magnet , using a 25 mm rf quadrature coil . t1- and t2-weighted images provide anatomical guidance as well as some localization of the nu4mns and were acquired with a fat suppressed spin echo sequence ( rapid acquisition with relaxation enhancement , rare ) with the following parameters for t1-weighted ( tr=1000 ms , teeff=13.2 ms , rare factor 2 , number of excitations , nex=4 ) and for t2-weighted ( tr=3500 ms , teeff=58.5 ms , rare factor 4 , nex=4 ) . t2 * -weighted imaging provides more of the localization of the nu4mns as the iron causes local changes in magnetic susceptibility which t2 * weighted images can be sensitive to . a gradient echo sequence was used with the following parameters ( gradient echo fast imaging , gefi ; tr=1200 ms , te=5.6 ms , flip angle 35 and nex=4 ) .

one way to image the molecular pathology in alzheimer s disease ( ad ) is by positron emission tomography using probes that target amyloid fibrils . however , these fibrils are not closely linked to the development of the disease . it is now thought that early stage biomarkers that instigate memory loss comprise of a oligomers ( aos ) . here we report a sensitive molecular magnetic resonance imaging ( mri ) contrast probe that is specific for aos . we attach oligomer - specific antibodies onto magnetic nanostructures and show the complex is stable and it binds to aos on cells and brain tissues to give a mri signal . when intranasally administered to an ad mouse model , the probe readily reached hippocampal aos . in isolated samples of human brain tissue , we observed an mri signal that distinguished ad from controls . such nanostructures that target neurotoxic aos are potentially useful for evaluating the efficacy of new drugs and ultimately for early - stage ad diagnosis and disease management .

retinoblastoma is the most common intraocular malignancy in childhood , and represents about 3% of all pediatric disease . retinoblastoma , a diagnosis that affects not only vision but also survival for many individuals , is also the most common intraocular tumor of childhood . the incidence of retinoblastoma is generally the same worldwide , at 1 case per 15 000 to 20 000 live births , corresponding to about 9000 cases every year . the mortality rate of retinoblastoma in children is about 4070% in asia and african countries , and 35% in europe , canada , and the usa . early retinoblastoma damage to the visual system may include pathophysiologic changes in the retina and corneal nerve head , and orbital corneal nerve damage . therefore , we speculate that the change in or injuries of the cornea may reflect or correlate with retinoblastoma status . in this study we focused on biomarkers obtained from electroencephalography ( eeg ) recordings to reflect corneal nerve injury in retinoblastoma in the eyes - closed resting state . previous studies have also investigated corneal nerve injury in eyes of humans and animals . several classical eeg biomarkers have recently been identified for predicting prognosis of retinoblastoma patients , including 5 different frequencies and powers . according to our clinical observations , eeg band power may be correlated with corneal nerve injury in retinoblastoma patients . thus , in this study we compared clinical eeg recordings with cornea confocal microscope results , and explored the use of eeg in retinoblastoma patients . this research was approved by the ethics committee of weifang medical university . written informed consent was obtained from parents or legal guardians for participation in the study . twenty consecutive patients who had been referred due to retinoblastoma to the department of ophthalmology , affiliated hospital of weifang medical university between 2010 and 2014 participated in this study . these 20 patients all had nonhereditary unilateral retinoblastoma presenting as intraocular tumor requiring primary enucleation . retinoblastoma in this study was diagnosed according to the method of dean et al . . the visual analysis was performed on standard bipolar montages with electrode positions according to the 10 to 20 international systems . the detailed processes of the eeg examination were performed as reported by zeng et al . . to observe the status of the subbasal never plexus , more than 3 representative images were evaluated for analysis . three masked and independent investigators evaluated the morphology of the corneal nerve and the subbasal nerve plexus , performed according to a previous report . the evaluation of the total length of nerve fibers represents the nerve density in the micrometers / frame ( 158 , 700 m ) . we designated the number of main nerves under a microscope as the main nerve trunks . we designated the number of nerve branches as the nerve branching under a microscope . using a microscope , we designated all of the nerves as the total nerves , including nerve branches and the main nerve trunks . this research was approved by the ethics committee of weifang medical university . written informed consent was obtained from parents or legal guardians for participation in the study . twenty consecutive patients who had been referred due to retinoblastoma to the department of ophthalmology , affiliated hospital of weifang medical university between 2010 and 2014 participated in this study . these 20 patients all had nonhereditary unilateral retinoblastoma presenting as intraocular tumor requiring primary enucleation . retinoblastoma in this study was diagnosed according to the method of dean et al . . the visual analysis was performed on standard bipolar montages with electrode positions according to the 10 to 20 international systems . the detailed processes of the eeg examination were performed as reported by zeng et al . . to observe the status of the subbasal never plexus , more than 3 representative images were evaluated for analysis . three masked and independent investigators evaluated the morphology of the corneal nerve and the subbasal nerve plexus , performed according to a previous report . the evaluation of the total length of nerve fibers represents the nerve density in the micrometers / frame ( 158 , 700 m ) . we designated the number of main nerves under a microscope as the main nerve trunks . we designated the number of nerve branches as the nerve branching under a microscope . using a microscope , we designated all of the nerves as the total nerves , including nerve branches and the main nerve trunks . a total of 20 patients ( 11 girls and 9 boys ) were included , with a median age of 58 months ( range , 43 to 127 months ) . according to the intraocular international retinoblastoma classification ( iirc ) , there were 0 patients with extreme low risk ( group a ) , 1 patient with low risk ( group b ) , 1 patient with intermediate risk ( group c ) , 16 patients with high risk ( group d ) , and 2 patients with extreme high risk ( group e ) . figure 1 shows the changes of the dominant power bands within delta and theta bands . the eeg pattern of the retinoblastoma patients was characterized by a distinct increase in the delta ( figure 1 , p<0.01 ) and significant decrease in the theta power ( figure 1 , p<0.05 ) . the results of cornea confocal observation showed that the corneal branch nerve fiber density was decreased significantly compared to the normal individuals . however , the density of the tumid and dendritic nerves was significantly increased compared to the normal individuals . the basal epithelium curving nerves were also significantly increased in the eyes of retinoblastoma patients ( figure 2a ) . however , the subbasal nerve plexus remained normal ( figure 2b ) . these results show that the corneal nerve was significantly damaged in eyes of retinoblastoma patients . in order to investigate the correlation between corneal nerve injury and the eeg spectra power , the results indicated that corneal nerve injury was positively correlated with the delta eeg spectra power ( figure 3a , p<0.05 ) and the corneal nerve injury was negatively correlated with the theta eeg spectra power ( figure 3b , p<0.05 ) . this suggests that the eeg spectra power changes may reflect corneal nerve injury in retinoblastoma patients . we found that the sensitivity and specificity of the increased delta power and decreased theta power in the diagnosis of retinoblastoma patients were different . in increased delta power ( delta > 3.8 ) , sensitivity and specificity were 75% and 85% , and in decreased theta power ( theta < 4.3 ) sensitivity and specificity were 80% and 80% , respectively ( table 1 ) . if compounded in parallel , the sensitivity of detecting corneal nerve injury was 75% and the specificity was 88% . if compounded in series , the sensitivity and specificity were 85% and 67% , respectively ( table 1 ) , which is appropriate for the diagnosis of corneal nerve injury in retinoblastoma patients . a total of 20 patients ( 11 girls and 9 boys ) were included , with a median age of 58 months ( range , 43 to 127 months ) . according to the intraocular international retinoblastoma classification ( iirc ) , there were 0 patients with extreme low risk ( group a ) , 1 patient with low risk ( group b ) , 1 patient with intermediate risk ( group c ) , 16 patients with high risk ( group d ) , and 2 patients with extreme high risk ( group e ) . figure 1 shows the changes of the dominant power bands within delta and theta bands . the eeg pattern of the retinoblastoma patients was characterized by a distinct increase in the delta ( figure 1 , p<0.01 ) and significant decrease in the theta power ( figure 1 , p<0.05 ) . the results of cornea confocal observation showed that the corneal branch nerve fiber density was decreased significantly compared to the normal individuals . however , the density of the tumid and dendritic nerves was significantly increased compared to the normal individuals . the basal epithelium curving nerves were also significantly increased in the eyes of retinoblastoma patients ( figure 2a ) . however , the subbasal nerve plexus remained normal ( figure 2b ) . these results show that the corneal nerve was significantly damaged in eyes of retinoblastoma patients . in order to investigate the correlation between corneal nerve injury and the eeg spectra power , spearman rank correlation analysis was performed . the results indicated that corneal nerve injury was positively correlated with the delta eeg spectra power ( figure 3a , p<0.05 ) and the corneal nerve injury was negatively correlated with the theta eeg spectra power ( figure 3b , p<0.05 ) . this suggests that the eeg spectra power changes may reflect corneal nerve injury in retinoblastoma patients . we found that the sensitivity and specificity of the increased delta power and decreased theta power in the diagnosis of retinoblastoma patients were different . in increased delta power ( delta > 3.8 ) , sensitivity and specificity were 75% and 85% , and in decreased theta power ( theta < 4.3 ) sensitivity and specificity were 80% and 80% , respectively ( table 1 ) . if compounded in parallel , the sensitivity of detecting corneal nerve injury was 75% and the specificity was 88% . if compounded in series , the sensitivity and specificity were 85% and 67% , respectively ( table 1 ) , which is appropriate for the diagnosis of corneal nerve injury in retinoblastoma patients . the eeg method of prognosis used in this study agrees with previous reports on use of the corneal nerve for disease diagnosis and prognosis [ 1315 ] . however , using eeg to diagnose corneal nerve injury in retinoblastoma patients has not been explored in previous studies . the 3 goals of retinoblastoma diagnosis , treatment , and improvement are saving the patient s life , eyes , and vision . in our previous study , we examined the eeg power bands and analyzed their relationship with corneal nerve injury . we observed not only increased power in the prevailing eeg power bands , but also a moderate shift of the dominant frequency peak toward faster theta and delta . this might be because retinoblastoma afflicts the corneal nerve , which is related with visual activity . our results show that the delta power was increased and theta power was decreased in retinoblastoma patients compared to normal individuals . in clinical ophthalmology , the pathogenesis of many diseases , such as herpes simplex keratitis , glaucoma , and retinoblastoma , may affect the corneal nerves [ 5,1820 ] . the cornea is the most densely innervated part of the human body , containing myelinated a and unmyelinated c fibers derived from the ophthalmic division of the trigeminal nerve . therefore , corneal confocal microscope analysis is always used in the diagnosis of corneal nerve damage . our results indicate decreased corneal branch nerve fiber density , as well as the appearance of the dendritic particles and tumid neurons , which suggests that retinoblastoma might induce corneal nerve injury . therefore , we analyzed the correlation between corneal nerve injury and the delta and theta power of eeg . the results indicated that the corneal nerve injury was positively correlated with the delta power and negatively correlated with the theta power of eeg . actually , the changes in eeg theta and delta power was very slight and short - term . our study indicated that the theta and delta power were mainly observed in the high - risk stage ( group d ) of retinoblastoma , according to the iirc classification and that the changes in eeg powers can not be obtained in many patients or at other stages . therefore , we speculate that the changes in delta and theta power are transient and are related to iirc stage for the retinoblastoma . in our study , the sensitivity of the increased delta power and decreased theta power in the diagnosis of corneal nerve injury were 75% and 80% , respectively and the specificity was 85% and 80% , respectively . if compounded in parallel , the specificity of corneal nerve injury was 88% and if compounded in series , the sensitivity was 85% , and it was obviously higher than in other groups . if this method can help determine whether the corneal nerve is already injured in retinoblastoma patients , it will provide useful a reference for follow - up , adjunctive therapy , and the prognosis of retinoblastoma patients . the changes in delta and theta of eeg appear to be associated with occurrence of corneal nerve injury . useful information can be provided for evaluating corneal nerve damage of retinoblastoma patients through analyzing eeg power bands .

backgroundin our clinical experience we discovered that eeg band power may be correlated with corneal nerve injury in retinoblastoma patients . this study aimed to investigate biomarkers obtained from electroencephalography ( eeg ) recordings to reflect corneal nerve injury in retinoblastoma patients.material/methodsour study included 20 retinoblastoma patients treated at the department of ophthalmology , affiliated hospital of weifang medical university between 2010 and 2014 . twenty normal individuals were included in the control group . eeg activity was recorded continuously with 32 electrodes using standard eeg electrode placement for detecting eeg power . a cornea confocal microscope was used to examine corneal nerve injury in retinoblastoma patients and normal individuals . spearman rank correlation analysis was used to analyze the correlation between corneal nerve injury and eeg power changes . the sensitivity and specificity of changed eeg power in diagnosis of corneal nerve injury were also analyzed.resultsthe predominantly slow eeg oscillations changed gradually into faster waves in retinoblastoma patients . the eeg pattern in retinoblastoma patients was characterized by a distinct increase of delta ( p<0.01 ) and significant decrease of theta power p<0.05 ) . corneal nerves were damaged in corneas of retinoblastoma patients . corneal nerve injury was positively correlated with delta eeg spectra power and negatively correlated with theta eeg spectra power . the diagnostic sensitivity and specificity by compounding in the series were 60% and 67% , respectively.conclusionschanges in delta and theta of eeg appear to be associated with occurrence of corneal nerve injury . useful information can be provided for evaluating corneal nerve damage in retinoblastoma patients through analyzing eeg power bands .

disrupted in schizophrenia 1 ( disc1 ) was initially discovered at the breakpoint in a balanced chromosomal translocation t ( 1 ; 11 ) segregating with major mental conditions , such as schizophrenia , bipolar disorder , and major depression in a scottish pedigree ( millar et al . , 2000 ) . since then , accumulating evidence from genetic studies indicated that disc1 is not only associated with schizophrenia and mood disorders , but also other psychiatric disorders of neurodevelopmental origin , such as autism , asperger syndrome , and agenesis of the corpus callosum ( hennah et al . , 2003 ; hodgkinson et al . , 2004 ; callicott et al . , 2005 ; kilpinen et al . , 2008 ; song et al . , 2008 , 2010 ; although recent genome wide association studies ( gwas ) have not found disc1 as a key genetic risk factor for patients met the current diagnostic criteria for schizophrenia ( purcell et al . , 2009 ; stefansson et al . , 2009 ; mathieson et al . , 2011 ) , it is noted that variations of disc1 influence anatomical and functional endophenotypes even in control subjects ( thomson et al . , 2005 ; di giorgio et al . , 2008 ; prata et al collectively , genetic variation of disc1 may confer vulnerabilities to a wide range of neurodevelopmental psychiatric conditions by affecting brain maturation , thereby modifying brain function . consistently , extensive biological studies indicate that disc1 plays a role in multiple cellular processes during and after brain development ( chubb et al . , 2008 ; brandon and sawa , 2011 ) . in fact , many protein binding partners of disc1 are associated with various molecular pathways that regulate fundamental cellular processes for brain development and function ( table 1 ) . nonetheless , it is still unknown which functional aspects of disc1 directly affect molecular mechanisms underlying disease susceptibility . how can we utilize accumulating biological data of disc1 to discover novel therapeutic targets and biological markers for major mental conditions ? here , we will review disc1-associated molecular pathways which have the potential to be novel therapeutic targets , with particular focus on well documented disc1 pathways involved in cerebral cortex development and function ( figure 1 ) . we will also discuss the potential link of disc1 pathways and environmental factors , such as immune / inflammatory responses , to explore therapeutic interventions based on understanding disease mechanisms of genetic and environmental interaction . disc1 may function as an anchoring molecule to regulate various molecular pathways via interaction with said protein interactors in a context dependent manner . various disc1-mediated pathways with many binding partners and environmental factors synergistically affect proper cerebral cortex development and function . for reviews of the other disc1 interactors , disrupted in schizophrenia 1 plays a critical role for the regulation of cell proliferation in the developing cerebral cortex via the canonical wnt signaling pathway ( mao et al . , 2009 ) . the data suggested that disc1 inhibits the activity of glycogen synthase kinase 3 beta ( gsk3 ) via protein interaction , thereby stabilizing -catenin which is required for proper progenitor proliferation through wnt pathway . the same group later reported that dix domain containing-1 ( dixdc1 ) , a homolog of the wnt signaling genes disheveled axin , interacts with disc1 to co - modulate gsk3/-catenin signaling for proper cell proliferation ( singh et al . , 2010 ) . accumulating evidences have shown that gsk3 signaling may be involved in various neuropsychiatric disorders , such as schizophrenia , autism , and alzheimer s disease , suggesting that gsk3 appears as a prominent therapeutic target for mental disorders ( bachmann et al . in fact , lithium , the mood stabilizer which is commonly used for the treatment of bipolar disorder , is known to inhibit gsk3 activity ( stambolic et al . , 1996 ) . the other psychoactive drugs , such as clozapine , risperidone , and valproic acid , have also been reported to affect gsk3 activity ( stambolic et al . , 1996 ; kang et al . , 2004 ; li et al . , 2007 ; rowe et al . , 2007 ) . nonetheless , since gsk3 regulates various downstream effectors , which are not only implicated in the wnt pathway , but also other signaling required for cellular development , such as sonic hedgehog and notch signaling pathways ( hur and zhou , 2010 ) , it is important to examine specific gsk3-mediated pathways relevant to disease mechanisms to find novel therapeutic strategies . in this regard , it may be ideal to focus on disc1-mediated gsk3 pathways , especially those in association with other genetic risk factors , to explore disease - associated molecular mechanisms . for instance , collapsin response mediator protein-2 ( crmp-2)/dihydropyrimidinase - like-2 ( dpysl2 ) , a susceptibility gene for schizophrenia ( nakata et al . , 2003 ) , is reported to be a potential protein interactor of disc1 by yeast - two - hybrid screening ( camargo et al . , 2007 ) . interestingly , crmp-2/dpysl2 is known to be phosphorylated by gsk3 for the regulation of axon outgrowth ( yoshimura et al . , 2005 ) . many groups have consistently reported that knockdown of disc1 using rna interference ( rnai ) impaired radial neuronal migration in the developing cerebral cortex ( kamiya et al . , 2005 , 2008 ; , 2010 ; singh et al . , 2010 ; young - pearse et al . , 2010 ; ishizuka et al . , findings from these studies suggest that disc1 , along with many protein binding partners , regulate neuronal migration via centrosome and microtubule - dependent mechanisms . of note , some of these binding partners are known as risk or causative genes for various neuropsychiatric disorders . these include nuclear distribution element - like ( ndel1 ) and pericentriolar material 1 ( pcm1 ) , risk genes for schizophrenia , and bbs4 , a causative gene for bardet biedl syndrome that frequently accompanies impaired cognition , mental retardation , and psychosis ( burdick et al . , 2008 ; kamiya et al . , 2008 ; amyloid precursor protein ( app ) also interacts with disc1 to recruit disc1 to the centrosome for regulation of neuronal migration ( young - pearse et al . , 2010 ) . furthermore , disc1 is a component of the lis1/dynein motor complex ( kamiya et al . , 2005 ) . mutations in human lis1 gene cause classical lissencephaly resulting in mental retardation ( pilz et al . , 1998 ) . consistently , lis1 heterozygous knockout mice in which lis1 expression is reduced , display disorganization of proper cortical layer formation and behavioral abnormalities , such as impaired spatial learning and motor function , indicating that this is a good animal model for human lissencephaly caused by lis1 haploinsufficiency ( hirotsune et al . , 1998 ) . interestingly , the prenatal administration of alln , a calpain inhibitor which prevents the degradation of lis1 , is effective to ameliorate neuronal migration defect and improve motor coordination in this animal model ( yamada et al . , 2009 ) . although mental disorders undoubtedly have genetic complexities and could not be explained by the simple haploinsufficiency model as the case of lissencephaly , elucidation of risk genes , and/or molecules in their interactome , specifically ones with enzymatic activity , may offer hope for novel treatment interventions for neuropsychiatric disorders . in this regard , endo - oligopeptidase activity of ndel1 is quite interesting from a drug discovery viewpoint ( hayashi et al . , 2005 ) . as a matter of fact , inhibitors of angiotensin - converting enzyme ( ace ) , an exopeptidase , are currently being used to treat hypertension and renal disease ( izzo and weir , 2011 ) , making peptidase activity an attractive drug target . although endogenous substrates for ndel1-oligopeptidase in brain development remain unknown , in vitro experiments identified several oligopeptides , such as neurotensin and bradykinin , as potential targets for ndel1 ( camargo et al . , 1983 ) . interestingly , neurotensin has a modulatory effect on neurotransmitter systems , including dopaminergic neurons , which may be involved in the pathophysiologies of schizophrenia ( boules et al . , 2007 ) . posttranslational modifications , which affect the functional diversity of target proteins , could also have potential as novel drug targets and biological markers in the disc1 pathways . we have recently reported that phosphorylation of disc1 at serine 710 is a molecular switch signaling from cell proliferation to neuronal migration in the developing cerebral cortex ( ishizuka et al . , 2011 ) . by utilizing in utero electroporation , this study has shown that a phosphor - dead mutant disc1 can rescue only the proliferation defect elicited by disc1 knockdown , whereas a phosphor - mimic mutant of disc1 can exclusively recover impaired migration . the question arises whether the phosphorylation of disc1 at serine 710 may be involved in the pathophysiologies of major mental disorders , such as schizophrenia . it is obviously impractical to investigate the phosphorylation status of disc1 in the developing human brain from subjects at risk of developing schizophrenia . nonetheless , recent progress in induced pluripotent stem ( ips ) cell technology will open new avenues to characterize such findings from preclinical studies using patient - derived neuronal cells , which might in turn identify biological markers for major mental disorders . disrupted in schizophrenia 1 impacts upon brain development may be a challenge for treatment intervention . however , synaptic deficits revealed by the disc1 pathway offer some potential for development of targeted pharmacologic intervention . subsequent findings underline roles for disc1 in regulating dendritic spines of the glutamate synapse ( hayashi - takagi et al . , 2010 ) . rac1 is activated by karilin-7 , leading to increased spine size following nmda glutamate receptor activation . however , disc1 appears to interact with karilin-7 , preventing access to and activation of rac1 until nmda receptor activation promotes release of kal-7 and spine enlargement . pharmacologic tools to modulate the karilin-7/disc1 interaction might be a means to regulate spine maintenance . traf2- and nck - interacting kinase ( tnik ) represents another potential pharmacological target in the disc1 protein interaction network . tnik is found in postsynaptic densities and regulates c - jun kinase , the actin cytoskeleton and a number of wnt pathway effectors ( fu et al . , 1999 ; genetic association studies have found single - nucleotide polymorphisms of tnik associated with schizophrenia ( potkin et al . , 2009 ; shi et al . , 2009 ) . tnik mrna expression was increased in the dorsolateral prefrontal cortex of schizophrenia subjects ( glatt et al . , 2005 ) and in lymphoblasts of monozygotic twins discordant for bipolar disorder ( matigian et al . , 2007 ) . a yeast - two - hybrid screen using disc1 as bait identified tnik as an interactor ( camargo et al . , 2007 ) . subsequently , tnik and disc1 were shown to interact in mouse brain ( wang et al . , 2011 ) . disc1 was found to inhibit the kinase activity of tnik , an action that could be reproduced by a small peptide derived from the disc1 interaction site . this disc1 peptide led to increased actin polymerization and decreased expression of a number of postsynaptic density proteins , including psd95 , stargazin , ampa receptor subunit glur1 and tnik , itself ( wang et al . , 2011 ) . microbial infections have been recognized as environmental factors responsible for the increased incidence of schizophrenia and associated disorders ( brown and derkits , 2010 ; sham et al . , 1992 ; torrey and yolken , 2003 ) . these reports have been supported by the epidemiological findings of an association between elevated cytokines in maternal serum and schizophrenia in the offspring ( delisi and wyatt , 1982 ; patterson , 2007 ; miller et al . , 2009 ) . subsequently , it has been demonstrated that it is the maternal immune response to a microbe that may contribute to the increased risk of schizophrenia . the role of cytokines in innate immune response makes them promising candidates for studying their functions in disruption of fetal brain development in vulnerable individuals ( dantzer et al . , 2008 ) . most studies with prenatal immune activation have thus far used wild - type mice and rats . however , recently , there have been several reports on developing and characterizing animal models based on combining prenatal immune activation with genetic mutations relevant to schizophrenia ( ibi et al . , 2010 ; ehninger et al . , we have been studying possible roles for disc1 in modulation of poly i : c - induced immune activation in pregnant mice to mimic prenatal in utero exposure to viruses as a model of gene environment interactions relevant to schizophrenia ( abazyan et al . , our findings have suggested that disc1 may be involved in mediating neuroimmune interplay in this mouse model . given the extended interactome of disc1 , it is not surprising that this protein is at the crossroads of the signaling transduction pathways activated by immune factors . one can envision multiple interactions between the pathways impacted by mutant disc1 and activated by cytokines and/or bacterial lipopolysaccharide ( lps ) and poly i : c itself via cytokine receptors or toll - like receptors ( tlr ) expressed by neurons or glia cells , respectively . one of the major common pathways is the phosphoinositide-3 kinase / akt - signaling network ( pi3k / akt ) that is activated by cytokines and poly ic and has been demonstrated to interact with disc1 partners ( camargo et al . , 2007 ) . another example is interactions with gsk3 , a key regulator of the host inflammatory response and the production of pro- and anti - inflammatory cytokines ( hayden et al . , 2006 ) . as described above , disc1 inhibits gsk3 activity through a direct interaction ( mao et al . , 2009 ) . we also found altered poly i : c - induced phosphorylation of gsk3 in mutant disc1 newborn mice that might at least in part explain altered basal and poly i : c - induced production of cytokines in fetal brains and resultant affective behaviors in adult offspring ( abazyan et al . , 2010 ) . these observations are consistent with an emerging role for gsk3 in inflammation - associated depression and anxiety ( jope , 2011 ) . many immune effects of gsk3 are related to its regulation of critical transcription factors , including nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ; hayden et al . , 2006 ) . a family of tlrs acts as primary sensors that detect a wide variety of microbial components and elicit innate immune responses . all tlr signaling pathways culminate in activation of nf-b , which controls the expression of an array of inflammatory cytokine genes . stimulation with tlr ligands triggers the rapid phosphorylation of specific serine residues of inhibitor of b ( ib ) proteins by the ib kinase ( ikk ) complex . phosphorylated ib proteins are subsequently polyubiquitinated and degraded , allowing nf-b to move into the nucleus . this so - called canonical pathway is involved in tlr - mediated induction of inflammatory cytokines such as tumor necrosis factor- ( tnf- ) and interleukin-6 ( il-6 ; hayden et al . , prior studies with disc1 have demonstrated that disc1 , particularly a nuclear isoform of the protein , can play an important role in regulation of transcription activity in the nucleus ( sawamura et al . , 2008 ) . we found that expression of mutant disc1 in n2 a neuronal cells led to delaying a recovery of ib after tnf--induced phosphorylation and ubiquitination of ib. this prolonged degradation due to expression of mutant disc1 seems to suggest that perturbation in functions of disc1 could also affect ( e.g. , stimulate ) pro - inflammatory signaling transduction cascades in neurons . in addition to immune signaling pathways , disc1 and perhaps other candidate genes can play a significant role in the cellular processes utilized by microbes during their life cycles ( carter , 2009 ) . it has been proposed that the involvement of disc1 in the control of the microtubule network might be important both in viral traffic and in the rerouting of microtubules to the vacuoles formed by t. gondii ( carter , 2009 ) . recent clinical trials of anti - inflammatory add - on therapy in schizophrenia have demonstrated superior beneficial treatment effects when antipsychotics were co - administered with anti - inflammatory compounds , as compared with treatment outcomes using antipsychotics alone ( meyer et al . , 2011 ) . however , a broad non - specific anti - inflammatory or immunosuppressive treatments that may have several unwanted effects such as increased sensitivity to infections ( meyer et al . , 2011 ) . ultimately , future therapeutic approaches will result from deciphering intracellular pathways that underlie convergence of environmental influences and genetic predisposition and their influence on neurodevelopmental processes . disrupted in schizophrenia 1-mediated pathways play multiple roles for critical cellular processes through many protein binding partners in a context dependent manner . nonetheless , it is still unknown which functional aspect of disc1 directly affects molecular mechanisms underlying disease susceptibility . are all disc1 functions in such cellular events implicated in disease processes or are only some specific functional aspects critical ? this is a tremendously difficult question , because the molecular disposition of disc1 is complex as reflected by multiple isoforms at both mrna and protein levels ( ishizuka et al . , 2006 nonetheless , biological functions of disc1 are currently being explored without waiting for the complete identification of disc1 isoforms , resulting in the identification of multiple roles of disc1 in various functional contexts . in fact , in addition to the roles in cerebral cortex we reviewed here , disc1 also contributes to brain development and function in other brain regions , such as hippocampal regions ( enomoto et al . , 2009 ; kim et al . , 2009 ; meyer and morris , 2009 ) . further investigations with advanced genetic engineering techniques , which allow us to dissect region and cell type - specific disc1 functions in a temporal manner , might contribute to more clearly elucidate disc1 functions relevant to psychiatric disorders . as complete functional recovery is unlikely for neurodevelopmental disorders , such as schizophrenia , developing preventive strategies is particularly important . indeed , if the findings on microbial etiologies and resultant immune dysfunction are replicated , simple public health measures may prove beneficial in diminishing the incidence of infections during pregnancy to prevent an appreciable proportion of schizophrenia cases . for example , influenza vaccination , improved hygiene to prevent t. gondii infection , and antibiotics to treat genital / reproductive infections are feasible strategies already employed ( brown and derkits , 2010 ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

genetic risk factors for major psychiatric disorders play key roles in neurodevelopment . thus , exploring the molecular pathways of risk genes is important not only for understanding the molecular mechanisms underlying brain development , but also to decipher how genetic disturbances affect brain maturation and functioning relevant to major mental illnesses . during the last decade , there has been significant progress in determining the mechanisms whereby risk genes impact brain development . nonetheless , given that the majority of psychiatric disorders have etiological complexities encompassing multiple risk genes and environmental factors , the biological mechanisms of these diseases remain poorly understood . how can we move forward to our research for discovery of the biological markers and novel therapeutic targets for major mental disorders ? here we review recent progress in the neurobiology of disrupted in schizophrenia 1 ( disc1 ) , a major risk gene for major mental disorders , with a particular focus on its roles in cerebral cortex development . convergent findings implicate disc1 as part of a large , multi - step pathway implicated in various cellular processes and signal transduction . we discuss links between the disc1 pathway and environmental factors , such as immune / inflammatory responses , which may suggest novel therapeutic targets . existing treatments for major mental disorders are hampered by a limited number of pharmacological targets . consequently , elucidation of the disc1 pathway , and its association with neuropsychiatric disorders , may offer hope for novel treatment interventions .

the erythrocyte sedimentation rate ( esr ) is an acute phase reactant ( apr ) . the rate of sedimentation in a period of one hour called esr and also biernacki test . esr test is a common hematologic nonspecific indicator of inflammation . to perform a test , nonclothing blood is placed in a vertical tube ( westergren ) and erythrocyte sedimentation rate is measured and is reported in units of mm / h . the best way to test was presented in 1921 by westergren , and it is still the golden standard method for measuring erythrocyte sedimentation rate . this method is considered to be simple and cheap , accessible , and accurate . esr is a valuable laboratory tool in evaluation of infectious , inflammatory , and malignant diseases [ 1 , 3 ] . red blood cells in outside from the body precipitate due to their higher density than the plasma ; in normal state these cells reject each other because of their negative surface charges and prevent rolex formation . in order to overcome the negative charge of the red cells it was shown that several factors such as ph levels of plasma other than the size of molecules or rolex formation contribute to erythrocyte sedimentation . esr levels increase with age and are higher in women [ 1 , 4 , 5 ] , anemia , and the black people . clinical factors that do not influence the esr are [ 1 , 4 ] obesity , body temperature , recent food , and nsaid [ 1 , 4 , 5 ] . ivig with the half - life of 3 - 4 weeks was first produced in 1960 [ 6 , 7 ] ; ivig in high doses is used for the treatment of many autoimmune diseases including autoimmune thrombocytopenia , chronic inflammatory polyneuropathy , kawasaki disease , and guillain - barr syndrome [ 79 ] . high - dose ivig effects on various proteins , including inflammatory profiles in 63 children with kawasaki disease , were studied . all children had clinical manifestations of kawasaki and received 2 gr / kg ivig and aspirin during 12 hr . serial testing was carried out before receiving ivig , 24 hours and 7 days later . after ivig infusion , mean esr was 12.7 6.46 mm / h before receiving ivig , 53.3 11.9 mm / h in 24 h , and 48.8 15.2 mm / h in the 7 days after the ivig infusion . iga and igm immunoglobulin levels did not change 24 hours and 7 days after injection ; however , igg is significantly increased . the result is that the high dose of ivig leads to rapid decreased changes of various proteins except for iga and igm and esr . in the same study on patients with myasthenia gravis and guillain - barr syndrome , we try to answer this question too , is esr a valuable apr marker in evaluation of inflammatory response to treatment when ivig is used previously ? total dose of ivig was 2 gr / kg ( 400 mg / kg in five days or 2 gr / kg in single dose ) . esr before infusion of ivig and within 24 hours after administration of the last dose of ivig was checked ( 1 - 2 gr / kg/12 h or 400 mg / kg dose ivig ) . the erythrocyte sedimentation rate was measured by westergren method ; ivig side effects were not observed in any patient . results have been shown in different age groups , neonatal , infancy , childhood , and school age . spss 16 and paired t - test were used to statistical software analysis ; significance level of less than 0.05 was considered meaningful . 23 ( 46% ) patients were males and 27 ( 54% ) were females ( table 1 ) . the mean and median esr before and after receiving ivig the mean of esr before ivig was 31.8 29.04 and after ivig was 47.2 36.9 ; this difference was meaningful ( p = 0.05 ) . in male group the mean esr before ivig was 34.6 33.5 and after ivig was 49.1 36.2 ; the difference with ( p = 0.003 ) is statistically significant . before receiving ivig the mean esr was 29.4 25.2 in females and after receiving ivig it was 45.6 38.04 results of different age groups , 6 patients less than 28 days , 13 patients from 1 month to 1 year , 20 patients from 1 to 6 years old , and 11 patients from 6 to 12 years , have been shown in table 3 . esr is known as an important factor in the evaluation of infectious and inflammatory processes . important point for the use of the esr as an apr is its role in the evaluation of response to treatment ; decreasing levels of esr are considered as a marker of response to therapy . plasma fibrinogen and globulins are the major factors affecting esr [ 1 , 2 ] . alpha and gamma globulin provide half the ability of fibrinogen and albumin has the lowest ability in the sediment of red cells . however , as mentioned , ivig as an intravenous immune globulin is used in many diseases [ 6 , 7 ] . whether the administration of ivig , influences the esr value in monitoring of the response to therapy ? in review of the literature generally two studies were found about the effect of ivig on esr . one of them has been done on kawasaki patients and has discussed numerous parameters of protein and also esr affected by ivig and the subsequent study has been limited to patients with myasthenia gravis and guillain - barr syndrome showing numerous parameters and blood esr affected by ivig , but this study has discussed 16 different diseases following the administration of ivig and has focused on the changes in esr . ivig as an immunoglobulin , mostly igg , has aggregator effect on red cells and prevents their negative discharge forces . although ivig has many anti - inflammatory effects and it is expected to reduce esr rate , in vivo its biologic effect is more effective than the immune modulation effect . the mean esr was 31.8 29.04 before receiving ivig and after ivig it was 47.2 36.9 , with significant differences ( p 0.05 ) . these changes were statistically significant in both sexes ; it could be interpreted because of lack of physiologic difference in children . in different age groups except the neonatal period , 1 month to 1 year ( 13 patients ) , 1 to 6 years ( 20 patients ) , 6 to 12 years ( 11 patients ) , the mean esr before ivig and after receiving ivig increased with significant differences ; p values were , respectively , p = 0.001 , p = 0.025 , and p = 0.006 . rapid physiological changes in various neonatal plasma proteins such as albumin and fibrinogen are the variables leading to these results . different immune regulatory functions of ivig through its interaction with innate and adaptive immune system and immune homeostasis in neonatal period could be another reason . among the apr , the esr is the most valuable criteria for evaluating and monitoring of response to inflammation ; on the basis of this study on patients who are receiving ivig as a therapy , esr increased falsely ( noninflammatory rising ) ; therefore use of esr for monitoring of response to treatment may not be reliable . based on the knowledge of authors and review of the literature this study seems to be the only work about esr and ivig administration effect in children . although these results do not apply to neonatal group we suggest that , in patients who receive ivig , interpretation of esr should be used cautiously on the followup process .

background . erythrocyte sedimentation rate ( esr ) is a valuable laboratory tool in evaluation of infectious , inflammatory , and malignant diseases . red blood cells in outside from the body precipitate due to their higher density than the plasma . in this study we discuss the ivig effect on esr in different diseases and different ages . methods and materials . fifty patients under 12 years old who had indication to receive ivig enrolled in this study . total dose of ivig was 2 gr / kg ( 400 mg / kg in five days or 2 gr / kg in single dose ) . esr before infusion of ivig and within 24 hours after administration of the last dose of ivig was checked . results . 23 ( 46% ) patients were males and 27 ( 54% ) were females . the mean of esr before ivig was 31.8 29.04 and after ivig it was 47.2 36.9 ; this difference was meaningful ( p = 0.05 ) . results of esr changes in different age groups , 6 patients less than 28 days , 13 patients from 1 month to 1 year , 20 patients from 1 to 6 years old , and 11 patients from 6 to 12 years have been meaningful ( p = 0.001 , p = 0.025 , and p = 0.006 , resp . ) . conclusion . in patients who are receiving ivig as a therapy , esr increased falsely ( noninflammatory rising ) ; therefore use of esr for monitoring of response to treatment may be unreliable . although these results do not apply to neonatal group , we suggest that , in patients who received ivig , interpretation of esr should be used cautiously on followup .

indications for covered self - expandable metallic airway stent removal include recurrent mucous plugging of the stent , excessive or recurrent granuloma formation , migration of the stent , stent infection , recurrence of stenosis , stent failure , stent fractures and accomplishment of treatment . usually , it is tedious to remove a stent because of their embedding in the bronchial mucosa and epithelization of the inner surface . granuloma formation at the extremities is a relative common occurrence in covered metallic stents possibly resulting from the radial force applied against the airway wall and the friction exerted . we report a case of covered self expandable metallic stent removal with a rigid bronchoscope under general anesthesia . the case is unique because it had almost all the common complications associated with the stent placement but it was successfully removed without much ado . to the best of our knowledge this is the first case of successful removal of a complete stent reported from india . a 21 year old female presented with complaints of increasing dyspnoea and cough with pain chest for last two weeks . patient had a past history of benign tracheal stenosis which was opened up by endobronchial electro surgery followed by placement of a self expandable metallic airway covered stent . x - ray chest done one and a half year back showed stent well in place [ figure 1 ] . x - ray chest done one and a half year back showing stent well in place bronchoscopy was done under local anesthesia and a stenosis was observed well above the level of the stent [ figure 2 ] with granulation tissue inside the stent [ figure 3 ] , lower end of the stent was fractured , carina was not visualized and stent was seen entering upper part of right main bronchus [ figure 4 ] . the beveled edge of the rigid scope was advanced between the stent wall and the airway mucosa thus creating a space between them . the circular extraction loop at the upper end of the metallic stent was grasped with a rigid optical alligator forceps and pulled in the lumen of rigid bronchoscope with continuous winding movements gradually separating the stent from the tracheal wall . immediately after that this is one of the rare cases of successful removal of self expandable metallic airway covered stent in benign tracheal stenosis . stenosis above the level of the stent granulation tissue inside the stent and at the margins fracture of stent at its lower end ( stent is seen at upper part of right main bronchus , carina is not visualized ) the stent after removal the management of patients with tracheal / bronchial strictures of benign etiology can be quite challenging . though surgical tracheal sleeve resection and reconstruction remains the gold standard for most benign airway strictures , non - surgical modalities such as laser photocoagulation and resection , balloon dilatation , electrocautery and stenting of the airway have been developed to deal with airway stenosis . various inoperable benign airway disorders are considered for airway stenting , like post intubation tracheal stenosis , tracheal burn or trauma , tracheo - broncho - malacia and extrinsic compression of trachea . though silicone prostheses are considered to be first choice in benign diseases their use is less suited , especially in airway wall malacia or distal and angular stenosis where sems are preferred . the disadvantages of the silicone stents are high migration rate , small lumen to wall thickness ratio and difficulty in clearing the secretions . on the other hand , the major drawback for placement of sems in benign situations is their difficult repositioning and removal in situations like excessive granulation tissue , stent fractures and stent migration . the complications of stent removal include significant oozing , tracheal mucosal dehiscence , tracheal puncture requiring thoracotomy , retained stent pieces , need for restenting , respiratory failure , tracheotomy , and even death . in our case , various authors have come up with different methods of removal of the stent , like the technique of nashef et al . , which was similar to that of rolling spaghetti on a fork , but much more difficult and at least equally messy . they further observed that there may be several fractures of the stent , which have to be removed piece by piece . in our case filler et al . , used a metal suction catheter to dissect the stent from the airway wall before extraction with the forceps . zakaluzny et al . , employed a rigid suspension laryngotracheoscope and used optical forceps to dissect the stent from the airway wall . alligator forceps were then employed to grasp and extract the stent through the rigid scope while the scope was advanced further into the airway . we first separated the stent from the tracheal wall and then removed all the works ( scope , forceps and the stent ) in one piece . to conclude , this case is unique for many reasons , firstly , it had almost all of the complications that can be associated with stent placement ( stenosis of trachea at the upper end of the stent , extensive granuloma formation , migration of the stent downwards and fracture of the stent at its lower end ) , secondly , we were able to remove the stent in toto [ figure 5 ] and lastly , there were no complications after removal of the stent . further studies are needed to re evaluate their removal successfully and safely and to produce better designed and technically superior stents like bio - absorbable stents which may not have to be removed at all .

covered self expandable metallic airway stents ( sems ) have been used for benign tracheal stenosis , post intubation tracheal stenosis , tracheal burn or trauma , tracheo - broncho - malacia , and extrinsic compression of trachea . their placement is considered to be permanent , with open surgery the only way to remove the stent , though there are few cases reports of their removal with the bronchoscope , but the complications after their removal are very high . in our patient , one and a half years after placement of sems , she developed cough with dyspnoea , video bronchoscopy showed stenosis above the level of stent with granulation tissue inside the stent , stent fracture in lower part and stent migration to right main bronchus , thus she had all conceivable complications of stent placement . the stent was removed with the help of rigid bronchoscope under general anaesthesia . she was discharged the following day . the case is being reported because it was unique in having all the possible complications of stent placement , and rare as we could take out the stent in toto . thirdly , the stent could be removed without any complication .

metastatic lesions comprise approximately 1% of the tumors in the sellar / parasellar ( sps ) area for which patients undergo transsphenoidal surgery ( tss ) [ 1 , 2 ] ; however , it has been reported in autopsy series that the rate of metastasis to these areas could be as high as 28% . breast and lung cancer are the two most common types of malignant tumors that metastasize to the sps region , with respective rates of 40% and 33% . metastases of prostate , renal cell , gastrointestinal , thyroid [ 8 , 9 ] , and pancreatic cancers , and lymphoma , leukemia , melanoma , and plasmocytoma have also been reported . despite the advancement in the imaging modalities , tumors that have metastasized to the sps areas may still be difficult to differentiate from pituitary adenoma on radiographic studies [ 2 , 14 , 15 ] . thickening of the pituitary stalk and invasion of the cavernous sinus may be suggestive of such lesions , but invasion of the cavernous sinus may commonly occur with pituitary adenomas . this distinction is also clinically challenging , although there are very few symptoms that suggest a metastatic lesion . management options are multimodal and vary depending on whether a primary source is known or on the likely differential diagnoses based on the clinical and radiological findings . multimodal options include radiation therapy , chemotherapy , and/or surgery [ 16 , 17 ] , although the tumor invasiveness renders surgical resection limited . although surgical series have not shown any survival benefits , the patient 's quality of life may be improved [ 10 , 14 ] . in this paper , we review the clinical , endocrine , and radiological features of the metastatic sps tumors with currently accepted therapeutic options based on the pertinent literature . in addition , we report six cases from our institution and discuss their management with long - term clinical outcome . a systematic review of the literature was performed using pubmed and the bibliographies of reviewed articles . the medical records of six patients admitted to the university of utah health sciences center between 2001 and 2011 were reviewed retrospectively . clinical presentation , radiographic studies , treatment , histopathological confirmation , outcome , and prognosis were recorded ( table 1 ) . a 77-year - old man with known prostate cancer presented with a four - month history of left retro - orbital pain followed by left eye ptosis . at presentation , he had complete left third nerve palsy . his visual acuity was intact in both eyes , with full visual fields to confrontation . brain magnetic resonance ( mr ) imaging ( figure 1 ) showed a heterogeneously enhancing mass lesion that measured 29 17 29 mm involving the sella , with invasion of the left cavernous sinus and the upper clivus . the lesion extended to the inferior orbital fissure and was centered in the sella turcica and the cavernous sinus . he subsequently underwent chemotherapy and focused radiation to the sellar region and was noted to have stable neurological examination findings two months after surgery , with no change in his ophthalmoplegia . an 82-year - old woman with known history of breast cancer presented with several weeks ' complaint of progressive left - sided hearing loss as well as facial pain and numbness in the first and the second divisions of the trigeminal nerve , respectively . on neurological examination , left - sided hearing loss and facial numbness along the v1 and v2 distributions were confirmed . mr imaging showed evidence of a heterogeneously enhancing mass in the left petrous apex that extended to involve meckel 's cave , the lateral cavernous sinus , and the internal auditory canal ( figure 2 ) . she was discharged home on postoperative day three in stable condition for followup with oncology . a 79-year - old man with known history of prostate cancer presented with several weeks ' history of progressively worsening double vision and eventual right eye ptosis . mr imaging disclosed an enhancing soft tissue mass involving the clivus , pituitary fossa , cavernous sinus , and posterior nasal cavity ( figure 3 ) that surrounded both internal carotid arteries in the cavernous sinuses . an endonasal transsphenoidal approach to the sphenoid sinus was carried out to obtain a biopsy of the lesion . a histopathological diagnosis of metastatic melanoma was made , and evaluation was undertaken by the oncology team . ) a 21-year - old man with a remote history of osteosarcoma and newly diagnosed metastatic renal cell carcinoma had complaints of worsening vision and facial pain . because there was a discrepancy in the pupillary size between his eyes , he underwent a computed tomography ( ct ) scan of the head , followed by craniofacial mr imaging , which revealed an enhancing mass in the right sphenoid sinus with adjacent extension . on neurological examination , he was noted to have complete hemifacial numbness and horner 's syndrome on the right side . mr imaging of the face demonstrated a homogeneously contrast - enhancing lesion centered within the right sphenoid sinus measuring 33 20 27 mm ( figure 4 ) . the lesion extended into the carotid canal , pterygopalatine fossa , and optic nerve canal with destruction of the vidian canal and foramen rotundum on the right . a 42-year - old woman with several weeks of frontal headaches initially presumed to be secondary to a sinus infection underwent mr imaging after antibiotic medications failed to alleviate her symptoms . brain mr imaging demonstrated a lobulated , 25 20-mm sellar and suprasellar lesion with extension into the right cavernous sinus and encasement of the right internal carotid artery . this lesion was isointense on t1- and t2-weighted images and heterogeneously enhancing with gadolinium administration . a ct scan was consistent with a lytic lesion involving the central skull base extending laterally and posteriorly to involve the bilateral medial sphenoid wings and the clivus , respectively . histopathologic evaluation was consistent with a diagnosis of diffuse large b - cell lymphoma ( figure 5 ) . a 53-year - old woman presented with a known diagnosis of breast cancer and a one - year history of worsening left eye vision with a more precipitous decline in the last month . mr imaging of the brain demonstrated a skull base lesion involving the left sphenoid bone , the anterior clinoidal process , and the cavernous sinus . on neurological assessment , the patient was noted to have an acutely diminished visual acuity in her left eye to a level of finger counting with left temporal visual field cut . mr imaging ( not shown ) showed a homogenously contrast - enhancing lesion of the skull base involving the greater sphenoid wing and the anterior clinoidal process with encasement of the optic nerve on the left . a left frontotemporal craniotomy was performed for biopsy of the lesion and to decompress the optic nerve and the cavernous sinus with a subtotal resection . one year after surgery , her neurological findings were unchanged , and her systemic disease was under control . neoplasms originating from a multitude of sites have been reported to metastasize to the sps region . breast and lung cancer account for approximately two - thirds of sps metastases , being the most common sources in women and men , respectively , [ 11 , 1821 ] . histological examinations of the tissue samples obtained during palliative hypophysectomy performed in patients with end - stage breast cancer and from autopsy series have documented metastasis to the sps region in 6% to 29% of cases [ 3 , 2225 ] . one hypothesis put forth to explain this prevalence is that the prolactin - rich environment of the pituitary enhances the proliferation of breast tumor cells . after carcinoma of breast and lung , lymphoma and prostate cancer have been reported to be the most common sources of metastasis to sps region . liver , renal cell , colon , and thyroid cancers and melanoma are rare sources of distant metastases to this region . the relatively rarer occurrence of our other cases , prostate , melanoma , renal cell , and lymphoma , is consistent with the literature . most cases are found in the sixth or the seventh decade of life as a part of a generalized metastatic spread , commonly associated with multiple , particularly osseous metastases [ 18 , 20 ] ; however , metastases can occur in young patients . the age of presentation ranged from 21 to 82 years in our small series , with an average age of 60 years . very occasionally , these lesions are the first manifestation of an occult cancer or the only site of metastasis [ 14 , 20 ] . thus , in a patient without any prior history of cancer , an sps lesion can not be assumed to be an adenoma , just as in a patient with a known primary cancer , it is not always metastatic . clinically , metastasis is generally suspected in patients with rapid onset and progressive symptoms , irrespective of a history of malignancy . the possible metastatic pathways to the pituitary and parasellar region include direct blood - borne metastasis to the posterior pituitary lobe , pituitary stalk , clivus , dorsum sellae , or cavernous sinus or leptomeningeal spread with involvement of the pituitary capsule [ 10 , 26 , 27 ] . there has been some controversy regarding the most common location of metastasis within the pituitary gland . authors of early series have reported that the majority of pituitary metastasis occurs in the posterior pituitary , but some dispute this claim . teears and silverman reported that 57% of the lesions localized to the posterior pituitary alone , 13% to the anterior pituitary alone , 12% to both lobes , and the remaining 18% to the capsule or stalk . they hypothesized that the posterior pituitary , by receiving direct arterial supply , is more likely to develop metastases than the adenohypophysis , which receives its blood supply from the hypophyseal portal system . the posterior lobe has a larger area of contact with the adjacent dura , which may be another contributing factor [ 4 , 18 ] . metastatic inoculation in the anterior lobe is usually the result of contiguous spread from the posterior lobe . these may be isolated , such as diplopia or ptosis , with the third ( oculomotor ) and the sixth ( abducens ) nerves being the most commonly involved , followed by the fourth ( trochlear ) nerve [ 14 , 2931 ] , or they may appear in a constellation of symptoms characterized by unilateral , rapidly progressive ophthalmoplegia with retroorbital pain . this latter presentation is the usual presentation of the cavernous sinus syndrome , also known as parasellar syndrome . if the branches of the trigeminal nerve are affected , alteration in the facial sensation , facial pain , or dysesthesia occurs . headache has been reported as a rather common symptom , with an incidence as high as 70% [ 14 , 34 , 35 ] ; however , the majority of pituitary metastases are clinically silent . in the autopsy study by teears and silverman , these metastases are often seen in patients with terminal malignancy who present with malaise , generalized pain , central nervous system involvement , or treatment - associated symptoms , although symptoms of pituitary insufficiency may be masked . several studies have indicated that diabetes insipidus ( di ) was the most common symptom [ 10 , 20 , 24 , 36 , 37 ] . in the series of mccormick et al . , di developed in 70% of patients ; however , if the anterior pituitary function is compromised , di may be concealed by reduced mineralocorticoid function . in some recent series , di has not been reported , which is likely because modern imaging techniques are able to detect abnormalities earlier than the timeframe required for di development . other rarer hormonal findings may be hypothyroidism and hypoadrenalism , hypogonadism , or overproduction of adrenocorticotropic hormone ( acth ) , growth hormone ( gh ) , or prolactin [ 10 , 3840 ] . because of their invasiveness , pituitary metastatic lesions have a high potential to cause visual deficits from suprasellar extension , with an incidence as high as 50% reported by branch jr . and laws jr . and others [ 10 , 34 , 41 ] . in both the series by chiang et al . and that of sioutos et al cranial nerve palsies involving the third and the sixth nerves and facial pain with numbness ( trigeminal origin ) were the most common presentation ( 33% ) in our cases with cavernous sinus involvement . retro - orbital pain , vision compromise , gait instability , horner 's syndrome , and hearing loss were infrequent and associated with petroclival and sphenoorbital extension of the lesions . all these symptoms were experienced with a relatively rapid onset from a few weeks to a few months that suggested the aggressive character of the lesions . symptoms strongly suggesting metastasis in the parasellar or sellar space include painful ophthalmoplegia in association with the sudden onset of di [ 10 , 26 , 34 , 42 ] . the pain may be retro - orbital or may be due to trigeminal dysfunction [ 4345 ] . in our series , 5 of the 6 ( 83% ) patients had a previous history of malignant disease . one of the patients that had prior history developed a malignancy ( melanoma ) other than the original one . only one patient without a prior history of malignancy first presented with lymphoma metastasis to the sps region . because pituitary adenomas also present with invasion of the sellar floor , cavernous sinus , or clivus , no specific neuroimaging criteria to define metastatic lesions in sps region have been reported . the diagnostic imaging tools for sps metastasis mainly include high - resolution ct and mr imaging . although ct is superior to mr imaging in detecting the bone involvement , the latter is preferable to determine the relationship of the lesion to the surrounding neurovascular structures . although nonspecific , the characteristics of these lesions on mr imaging are an iso- or hypointense mass on t1-weighted imaging with a usually hyperintense signal on t2-weighted imaging , and homogeneously enhancing mass in images obtained after the administration of contrast agent . invasion of the cavernous sinus , sclerotic changes around the sella turcica and clivus , isointense signal on both t1- and t2-weighted imaging , and loss of high - intensity signal in the posterior pituitary have been reported to be helpful in differentiating metastatic lesions from benign ones [ 15 , 30 , 48 ] . . found that thickening or enhancement of the infundibulum was the most characteristic ct or mr imaging feature . schubiger and halter reported that the invasion of the infundibular recess by a suprasellar mass is suggestive of metastasis . because of the rapid growth of metastatic lesions , a dumbbell - shaped intra- and suprasellar tumor with indentation at the diaphragm level is generally indicative for these cases [ 20 , 37 , 49 ] . the radiodiagnostic findings that suggested a malignant / metastatic process in our cases were the involvement of multiple compartments in the anterior , middle , posterior cranial fossae , extension to the infratemporal and pterygopalatine fossae , sphenoid sinus , and nasal cavity with bony destruction in the cranial base and asymmetric or bilateral invasion into the cavernous sinus . the management of sps metastases is multimodal , including surgical resection , radiation therapy , and chemotherapy . treatment is mainly palliative and depends on the symptoms and the extent of systemic disease [ 10 , 51 ] . because of the invasiveness and the high vascularity of the tumor , total surgical resection is generally not undertaken [ 1 , 20 ] . therefore , surgical treatment should aim for symptomatic relief and the preservation of visual function , even in patients with widespread primary disease , and should be followed by local radiation treatment and/or chemotherapy [ 10 , 51 ] . the body of evidence is inconclusive on the effect of the latter two modalities on survival [ 14 , 52 ] . laws jr . reported improvement in symptoms , especially in pain and visual field defects , with no difference in survival after complete resection compared with subtotal or partial resection [ 10 , 14 ] . on the other hand , others have supported the concept of improvement in survival after surgical resection of the lesion [ 5355 ] . surgical exploration is also essential if tissue diagnosis is likely to affect therapy in patients with no known primary malignancy . resection is most commonly done via transsphenoidal route , although subfrontal or pterional approaches are also options depending on the location and the extension of the lesion . four out of six patients in our series underwent surgical biopsy through either transsphenoidal or transcranial route and then underwent subsequent radiation / chemotherapy . one patient who had visual compromise had subtotal resection for palliation followed by radiation / chemotherapy . one patient who had renal cell carcinoma was directly referred to radiation oncology for immediate radiation and subsequent chemotherapy . besides its role as an adjunct after surgery [ 14 , 42 , 56 ] , radiosurgery or conventional radiation is recommended as the initial course of treatment in patients with systemic disease out of control , recurrence in the systemic disease with concomitant sps metastasis , or medical comorbidities that put the patient at risk for a surgical intervention [ 2 , 7 , 18 , 41 , 5762 ] . radiosurgery , which is considered less invasive than conventional radiation , has been reported to achieve good tumor control [ 63 , 64 ] . in a series of 23 patients by iwai et al . , the rates of tumor control and symptom improvement were 67% and 53% , respectively ; however , radiosurgery to the sps region is limited by its potential to cause radiation injury to the surrounding neurovascular structures such as optic apparatus , pituitary gland , or cranial nerves coursing in cavernous sinus . doses reported in the literature for these structures range from 8 to 40 gy [ 63 , 6568 ] , and the optimal dose may be quite variable depending on the proximity of the lesion . furthermore , the debate about whether the radiation should be directed to the sps region alone or to the entire brain continues . chemotherapy is commonly used alone or along with radiation therapy mostly for palliation in the treatment of metastatic disease in sps region . its value has not been adequately studied and reported in the literature . the prognosis of patients with metastases to the sps region is grim in a majority of cases because of the aggressive character of the primary disease . even in patients with no other metastasis at the initial evaluation , the prognosis remains poor because of radiologically undetectable microscopic metastases ; however , it has been suggested that the extent of systemic disease affects survival in these patients . patients with a single sps region metastasis may have a better outcome [ 10 , 20 ] . median survival is less than 2 years independent of the management strategy [ 10 , 14 , 70 ] . the records on the long - term follow - up of the majority of our patients are lacking because the patients were not monitored for surgical outcome , or the follow - up periods were too short . the only patient that had palliative surgery for vision compromise was seen at one year after the surgery with stable neurological findings . suggestive symptoms include rapid onset of progressive ophthalmoplegia with retro - orbital or facial pain , visual impairment , and/or di . management varies depending on whether a primary source is identified , the symptomatology , the location and extent of the lesion , the stage of the primary disease , and the medical comorbidities . subtotal or partial surgical resection is aimed mainly for symptom relief . a multimodal approach involving subtotal resection of the lesion followed by radiation and/or chemotherapy is widely accepted , especially in symptomatic patients whose primary disease is under control . radiation with or without chemotherapy is generally recommended as first - line treatment in patients with advanced primary disease or in those with high - risk medical comorbidities . a biopsy usually precedes radiation therapy if the primary source of the metastasis is unknown . the prognosis for patients is generally poor , independent of the therapeutic modality , and the overall survival is less than two years .

the sellar and parasellar ( sps ) region is a complex area rich in vital neurovascular structures and as such may be the location of first manifestation of a systemic malignancy . metastases to this region are rare ; breast cancer is the most common source among those that metastasize to the sps region . ophthalmoplegia , headache , retroorbital or facial pain , diabetes insipidus , and visual field defects are the most commonly reported symptoms . lack of specific clinical and radiological features renders sps metastases difficult to differentiate from the other frequently encountered lesions in this area , especially when there is no known history of a primary disease . currently accepted management is multimodality therapy that includes biopsy and/or palliative surgical resection , radiation , and chemotherapy . although no significant survival benefits have been shown by the surgical series , surgical resection may improve quality of life . here we review the relevant literature and present six illustrative cases from our own institution .

when a septal deviation ( sd ) is present during the endoscopy performed before a transcanalicular diode laser - assisted dacryocystorhinostomy ( tcl dcr ) , one tends to think that this anatomical alteration could influence the final result , due to technical difficulties and alterations in osteotomy healing . dacryocystorhinostomy ( dcr ) is the surgical treatment for opening up lacrimal pathways [ 1 , 2 ] . there are three types of dcrs : external , endoscopic , and transcanalicular approach [ 36 ] . we use the transcanalicular approach ( tcl dcr ) for all patients , except recurrences , which we treat externally . endoscopically nasosinusal anatomical variations may appear , such as septal deviation ( sd ) , inferior turbinate hypertrophy , and concha bullosa [ 79 ] . we designed the present study in order to investigate the influence of nasal septal deviation on the surgery of lacrimal pathways . if significant influence can not be proved , then previous or concomitant septoplasty could be avoided in candidates for surgery of the lacrimal pathways ( lp ) . from january 1 , 2008 , until december 31 , 2010 , one hundred and twenty - four patients were considered candidates for tcl dcr surgery , so they were included in this study . a protocol was designed , including personal data , medical history , and degree of epiphora . in order to quantify and standardize the degree of epiphora candidates for tcl dcr surgery had to meet the following conditions : need to dry tears more than 5 times a day ( munk score : 35 ) , blockage of the vertical part of the lacrimal pathways , presence of lacrimal sac proved by dacryocystography , symptoms ( epiphora ) , chronic dacryocystitis , and/or a history of acute episodes . the patients were seen by the otorhinolaryngologist and the ophthalmologist in the same follow - up visit , in order to assess the feasibility of tcl dcr ; septal deviations were noted . during this multidisciplinary consultation , we chose a classification based on the distance separating septum from lateral nasal wall : mild ( less than 50% the distance from septum to nasal wall ) ; moderate ( greater than 50% the said distance ) ; and severe ( septum touched the lateral nasal wall ) . patients younger than eighteen , with previous dcr ( whatever the technique ) , with other disorders of ocular annexes , or lacking in motivation ( they interfere with the munk score ) were excluded on this study . all patients signed the consent form for tcl dcr to participate in this clinical trial and were operated on without taking into consideration the presence of sd or the lack of it . this device uses an ingaasp diode laser generator and a semiconductor with a wavelength of 980 nm ( 5 ) with a maximum input power of 20 w. the corresponding silica optical fiber was sterile and disposable , measuring 600 microns . the ent used a karl storz tube with an optical angle of 0 degrees . during the postoperative period topical treatment twenty - four hours after surgery nasal irrigation with saline water and topical nasal applications of fluticasone furoate were prescribed . during the follow - up revisions endoscopies ( one month , three months , and six months ) were performed and traces of fibrin were removed ; an assessment was also carried out , taking into account presence of epiphora ( munk scale ) , positive or negative nasal syringing with fluids , and endoscopic appearance of the osteotomy site . surgery was deemed success when the patients scored 0 or 1 on the munk scale , in which they had to dry tears twice or less than twice a day , six months after the operation . therefore , we considered failure when they scored 2 to 5 on the munk scale . we divided the patients who had undergone tcl dcr into two groups : one group included lp with no anatomical nasosinusal variants and the other lp group was with sd or other nasosinusal alterations . this prospective , nonexperimental clinical study was carried out correlating clinical features with a longitudinal analysis . dichotomous variable presence or absence of sd has been studied and compared in both groups . contrasts referred to equality of means of anova like duration of procedure , age , and duration of epiphora in years , pearson 's correlation coefficient like age and duration of epiphora , or modeling explanatory ability of those predictor variables acting on probability of success , using the logistic regression model like successful syringing at 3 and 6 months after operation , presence of granulomas , presence of synechiae , presence of postoperative granulomas , and presence of sd , have been used . the study included 124 patients , in which 159 lp operations were performed , 102 lp ( 64.15% ) did not have any anatomical variants , and 57 lp ( 35.84% ) had anatomical variants . out of these 57 lp with anatomical variants , 39 had sd ( 68.42% ) : in 21 of them it was mild ( 53.84% ) , in 15 it was moderate sd ( 38.46% ) , and in 3 it was severe sd ( 7.6% ) . other anatomical alterations were found in the remaining 18 lp ( 31.57% ) : one lp with concha bullosa ( 0% ) and 17 lp with hypertrophic inferior turbinate ( 51% ) . in the group with no anatomical alterations , 69 lp had a successful postoperative outcome ( 67.6% ) . in the sd group , 26 lp had a successful postoperative outcome ( 66.7% ) . according to the logit method , the difference between both groups in postoperative outcome was not significant ( p > 0.05 ) . if we compare the success rate of the sd group ( 66.7% ) and other anatomical alterations ( 44.1% ) in our study , the difference is statistically significant ( p < 0.05 ) . we also compared success rates in each sd group ( mild , moderate , and severe ) . the success rate for each group was as follows : 66.67% for mild sd ; 66.60% for moderate sd ; and 66.66% for severe sd . in patients with severe sd ( 3 ) , a previous septoplasty was necessary in order to make a correct tcl dcr possible later on , as a part of the same surgical procedure . there were no complications during or after these procedures , no intraoperative bleeding , and no postoperative infection and all patients were discharged 4 - 5 hours after the operation . the demographic data collected for our sample agree with that found in worldwide medical literature , as far as sex [ 1315 ] , age [ 1316 ] , and race are concerned . there was a higher success rate in the sd group ( 66.7% ) compared to the rest of anatomical alterations ( success in this latter group amounted to 44.1% ) , but the former had a lower success rate when compared to the group with no anatomical alterations ( 67.6% ) . most of septal deviations ( 69.6% ) are inferior ridges or posterior deviations , so they did not need surgery . there is no need for previous or concomitant septoplasty in some cases like mild and moderate sd , because the success rate was similar . all severe sd were operated on during the same procedure ; therefore , access to the middle meatus in the nasal fossa presented no difficulties . one month and three months later , most patients scored 0 to 1 on the munk scale . it could change the scores from 0 to 2 over the course of the 6 months following surgery , so the success rate would decrease . we have not found articles referring to surgical success in patients with sd in scientific literature . in our study , we found no significant statistical differences between the success rate in the two groups . furthermore , we could avoid previous or concomitant septoplasty in some cases like mild and moderate sd because the success rate was similar to nonseptal deviation group .

purpose . the objective of the present study is to determine whether the success rate in transcanalicular diode laser - assisted dacryocystorhinostomy ( tcl dcr ) is influenced by the variant septal deviation ( sd ) . methods . patients were divided into two groups : one including operated lacrimal pathways ( lp ) with no anatomical nasosinusal variants and the other group of lp with sd . this study began on january 1 , 2008 , and ended on december 31 , 2010 , at morales meseguer hospital . variables were compared by means of anova and a logistic regression model ( logit ) . results . out of the 159 lp operated on , 102 had no nasosinusal anatomic variant , but 39 lp were associated with sd . the first group evidenced a success rate of 67.64% , while the second group evidenced a success rate of 66.7% . conclusion . we found no significant statistical differences between the success rates in the two groups ( with sd and no anatomical variants ) . so we could avoid previous or concomitant septoplasty in some cases ( mild and moderate sd ) .

when do such problems become abnormal and merit a specific psychiatric diagnostic label , remains a question under study with no clear cut answers . we present a case of a young girl for whom clinical attention was sought for transient eating problems . a 12-year - old young lady belonging to lower socioeconomic rural background was seen by the psychiatry services when referred for problems of eating since past 2 months . the patient had a history of delayed development and achieved milestones slower than other siblings . she would eat what was given to her by her mother , and ask for specific food items occasionally . around 2 years back , the patient developed generalized tonic clonic seizures during sleep which were followed by myoclonic jerks . the electroencephalograpm ( eeg ) findings were consistent with a diagnosis of juvenile myoclonic epilepsy ( jme ) . the episodes of generalized tonic clonic seizures were well controlled with valproate , but myoclonic jerks continued to recur . due to the occurrence of jerks at school , other children would tease her . she became irritable with her family members quite often , and would hit the siblings when provoked . she did not have depressive cognitions or change in sleep or appetite then . due to behavioral problems of refusal to go to school and irritability , intelligence quotient ( iq ) assessment was done and reported as 57 with a diagnosis of mild mental retardation . since around 2 months prior she would eat rice at the previous amounts , but would refuse to eat rotis , the staple diet of the family . if persuaded , she would get irritable and start crying and go to sleep without eating anything . at times she said that she was hungry and would ask for food , and would eat rice but refuse rotis . she did not have any associated loose stools , abdominal pain , vomiting , or regurgitation . she would not demand for specific food items like chocolates , etc . ; would not appear sad , withdrawn or fearful , or have any disturbances in sleep . over the period of 1 month , the weight reduced by about 1 kg . on further exploration , no preoccupation with body image , of previously fussiness of eating was reported by the family members . the child had a slow to warm up temperament and there was no history suggestive of eating inedible objects , delusions , hallucinations or repetitive behaviors , significant obstinacy , hyperactivity or inattentiveness . the general physical examination was within normal limits , the height and weight was over the 10 percentile and there were no signs of chronic malnutrition . rapport could be established with difficulty and the child answered briefly only after urging from the mother and therapist . when patient returned in 2 weeks , there was no further decrement in weight and the patient started to eat all kinds of food stuffs again and to the previous amounts . even on recovery , she did not elaborate the cause of decreased intake of certain foodstuffs and that of resumption . multiple possibilities can be entertained in this child while evaluating a child presenting with such eating problems . the age appropriateness of such behaviors , development level aptness , sociocultural background , parenting style , and medical conditions need to be ascertained before considering whether such a behavior can be assessed as abnormal . feeding habits in adolescents reveals deficits in calories and proteins in apparently healthy school - going children from relatively well - to - do backgrounds . with such a premise , is diagnosing the transient problems of eating as a separate disorder in this child a useful and relevant exercise , or whether these problems can be passed off as developmentally appropriate erratic behaviors ? even if a diagnostic label is required , where does the child fit in ? is the diagnosis of mental retardation sufficient to subsume these symptoms or an additional diagnosis is needed ? since the diagnostic categories of affective , psychotic , and anxiety spectrum disorders do not apply in this case , whether the problems can be better characterized as eating disorder not other specified ( eating disorder nos ) ? however , there was no evidence of body image disturbances . does feeding disorder of infancy and childhood better characterize this case , though the obvious atypicality being age of presentation and transient symptoms in this case ; or does this case remain a many researchers have proposed alternate pragmatic classification systems for characterizing the problems of eating in childhood . fox and joughin have described a variety of eating problems that are encountered in the childhood and adolescence . among these , the index case exhibits symptoms closest to food avoidance emotional disorder which occurs in the age range of 5 - 16 years and is characterized by avoidance of food and emotionality in the absence of preoccupation of body weight or body image . many children who present with behavioral problems of eating can not be pigeonholed into diagnostic class of present nosology , but still benefit with interventions . hence , we suggest that these disorders should be treated pragmatically after detailed assessment without being excessively concerned about the diagnostic label , at the same time not overlooking the obvious diagnostic possibilities .

eating problems are commonly encountered in childhood and adolescents , and may be manifestation of a variety of psychiatric disorders when medical causes are excluded . we present the case of a young lady presenting with problems of eating which presented with difficulties of ascribing a diagnostic label for eating problems . the patient , a 12-year - old girl with history suggestive of mild mental retardation and juvenile myoclonic epilepsy presented with selective eating of foodstuffs which improved spontaneously in 2 months . the different diagnostic possibilities entertained for the case are discussed .

adenoid cystic carcinoma ( adcc ) is a malignant salivary gland tumor that was first described by billroth in 1859 under the name cylindroma attributing to its cribriform appearance formed by the tumor cells with cylindrical pseudolumina or pseudospaces . the term adenoid cystic carcinoma was introduced by ewing ( foote and frazell ) in 1954 . adcc is a relatively rare malignant salivary gland tumor comprising less than 1% of all malignancies of head and neck . it is the 5 most common malignancy of salivary gland origin , representing 5 - 10% of all salivary gland neoplasms . the parotid and submandibular glands are the two most common sites for adcc accounting for 55% of the cases . among the major glands , adcc accounts for 8.3% of all palatal salivary gland tumors and 17.7% of malignant palatal salivary gland tumors ( afip series ) . the other less common sites are lower lip , retromolar tonsillar pillar area , sublingual gland , buccal mucosa and floor of the mouth . the nose and paranasal sinuses represent the next most common sites for minor gland adccs . the tumor is most often clinically deceptive by its small size and slow growth , which actually overlies its extensive subclinical invasion and marked ability for early metastasis making the prognosis questionable . the three recognized histological variants of adcc are cribriform , tubular and solid although , cribriform is the most commonest and solid is the least commonest . it is not uncommon to have more than one histopathologic pattern in a single neoplasm and rather all three patterns can be observed in the majority of tumors . the main reason for histological typing is to assess the prognostic difference between histologic types . tubular pattern ( well differentiated ) is believed to have the best prognosis compared to the cribriform pattern ( moderately differentiated ) and solid pattern ( poorly differentiated ) . a 60-year - old male patient reported to the department of oral and maxillofacial surgery with a chief complaint of pain and discharge in the upper left back region of the jaw since one and half years . according to the patient one year prior to our consultation , he got the maxillary molars extracted from a local dentist due to pain and mobility of the teeth in that area . upon consultation patient revealed that the pain continued to exist even after the extraction . the patient is a known hypertensive and is on medication since three years for the same . the patient had smoking habit , which he quit only a couple of years ago . intraoral examination revealed a solitary erythematous swelling with diffuse borders involving the left posterior portion of hard palate and was also seen involving the soft palate on the same side . the swelling was seen extending from mesial aspect of first premolar to 1 cm posterior to tuberosity on left side , involving the palatal area but not crossing the midline [ figure 1 ] . intraorally , the lesion was seen involving the hard and soft palate but not crossing the midline the swelling was nontender . the clinical differential diagnosis included a benign or malignant neoplasm of minor salivary glands , a neoplasm of maxillary sinus . ct scan showing complete obliteration of left maxillary sinus an incisional biopsy of palate and sinus lining was performed . islands of uniform cells arranged in cord - like pattern with hyperchromatic nuclei were seen enclosing round to oval pseudocystic spaces in the stroma . the tumor cells are also seen arranged in the form of solid islands [ figure 4 ] and ductal pattern [ figure 5 ] . focal areas showed small cords and longitudinal tubules of isomorphic cells set in a background of densely hyalinized stroma [ figure 6 ] . variable - sized cribriform spaces formed by small deeply basophilic cells imparting swiss cheese pattern solid pattern composed of nests of basaloid cells and mitotic figures tubular pattern characterized by ductal structures formed by layers of isomorphic basaloid cells tubules of isomorphic tumor cells set in a densely hyalinized stroma a diagnosis of adcc ( cribriform pattern ) was established . the patient was treated by wide surgical excision with clear margins and hemi - maxillectomy of left maxillary region with post - radiotherapy . the present case was staged as t4n0m0 based on american joint committee on cancer as a guide to prognosis . the patient was under regular follow - up and is free of the disease at one - year follow - up . adenoid cystic carcinoma most often presents a diagnostic and treatment challenge owing to the rarity of the lesion . most findings regarding adcc are actually based upon studies with a small number of patients and there is a need for further information regarding its clinical behaviour as well as treatment modalities and their results . adcc may occur at any age although in most cases the patients age ranged from 24 to 78 years . the age of patients affected with major salivary gland tumors has been shown to be younger ( mean 44 years ) compared to the age of those who developed tumors of the minor glands ( mean 54 years ) and shows female predilection ( f : m 1.2:1 ) . pain is a common and important finding , occurring early in the course of the disease before there is a noticeable swelling . palate is more commonly involved generally the area of greater palatine foramen . among the malignant neoplasms of minor salivary glands , the most common was mucoepidermoid carcinoma ( 21.8% ) followed by polymorphous low grade adenocarcinoma ( plga ) ( 7.1% ) and adccs of the minor glands have been reported to have worse prognosis than those of the major salivary glands . tumors involving the nose , paranasal sinuses and maxillary sinus have the worst prognosis as they are usually detected with higher stages at the time of diagnosis . tumors of minor salivary glands usually have the tendency to infiltrate extra glandular soft tissues and bone thereby allowing increased dissemination of the tumor . lymph node involvement is uncommon ( < 5% of cases ) and is usually due to contiguous spread rather than lymphatic permeation or embolization . the differential diagnosis of adcc includes plga , basal cell adenoma ( bca ) , mixed tumor and basaloid squamous cell carcinoma ( bsc ) . the cribriform pattern so typical of adcc may also rarely be seen in bca , mixed tumor and plga , and the distinction between plga and adcc is more challenging . a polymorphous architecture characterizes plga whereas adcc has a more limited range of histologic patterns with no more than three patterns . plga shows uniform cell population with cytologically bland , round or oval vesicular nuclei and pale eosinophilic cytoplasm where as cells in adcc have clear cytoplasm , angular , hyperchromatic nuclei and may show mitotic activity . the ki-67 index is reported to be 10 times higher in adcc compared to plga . smooth muscle markers of myoepithelial differentiation are positive in adcc but negative in plga . though plga may form solid areas , they lack the overall high - grade feel associated with adcc . occasional foci in pleomorphic adenoma ( pa ) can resemble adcc but the presence of typical myxochondroid matrix and plasmacytoid or spindle shaped cells helps to avoid confusion . cribriform structures may sometimes be observed in bca and such cases can be differentiated from adcc on the basis of gradual structural alteration from areas typical of bca . the peripheral palisading and focal squamous differentiation with whorling pattern present in bca are not usually encountered in adcc . the basement membrane material secreted by bsc tends to dissect between tumor cells rather than form crisp cribriform spaces seen in adcc . focal keratinization , attachment to rete pegs , and presence of surface squamous dysplasia or carcinoma in situ helps to distinguish it from adcc . immunohistochemical studies demonstrated that the pseudocysts are positive for periodic acid schiff reagent ( pas ) and alcian blue and contain basement membrane components such as type iv collagen , heparin sulfate and laminin isoforms . epithelial cells are positive for carcinoembryonic antigen ( cea ) and epithelial membrane antigen ( ema ) . duct lining cells are positive for c - kit ( cd117 ) and myoepithelial cells are positive for s-100 protein , calponin , p63 , smooth muscle actin and myosin . expression of s-100 , glial fibrillary acidic protein ( gfap ) and neural cell adhesion molecule have been correlated with the presence of perineural invasion . molecular genetic data : alterations in chromosomes 6q , 9p and 17p12 - 13 are the most frequent cytogenetic alterations reported . hypermethylation of the promoter region of the p16 gene was associated with higher histologic grades of malignancy . possible treatment modalities available for the treatment of adcc are surgery , radiotherapy , chemotherapy and combined therapy . it requires excision with the widest surgical margins possible as tumor cells extend well beyond the clinical and radiographic margins . neutron therapy , which involves larger particles of greater energy can achieve reasonable local control as a primary therapeutic modality . main factors associated with patient survival are anatomic location , primary lesion size , presence or absence of metastasis at the time of diagnosis , perineural invasion and histological variant . distant metastasis occurs in 25%-50% of patients and lung is the most common involved site . the 5-year survival rate after effective treatment is 75% , but long - term survival rates are low ( 10 years-20% and 15 years-10% ) . the present patient was recalled at every 6-month interval in view of the sinus involvement and propensity for distant metastasis . lesions involving the sinus tend to have a poor prognosis due to its infiltrative growth and distant metastasis .

adenoid cystic carcinoma is an uncommon , slow growing malignant salivary gland tumor that is characterized by wide local infiltration , perineural spread , propensity to local recurrence and distant metastasis . in this paper , the authors present a case of adenoid cystic carcinoma affecting the palate and involving the maxillary sinus in a 60-year - old male patient along with a brief review of literature .

interacting with other human beings is a basic element of daily life , yet not a trivial challenge . many joint actions between adults are highly sophisticated , but are performed with apparent ease ( knoblich and jordan 2003 ; sebanz et al . 2006 ) . cooking a meal with friends , lifting a heavy bag together , or dancing with others are only a few of numerous examples for joint actions . in the early years of life , however , children still have difficulties coordinating their actions with those of others ( brownell et al . the question arises as to which mechanisms underlie the development of joint action capabilities . for adults , it has been shown that a key factor for successful joint action is the involvement of the motor system of the brain . motor - related brain activity is not only observed during execution of one s own actions but is also important for predicting and incorporating a partner s actions ( see bekkering et al . recent findings by kourtis and colleagues indicate that the motor system in adults is more strongly activated when they predict actions of a joint action partner as compared to those of an individual actor . for adults , being engaged in a joint action thus has an effect on the involvement of their own motor system when observing the actions of another person ( kourtis et al . an interesting possibility to investigate the underlying neurocognitive mechanisms of action observation during joint action is to look at early stages of joint action in development . to date , it is unclear whether similar effects of motor system involvement can be observed when young children are engaged in a joint action . therefore , the aim of the current study was to investigate young children s brain activity around the age that they begin to incorporate others actions into their own action performance more successfully ( cf . more specifically , we examined whether being engaged in a joint action modulates the involvement of young children s own motor system when observing the actions of another person , and if so , how this motor involvement is associated with their joint action performance . early forms of joint action such as handing over a toy can be observed already in infancy ( carpenter 2009 ) . however , infants cooperation with adults often requires extensive scaffolding by the adult action partner ( see e.g. , warneken and tomasello 2007 ) . despite infants motivation to act jointly with another person ( see carpenter 2009 , for a review ) , the nature of their cooperation attempts is still not entirely mutual . it is during the second and third year of life that joint actions with peers and adults become more reciprocal and sophisticated ( brownell et al . , young children can cooperate successfully in simple interactive games like letting a ball bounce on a little trampoline by holding and moving the frame of the trampoline jointly ( warneken et al . children aged 2 years and older reliably solve simple cooperation tasks with peers ( brownell and carriger 1990 ) and show improvement in monitoring their partner s actions and integrating them into their own action performance ( brownell et al . , children have been shown to coordinate their actions in a sequential button - pressing task as accurately with an adult partner as when acting on their own ( meyer et al . , children reliably succeed in joint actions that involve complementary roles for the two action partners ( ashley and tomasello 1998 ) , something which appears to be difficult for younger children ( hunnius et al . , the ability to successfully cooperate with others is a skill that develops gradually during early childhood . from a neurocognitive perspective , successful joint actions require the brain to connect observed actions of others with their own motor system in order to adapt own actions accordingly . first developmental studies on action execution and action perception in young children reveal mechanisms of motor system involvement comparable to those found in adults ( see e.g. , hari and kujala 2009 ; rizzolatti and craighero 2004 , for adult studies ) : already during infancy , motor - related brain activity has been found for action execution as well as for the observation of another person s actions ( lepage and thoret 2006 ; marshall et al . 2010 ; nystrm 2008 ; shimada and hiraki 2006 ; southgate et al . motor involvement during one s own actions and during the observation of others actions can be studied by measuring oscillatory activity in the eeg signal . in both children and adults , motor activation has been associated with a power reduction in the mu- and beta - frequency bands above motor areas ( caetano et al . a recent eeg study investigated 14-month - old infants brain activity while they were pressing a button on their own or observing an adult doing the same ( marshall et al . decreased power was found in the infant equivalent of the mu - frequency range over central electrode sites . this is in line with previous findings of reduction in mu - power during action execution and observation in adults ( caetano et al . moreover , the involvement of the motor system during action observation was shown to be modulated by children s action experience ( van elk et al . more motor involvement indicated by less mu- and beta - power over motor areas was found during observation of an action that children had more experience with . until now , developmental brain imaging studies have focused on execution and observation of individual actions . however , how young children s brain activity is modulated in the context of real - time joint action is still unexplored . previous research in adults reveals that the involvement in a joint action has an effect on the neurocognitive processes associated with the other person s actions ( see bekkering et al . for instance , monitoring a person s action in a cooperative context showed an early component of error - related brain activation that was absent when the other person acted in a competitive context ( koban et al . 2010 ) . in a recent set of experiments , kourtis and colleagues ( 2010 ) investigated the involvement of the motor system when observing a joint action partner or an uninvolved actor . in one of their experiments , they created a social context in which two action partners were facing each other , while an individual actor was sitting next to them . in a go / no - go paradigm , motor - related brain activity was assessed using eeg . a stronger decrease in beta - power and changes in motor - related potentials , which reflected stronger anticipatory motor activation , were found for one s own action partner than for the individual actor ( kourtis et al . hence , the mere involvement in a joint activity with another person modulated the observer s motor activation when observing the partner s actions as compared to observing the actions of a person not involved in the joint action . as indicated by previous research , the involvement of one s own motor system plays a crucial role in understanding and predicting others actions ( see sebanz and knoblich 2009 , for a review ) . when acting jointly , it is especially important to be able to understand and predict the other person s actions so that it is possible to constantly adjust one s own actions to those of the partner . therefore , the activation of the motor system , which is thought to support action understanding and action prediction ( de lange et al . 2008 ; iacoboni et al . 2005 ; rizzolatti and sinigaglia 2010 ; southgate et al . 2010 ) , is of special relevance in joint actions . still , little is known about the role of the motor system in early joint action development . the current study is the first to investigate motor involvement in young children who are engaged in a joint action with another person . in particular , we were interested in whether motor - related brain activity while observing another person s actions is modulated by being involved in a common joint action . furthermore , the aim of this study was to shed light on the link between the activation of young children s motor system during action observation and their joint action performance . in order to investigate young children s motor involvement in joint action , we simultaneously assessed 3-year - olds brain activity and their performance in a joint action task . the experimental setup consisted of a simple computerized button - pressing game in which a cartoon figure had to be moved up a ladder by pushing two buttons alternately ( cf . the game was played in different conditions that varied the children s involvement in the joint action . in the joint action condition , children were playing together with an adult partner , taking turns to push the two buttons . in the joint action observation condition , the children watched the same adult play the game together with a third adult actor . to determine modulations in the child s motor system activation , we compared children s brain response during their partner s actions in the two conditions . finally , we correlated the outcome of the eeg analysis with children s joint action performance to examine the relation between their motor system involvement for the partner s actions and their own action performance . the final sample consisted of seven 3-year - old children ( 5 boys ) with a mean age of 36.7 months ( sd = .99 ) . seventeen of these 29 participants were excluded due to lack of or bad eeg recording traces resulting from insufficient time ( i.e. , limited by the little participants patience ) to lower impedances in the preparation phase . another twelve participants were excluded , either due to a lack of at least 8 movement- and artifact - free trials per condition ( n = 10 ) or due to experimental errors ( n = 2 ) . the high dropout rate in the current experiment is consistent with other developmental studies assessing electrophysiological recordings ( cf . 2007 ; southgate et al . 2010 ) . during a sequential joint action game , we recorded brain activity and performance accuracy of the 3-year - old children . figure 1 illustrates the experimental setup that consisted of a simple computerized button - pressing game proved to be suitable for children of this age in a previous study ( meyer et al . 2010 ) . in this game , a cartoon figure of a frog could be moved up a ladder by alternately pushing two buttons . as can be seen in fig . 1 , the visual stimuli were presented on a wide - screen that was tilted to increase the height of the presented ladder and thereby the number of steps required to reach the top . in total , the ladder consisted of 42 steps that were shown on the screen . at the top of the ladder , there was a target location for the frog represented by a cartoon figure of a pig , the frog s friend on a cloud . in front of the screen , we placed two custom - made buttons to control the game and a board with the contours of two hands indicating starting and resting positions of the hands . the two buttons were interconnected via a tilt mechanism such that pushing one button down caused the other button to move up . more precisely , a right button press triggered the frog to move up using its right leg and pressing the left button moved up the left leg of the frog , so that alternating left button presses also elicited a short beep tone ( 60 ms duration ) in order to keep the child s interest and attention . with each button press , eeg markers were sent such that button presses could be traced back in the eeg recordings . 1the experimental setup of the joint button - pressing game . in front of a tilted wide - screen , we positioned two chess - clock buttons and resting positions marked by hand contours . by pressing the two buttons alternately , a cartoon figure could be moved up a ladder on the screen the experimental setup of the joint button - pressing game . in front of a tilted wide - screen , we positioned two chess - clock buttons and resting positions marked by hand contours . by pressing the two buttons alternately , a cartoon figure could be moved up a ladder on the screen each participant was involved in three different conditions of the game : a joint action condition , a joint action observation condition , and an individual action condition . the focus of this paper is on children s motor - related brain activity during action observation with respect to joint actions . we were interested in whether the motor system of the 3-year - olds was activated more strongly while observing others actions when involved in a joint action as compared to watching two people acting jointly without being involved . the same person acted both as the child s joint action partner and together with a third actor in the joint action observation condition . in the current study , we concentrate on the results of the joint action and the joint action observation condition.1 in the joint action condition , we instructed the children to push the right button with their right hand in turns with their adult action partner ( actor1 ) who pushed the left button with her left hand . more specifically , the button - pressing action was supposed to start with the hand on the resting position , which was marked by drawings of hand contours on a board in front of the buttons ( see fig . 1 ) . starting from this position , the action was executed by pushing the respective button and ended when the hand was placed back on the resting position . we thereby aimed to prevent children from leaving their hands on the button throughout the joint play and introduced a standardized action pattern that was comparable across conditions . during the measurement , the children sat on their parent s lap on a chair to the right of actor1 . in the joint action observation condition , the children watched two adults ( actor1 and actor2 ) playing the same game together . while actor1 and actor2 were playing jointly , actor2 sat between the child and actor1 such that the child would have to move only minimally to the right . for all children , the children were not explicitly instructed where to look during the game , but in subsequent steps , only data of trials were included during which children looked at the experimenter , the buttons , or the screen ( see eeg data analysis section ) . video recordings of the entire measurement session were made and aligned with the experimental events on the screen , and children s eeg and button presses were recorded . for demonstration purposes , the joint action observation condition always preceded the joint action condition . depending on the attention span of the children , we additionally included another run of the joint action observation condition after the joint action condition . six out of the seven participants therefore watched actor1 and actor2 play both before and after they played together with actor1 . before pooling together the data of the observation condition from the two time points , we tested for order effects . to make sure that children s motor activation did not differ significantly between the two time points , we compared activity during action observation of actor1 s button press ( t = 450 to 0 ms ) . since no differences in mu- and beta - power were found between data collected before and after the children had played themselves ( for details , see eeg data analysis ) , the data of the joint action observation condition were subsequently pooled . electrophysiological recordings were conducted using child - sized eeg caps with 30 electrode sites on the scalp . the ag / agcl active electrodes were placed in an acticap ( brain products , munich ) , arranged in the 1020 system , and referenced to electrode fcz over the central midline . the signal was amplified using a 32-channel brainamp dc eeg amplifier , band - pass filtered ( .1125 hz ) , and digitized at 500 hz . we strived to keep all impedances below 60 k. we analyzed the data using fieldtrip , an open source matlab ( version 7.0 , themathworks , inc . ) toolbox developed at the donders institute for brain , cognition and behaviour ( http://www.ru.nl/neuroimaging/fieldtrip ) . the eeg data were locked to the button press of the first experimenter ( actor1 ) and determined 450 ms before and 450 ms after the button was pressed . during this time , the children were observing actor1 s actions and the effect on the screen when actor1 was either their joint action partner ( joint action condition ) or the joint action partner of actor2 ( joint action observation condition ) . by including exclusively data from actor1 , we kept the comparison between the two conditions constant . to examine the involvement of the motor system in these two conditions , we focused on electrodes c3 and c4 over motor cortices . as mentioned in the introduction , power decrease in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas is associated with motor activation ( cf . hari 2006 ) and thus is the focus of the current analysis . on the basis of the video recordings of the measurement session , trials were rejected if children moved their hands or did not pay attention to the game ( i.e. , when they looked at neither the experimenter , nor the buttons , nor the screen ) during the critical period of the experimenter s action ( i.e. , the hand movement toward the button ) . since it was an interactive game in which we relied on children s spontaneous behavior , many trials had to be excluded due to children moving during the window of interest ( t = 450 to 0 ms ) . participants with less than 8 trials per condition were excluded from the analyses ( see participants ) . we visually inspected the remaining trials to exclude eeg artifacts ( such as noisy channels or eye blinks ) . as a result , on average , 15 trials remained for the joint action condition ( range 836 ) and 35 trials for the joint action observation condition ( range 1860 ) . a dft filter2 was used to remove line noise from the data , and for each trial , we took out the offset by subtracting the mean signal of the entire trial . we then calculated time - resolved spectral power estimates using the fourier transform in combination with a hanning taper . for this , we used a 300-ms sliding time window that was advanced in steps of 50 ms . we obtained separate tfrs for the joint action condition and the joint action observation condition . to contrast children s brain response in these two conditions , we computed the normalized difference per time frequency sample between the two conditions ( [ tfr actor1 as joint partner tfr actor1 as partner of actor2]/[tfr actor1 as joint partner + tfr actor1 as partner of actor2 ] ) ( cf . the eeg data were locked to the button press of actor1 , which is denoted as zero . hence , children observed actor1 moving her hand toward the button from about 450 ms to 0 . at zero , the button press of actor1 made the frog on the screen move upward . in the period of 0450 ms , children were preparing to press the button themselves in the joint action condition , while it was actor2 s turn in the joint action observation condition . at the same time , actor1 was placing her hand back on the resting position in front of the button.fig . 2a time - resolved normalized difference in power at electrode site c3 . power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . at time 0 , actor1 pushed the button that moved up a cartoon figure on the screen . before time 0 , actor1 moved her hand toward the button . after time 0 , actor1 moved her hand back to the resting position , while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography ( see methods and results ) . b correlation between the individual beta - power difference and the percentage of errors children made during the joint game . c topography of the normalized beta - power difference , including the data points marked by the white box . power differences are displayed on seven electrodes ( only electrodes were used that were sufficiently noise - free for all seven children ) a time - resolved normalized difference in power at electrode site c3 . power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . at time 0 , actor1 pushed the button that moved up a cartoon figure on the screen . after time 0 , actor1 moved her hand back to the resting position , while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . white boxes indicate the time frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography ( see methods and results ) . b correlation between the individual beta - power difference and the percentage of errors children made during the joint game . c topography of the normalized beta - power difference , including the data points marked by the white box . power differences are displayed on seven electrodes ( only electrodes were used that were sufficiently noise - free for all seven children ) in the statistical evaluation of the electrophysiological data , we determined whether power estimates during observation of the goal - directed action of actor1 differed significantly between conditions . we used the window of 450 ms prior to the button press until the button press ( t = 450 to 0 ms ) and the frequency bands of 711 hz ( mu ) and 1721 hz ( beta ) . 2 ) were then averaged over the respective frequency range and time window . by means of one - sample t tests analogous to this analysis , we evaluated the data of the joint action observation condition collected before and after the joint action condition ( with t = 450 to 0 ms ; mu : 711 hz ; beta : 1721 hz ) . due to the relatively small sample size of 7 participants , it might be argued that effects in the mu- and beta - power could be driven by extreme outliers . to exclude this possibility and to provide an overview of the strength of the observed effect in both mu- and beta - power , we ran complementary analyses on an individual participant level . for this purpose , we estimated the power in the mu- and beta - frequency ranges as described earlier for each individual trial per condition and participant . to determine the average power in the two conditions for each participant , we averaged power estimates of mu - power of all trials per participant over the time window of 450 to 0 ms . using the same approach , we obtained average power values in the beta - frequency range for the two conditions per child . subsequently , we evaluated the difference in power between the two conditions ( separately for mu- and beta - power ) on an individual participant basis using independent - sample t tests with trials as units of observation . additionally to the general power difference on a group and individual level , we were interested in how time - locked these differences were to the actions of the joint action partner . in other words , we examined whether an increase in motor activation ( reflected by less power in the mu- and beta - band ) was specifically locked to actor1 s actions or rather reflected a general activation of the children s motor system . to test this , we compared the normalized difference in the mu- and beta - frequency range for the two conditions before and after actor1 pressed the button . if the children s motor system was generally more activated in the joint action condition , we would expect no difference between these two time periods of actor1 s action . however , if the motor activation was time - locked to the actions of children s joint partner , we would expect the power differences to be different before and after actor1 s button press . therefore , we also calculated the average normalized difference of mu - and beta - power for the time period after actor1 s button press ( t = 0450 ms ) . to evaluate the statistical difference between the two time periods , we used paired - sample t tests with time period ( before vs. after actor1 s button press ) as an independent factor , one for testing differences in the mu- and one for differences in the beta - frequency range . to further evaluate the relation of mu- and beta - power during observation of joint action with regard to children s own joint action performance , we correlated the eeg results with the behavioral button press data . on the basis of the significant power differences between conditions , we chose representative time frequency windows ( see white boxes in fig . 2 ) . frequency windows because they represent strongest continuous effect windows time - locked to the button press of actor1 . the normalized difference in power of the respective frequency and time was then averaged to obtain a representative effect value . we subsequently correlated those effect values with the percentage of errors children made when acting jointly . the percentage of errors indicates how often the 3-year - olds pushed their button when it was not their turn . the percentage was computed as number of incorrect button presses of the child ( i.e. , button presses when it was actor1 s turn to press ) divided by the total number of times the child pushed the button when playing the joint game . we used a pearson correlation across participants to test the relation between the effect value of each specified window ( see white boxes in fig . 2 ) and the percentage of errors . the final sample consisted of seven 3-year - old children ( 5 boys ) with a mean age of 36.7 months ( sd = .99 ) . seventeen of these 29 participants were excluded due to lack of or bad eeg recording traces resulting from insufficient time ( i.e. , limited by the little participants patience ) to lower impedances in the preparation phase . another twelve participants were excluded , either due to a lack of at least 8 movement- and artifact - free trials per condition ( n = 10 ) or due to experimental errors ( n = 2 ) . the high dropout rate in the current experiment is consistent with other developmental studies assessing electrophysiological recordings ( cf . during a sequential joint action game , we recorded brain activity and performance accuracy of the 3-year - old children . figure 1 illustrates the experimental setup that consisted of a simple computerized button - pressing game proved to be suitable for children of this age in a previous study ( meyer et al . 2010 ) . in this game , a cartoon figure of a frog could be moved up a ladder by alternately pushing two buttons . as can be seen in fig . 1 , the visual stimuli were presented on a wide - screen that was tilted to increase the height of the presented ladder and thereby the number of steps required to reach the top . in total , the ladder consisted of 42 steps that were shown on the screen . at the top of the ladder , there was a target location for the frog represented by a cartoon figure of a pig , the frog s friend on a cloud . in front of the screen , we placed two custom - made buttons to control the game and a board with the contours of two hands indicating starting and resting positions of the hands . the two buttons were interconnected via a tilt mechanism such that pushing one button down caused the other button to move up . more precisely , a right button press triggered the frog to move up using its right leg and pressing the left button moved up the left leg of the frog , so that alternating left button presses also elicited a short beep tone ( 60 ms duration ) in order to keep the child s interest and attention . with each button press , eeg markers were sent such that button presses could be traced back in the eeg recordings . 1the experimental setup of the joint button - pressing game . in front of a tilted wide - screen , we positioned two chess - clock buttons and resting positions marked by hand contours . by pressing the two buttons alternately , a cartoon figure could be moved up a ladder on the screen the experimental setup of the joint button - pressing game . in front of a tilted wide - screen , we positioned two chess - clock buttons and resting positions marked by hand contours . by pressing the two buttons alternately , a cartoon figure could be moved up a ladder on the screen each participant was involved in three different conditions of the game : a joint action condition , a joint action observation condition , and an individual action condition . the focus of this paper is on children s motor - related brain activity during action observation with respect to joint actions . we were interested in whether the motor system of the 3-year - olds was activated more strongly while observing others actions when involved in a joint action as compared to watching two people acting jointly without being involved . the same person acted both as the child s joint action partner and together with a third actor in the joint action observation condition . in the current study , we concentrate on the results of the joint action and the joint action observation condition.1 in the joint action condition , we instructed the children to push the right button with their right hand in turns with their adult action partner ( actor1 ) who pushed the left button with her left hand . more specifically , the button - pressing action was supposed to start with the hand on the resting position , which was marked by drawings of hand contours on a board in front of the buttons ( see fig . 1 ) . starting from this position , the action was executed by pushing the respective button and ended when the hand was placed back on the resting position . we thereby aimed to prevent children from leaving their hands on the button throughout the joint play and introduced a standardized action pattern that was comparable across conditions . during the measurement , the children sat on their parent s lap on a chair to the right of actor1 . in the joint action observation condition , the children watched two adults ( actor1 and actor2 ) playing the same game together . while actor1 and actor2 were playing jointly , actor2 sat between the child and actor1 such that the child would have to move only minimally to the right . for all children , the same first experimenter ( actor1 ) acted as their joint action partner . the children were not explicitly instructed where to look during the game , but in subsequent steps , only data of trials were included during which children looked at the experimenter , the buttons , or the screen ( see eeg data analysis section ) . video recordings of the entire measurement session were made and aligned with the experimental events on the screen , and children s eeg and button presses were recorded . for demonstration purposes , depending on the attention span of the children , we additionally included another run of the joint action observation condition after the joint action condition . six out of the seven participants therefore watched actor1 and actor2 play both before and after they played together with actor1 . before pooling together the data of the observation condition from the two time points , we tested for order effects . to make sure that children s motor activation did not differ significantly between the two time points , we compared activity during action observation of actor1 s button press ( t = 450 to 0 ms ) . since no differences in mu- and beta - power were found between data collected before and after the children had played themselves ( for details , see eeg data analysis ) , the data of the joint action observation condition were subsequently pooled . electrophysiological recordings were conducted using child - sized eeg caps with 30 electrode sites on the scalp . the ag / agcl active electrodes were placed in an acticap ( brain products , munich ) , arranged in the 1020 system , and referenced to electrode fcz over the central midline . the signal was amplified using a 32-channel brainamp dc eeg amplifier , band - pass filtered ( .1125 hz ) , and digitized at 500 hz . we analyzed the data using fieldtrip , an open source matlab ( version 7.0 , themathworks , inc . ) toolbox developed at the donders institute for brain , cognition and behaviour ( http://www.ru.nl/neuroimaging/fieldtrip ) . the eeg data were locked to the button press of the first experimenter ( actor1 ) and determined 450 ms before and 450 ms after the button was pressed . during this time , the children were observing actor1 s actions and the effect on the screen when actor1 was either their joint action partner ( joint action condition ) or the joint action partner of actor2 ( joint action observation condition ) . by including exclusively data from actor1 , we kept the comparison between the two conditions constant . to examine the involvement of the motor system in these two conditions , we focused on electrodes c3 and c4 over motor cortices . as mentioned in the introduction , power decrease in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas is associated with motor activation ( cf . hari 2006 ) and thus is the focus of the current analysis . on the basis of the video recordings of the measurement session , trials were rejected if children moved their hands or did not pay attention to the game ( i.e. , when they looked at neither the experimenter , nor the buttons , nor the screen ) during the critical period of the experimenter s action ( i.e. , the hand movement toward the button ) . since it was an interactive game in which we relied on children s spontaneous behavior , many trials had to be excluded due to children moving during the window of interest ( t = 450 to 0 ms ) . participants with less than 8 trials per condition were excluded from the analyses ( see participants ) . we visually inspected the remaining trials to exclude eeg artifacts ( such as noisy channels or eye blinks ) . as a result , on average , 15 trials remained for the joint action condition ( range 836 ) and 35 trials for the joint action observation condition ( range 1860 ) . a dft filter2 was used to remove line noise from the data , and for each trial , we took out the offset by subtracting the mean signal of the entire trial . we then calculated time - resolved spectral power estimates using the fourier transform in combination with a hanning taper . for this , we used a 300-ms sliding time window that was advanced in steps of 50 ms . we obtained separate tfrs for the joint action condition and the joint action observation condition . to contrast children s brain response in these two conditions , we computed the normalized difference per time frequency sample between the two conditions ( [ tfr actor1 as joint partner tfr actor1 as partner of actor2]/[tfr actor1 as joint partner + tfr actor1 as partner of actor2 ] ) ( cf . the eeg data were locked to the button press of actor1 , which is denoted as zero . hence , children observed actor1 moving her hand toward the button from about 450 ms to 0 . at zero , the button press of actor1 made the frog on the screen move upward . in the period of 0450 ms , children were preparing to press the button themselves in the joint action condition , while it was actor2 s turn in the joint action observation condition . at the same time , actor1 was placing her hand back on the resting position in front of the button.fig . power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . at time 0 , actor1 pushed the button that moved up a cartoon figure on the screen . before time 0 , actor1 moved her hand toward the button . after time 0 , actor1 moved her hand back to the resting position , while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography ( see methods and results ) . b correlation between the individual beta - power difference and the percentage of errors children made during the joint game . c topography of the normalized beta - power difference , including the data points marked by the white box . power differences are displayed on seven electrodes ( only electrodes were used that were sufficiently noise - free for all seven children ) a time - resolved normalized difference in power at electrode site c3 . power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . at time 0 , actor1 pushed the button that moved up a cartoon figure on the screen . before time 0 , actor1 moved her hand toward the button . after time 0 , actor1 moved her hand back to the resting position , while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography ( see methods and results ) . b correlation between the individual beta - power difference and the percentage of errors children made during the joint game . c topography of the normalized beta - power difference , including the data points marked by the white box . power differences are displayed on seven electrodes ( only electrodes were used that were sufficiently noise - free for all seven children ) in the statistical evaluation of the electrophysiological data , we determined whether power estimates during observation of the goal - directed action of actor1 differed significantly between conditions . we used the window of 450 ms prior to the button press until the button press ( t = 450 to 0 ms ) and the frequency bands of 711 hz ( mu ) and 1721 hz ( beta ) . 2 ) were then averaged over the respective frequency range and time window . by means of one - sample t tests analogous to this analysis , we evaluated the data of the joint action observation condition collected before and after the joint action condition ( with t = 450 to 0 ms ; mu : 711 hz ; beta : 1721 hz ) . due to the relatively small sample size of 7 participants , it might be argued that effects in the mu- and beta - power could be driven by extreme outliers . to exclude this possibility and to provide an overview of the strength of the observed effect in both mu- and beta - power , we ran complementary analyses on an individual participant level . for this purpose , we estimated the power in the mu- and beta - frequency ranges as described earlier for each individual trial per condition and participant . to determine the average power in the two conditions for each participant , we averaged power estimates of mu - power of all trials per participant over the time window of 450 to 0 ms . using the same approach , we obtained average power values in the beta - frequency range for the two conditions per child . subsequently , we evaluated the difference in power between the two conditions ( separately for mu- and beta - power ) on an individual participant basis using independent - sample t tests with trials as units of observation . additionally to the general power difference on a group and individual level , we were interested in how time - locked these differences were to the actions of the joint action partner . in other words , we examined whether an increase in motor activation ( reflected by less power in the mu- and beta - band ) was specifically locked to actor1 s actions or rather reflected a general activation of the children s motor system . to test this , we compared the normalized difference in the mu- and beta - frequency range for the two conditions before and after actor1 pressed the button . if the children s motor system was generally more activated in the joint action condition , we would expect no difference between these two time periods of actor1 s action . however , if the motor activation was time - locked to the actions of children s joint partner , we would expect the power differences to be different before and after actor1 s button press . therefore , we also calculated the average normalized difference of mu - and beta - power for the time period after actor1 s button press ( t = 0450 ms ) . to evaluate the statistical difference between the two time periods , we used paired - sample t tests with time period ( before vs. after actor1 s button press ) as an independent factor , one for testing differences in the mu- and one for differences in the beta - frequency range . to further evaluate the relation of mu- and beta - power during observation of joint action with regard to children s own joint action performance , we correlated the eeg results with the behavioral button press data . on the basis of the significant power differences between conditions , we chose representative time frequency windows ( see white boxes in fig . 2 ) . frequency windows because they represent strongest continuous effect windows time - locked to the button press of actor1 . the normalized difference in power of the respective frequency and time was then averaged to obtain a representative effect value . we subsequently correlated those effect values with the percentage of errors children made when acting jointly . the percentage of errors indicates how often the 3-year - olds pushed their button when it was not their turn . the percentage was computed as number of incorrect button presses of the child ( i.e. , button presses when it was actor1 s turn to press ) divided by the total number of times the child pushed the button when playing the joint game . we used a pearson correlation across participants to test the relation between the effect value of each specified window ( see white boxes in fig . 2 ) and the percentage of errors . to examine whether young children s involvement in joint action modulates their motor activation for others actions , we focused the eeg analysis on power differences in frequency bands ( mu : 711 hz , beta : 1721 hz ) and electrode sites ( c3 , c4 ) associated with motor activation in the brain ( cf . we were mainly interested in the contrast between children s brain activity while observing the actions of their joint action partner ( joint action condition ) and of the same person acting as the joint action partner of a third person ( joint action observation condition ) . figure 2 illustrates the difference in activity when children were observing actor1 acting as their own joint action partner and as the joint action partner of another person ( actor2 ) . more specifically , the figure shows the normalized difference in power estimated for frequencies 530 hz at electrode c3 . since results for electrode c4 did not show any significant difference in either of the frequency bands for the two conditions , the subsequent results only include data of electrode c3 ( see topography in fig . 2 ) . data of the joint action observation condition obtained before and after the joint action condition did not differ significantly ( all p > .05 ) . therefore , further reported results include pooled data of the joint action condition . cold colors in the tfr of fig . 2 represent less power for observing actor1 as their own joint action partner , whereas warm colors represent more power for observing actor1 as their joint action partner . the difference between the conditions is most pronounced in two frequency bands , namely around 10 hz and around 18 hz . in both bands , there is less power when children are observing their own joint action partner than when observing actor1 playing together with a third person ( actor2 ) . previous studies have associated less power in these frequency ranges with more motor activation ( cf . pfurtscheller and lopes da silva 1999 ) . consequently , the current findings of decreased power indicate more motor involvement in the joint action condition compared with the joint action observation condition . for statistical evaluation , we analyzed the power differences in the time before the button was pushed down . in both the mu- and the beta - frequency range , the normalized difference was significantly different from zero ( mu , t(6 ) = 3.49 , p = .013 , r = .81 ; beta , t(6 ) = 5.06 , this indicates that the 3-year - olds showed significantly more motor involvement when observing their own joint partner acting compared with observing the same person in joint action with another person . figure 3a and b show the resulting average power for each participant separately for the two conditions ( joint action observation condition : represented in blue ; joint action condition : represented in green ) . 3a . the same data pattern , namely lower average power for the joint action condition compared with the joint action observation condition , can be seen in six out of seven participants . using single - subject statistics , this tendency in mu - power differences reaches significance in two participants ( participant 1 : t(35.976 ) = 2.39 , p = .000 , r = .37 ; participant 2 : t(55 ) = 2.07 , p = .043 , r = .26 ) . all seven participants exhibit the tendency of more attenuated power when observing actor1 in the joint action condition than in the joint action observation condition , and this difference reaches significance in three out of seven participants using single - subject statistics ( participant 2 : t(52.525 ) = 3.29 , p = .002 , r = .41 ; participant 4 : t(52.592 ) = 3.55 , p = .001 , r = .43 ; and participant 6 : t(24 ) = 2.12 , p = .044 , r = .39).fig . 3power averaged over the time window of 450 to 0 ms in the a mu(711 hz)- and b beta(1721 hz)-frequency range displayed as a function of condition ( joint action observation ; joint action ) on an individual participant level . vertical black lines represent standard errors of the means power averaged over the time window of 450 to 0 ms in the a mu(711 hz)- and b beta(1721 hz)-frequency range displayed as a function of condition ( joint action observation ; joint action ) on an individual participant level . vertical black lines represent standard errors of the means shifting the focus of the analysis back to the grand average , fig . 2 shows that the enhanced motor activation in the mu - frequency range seems to be persistent throughout the entire time window ( 900 ms ) , whereas the beta - band effect appears to occur time - locked to actor1 s button press . we tested the time - specificity of the effect by comparing the time period of reaching toward the button ( t = 450 to 0 ms ) with a time period of the same duration after the button had been pressed ( t = 0450 ms ) . comparing the two time periods within the mu - frequency range did not show significant differences between the two time windows of observation in the grand average , t(6 ) = .35 , p = .737 , r = .14 . in contrast to this , the beta - power difference was more pronounced during the goal - directed action of actor1 than after actor1 had pressed the button , t(6 ) = 3.52 , p = .013 , r = .82 . thus , while activity in the beta - frequency range appears to be related specifically to the timing of the joint action partner s button press , the effect in the mu - range might be indicative of a general involvement of motor activation throughout the joint play compared with the mere observation of two people playing . finally , we examined the relationship between the mu- and beta - effects and children s joint action performance . to determine whether the enhanced motor activation in the two bands was related to how well children acted together with actor1 , we correlated the effects in the frequency bands with children s performance during joint play . for this purpose , the percentage of errors served as an indicator of performance quality . on average , the children pushed their own button about 10% of the times during the joint play ( range 030.6% ) when it was actually the turn of actor1 . based on the tfr effects ( i.e. , the difference between observing actor1 in the joint action condition and the joint action observation condition ) illustrated in fig . 2 , we selected time frequency windows that represent the strongest difference time - locked to actor1 s button press . results revealed a significant correlation between the effect in the beta - frequency range and the percentage of errors in children s joint action performance , r = .83 , p = .021 . no significant correlation was found between the effect in the mu - frequency range and performance , r = .465 , p > .5 . the present study is one of the first to explore the role of the motor system in young children s involvement in a naturalistic joint action . as hypothesized , involvement in joint action modulated activity in the motor system of 3-year - old children when observing the actions of another person . the results of the eeg analysis show significantly less power in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas when children observed actions of their own joint action partner as compared to when they observed the actions of the same person playing with someone else . since power decrease in these frequency bands is acknowledged to be associated with activation of the motor system , the current findings indicate enhanced motor system activation during action observation when the 3-year - old children were involved in the joint action . this data pattern was consistently observed over individual participants in both the mu- and beta - frequency range . moreover , the power decrease in the beta - frequency range appears to be time - locked to the action of children s joint action partner . after the button press of the joint action partner , power differences in this frequency band vanished . in contrast , power differences in the mu - band continued even when the partner moved her hand away from the button after having pushed it . to address possible relations between motor system involvement of the children and their joint action performance , we correlated the effects in both frequency bands with children s performance accuracy . the effect in the beta - frequency range correlated significantly with the percentage of errors children made during the joint action . this points to a negative relation between children s motor system involvement when observing their partner s actions and the amount of errors they made during the joint action : the less the child s motor system was activated during observation of their joint action partner , the more errors ( i.e. , erroneous button presses ) the child performed . the effect in the mu - frequency range , however , turned out to be not significantly correlated to the children s joint action performance . in general , motor activation during action observation is thought to facilitate the understanding and prediction of others actions ( cattaneo et al . 2007 ; de lange et al . 2008 ; iacoboni et al . 2005 ; rizzolatti and sinigaglia 2010 ; southgate et al . 2010 ; stapel et al . 2010 ) . during joint actions , it is particularly important to predict the timing and type of action to be performed by the action partner . anticipating what our joint action partner will do next facilitates our coordination with the other person and allows for a successful interaction ( see sebanz and knoblich 2009 , for a review ) . the current findings of enhanced motor activation for observing the own partner s actions as well as the correlation between motor system involvement and children s joint action performance might reflect children s predictive processing . however , it should be mentioned that the final sample size of the current experiment is rather small such that future research would be needed to further establish the observed effects . further research is also required to make causal inferences on the function of the motor system for children s prediction of their partner s actions . given children s motor system involvement and its link to joint action performance , the question arises of how exactly the motor system is involved in joint actions . does the motor activation reflect a precise spatial and temporal simulation of the other s actions ? with respect to the topography , enhancement in the motor system was found in an electrode over the left hemisphere ( c3 ) while children were observing left - hand movements . in adults , activation of left motor areas is associated with performance of right - side movements ( cf . , it therefore seems unlikely that the children simulated the actions of their partner on an effector level , which is in line with results of an action observation study with adults using meg ( kilner et al . one might speculate that while observing the action partner the motor system of the 3-year - olds represented actions of the right hand , which was also the effector they needed for their own actions . all in all , however , the spatial resolution of the current findings is not sufficient to answer this question properly and further research is required to allow conclusive interpretations . although the extent to which spatial aspects of the other s actions are integrated in children s motor system remains speculative , the findings show a clear integration of the action partner s timing as indicated by beta - power modulation . integrating temporal information about the partner s actions into one s own motor system points thereby to a certain purpose enhanced motor system involvement might serve , namely facilitating action prediction in joint actions . there may be various reasons why we found different patterns of effects for mu- compared with beta - power . one explanation for finding beta to be more time - locked to the partner s actions might simply be the nature of beta , which is a faster rhythm than mu . oscillations in a higher frequency range might be more effective and flexible in adapting to events for instance by recovering faster ( pineda 2005 ) . in fact , a recent study by van ede and colleagues showed that modulations of beta - oscillations during somatosensory anticipation were deployed with more temporal specificity than mu - oscillations ( van ede et al . the continuous suppression of mu - power throughout the action partner s turn might reflect a general activation of the motor system bridging the time that it is not the child s turn by keeping their motor system alert . alternative explanations might refer to differences in the function of these two rhythms as indicated by previous research . modulations in these frequency bands might be related to the type of action or the context in which actions are performed . while beta - power during action execution and observation in adults has been shown to be modulated by the correctness of actions ( koelewijn et al . 2008 ) , in 12-month - old infants mu - suppression has been reported to differ depending on whether the infants were observing ordinary or extraordinary actions ( stapel et al . more importantly , in adults , anticipatory suppression of the beta- but not mu - power was found to be stronger when observing actions of a partner than when observing actions of an individual actor ( kourtis et al . in accordance with this , our findings point to different modulations of mu- and beta - frequency range activity with regard to observing others actions depending on whether one is involved in a joint action game or merely observing others playing jointly . we have interpreted decreased power in the mu- and beta - frequency band as reflecting the activation of motor - related areas . however , the precise neural origin of modulations in the mu- and beta - frequency bands remains a matter of debate with some evidence suggesting origins in primary motor and premotor areas , whereas others suggest more posterior ( e.g. , somatosensory ) areas ( caetano et al . 2007 ; pineda 2005 ; salmelin and hari 1994 ; stancak and pfurtscheller 1996 ; van ede et al . 2010 although the precise neural sources of decrease in mu- and beta - power in the scalp - recorded eeg remain to be determined , studies in both children and adults consistently show that the execution and observation of actions is accompanied by power decreases in the mu- and beta - frequency bands ( caetano et al . 2010 ; muthukumaraswamy and johnson 2004 ; nystrm 2008 ; van elk et al . 2008 ) . in line with these findings , the present study clearly shows that the observation of an action resulted in a decrease in mu- and beta - power , which was stronger when the 3-year - olds were engaged in joint action with the observed person as compared to when they were not . being engaged in a joint action appears to result in stronger motor involvement during observation of others actions . what exactly makes a joint action situation so different from merely observing two people act together ? which factors might play a role in eliciting stronger motor involvement for others actions when involved in a joint action ? it can be speculated that motivational and attentional factors play a role here . being involved in a joint action implies that the actions of the partner gain relevance for one s own subsequent actions . in line with this , previous research has shown that different aspects of social relevance in the relation between actor and observer , such as the identity of the actor or eye contact between actor and observer , modulate motor activation during action observation ( see frith and frith 2010 , for a review ) . developmental research has shown that 3-year - olds , but not younger children , monitor their peer s actions in a selective manner ( see gauvain 2001 , for a review ) . more precisely , 3-year - olds direct their attention to their peer s attempts to solve a task , while children below the age of three pay social attention in general to their peers without a special focus on their task - solving activities ( see gauvain 2001 , for a review ) . an increased attentional focus on other s task performance might thus have elicited enhanced motor involvement during the joint action . however , the extent to which attentional and motivational factors contribute to the modulation in children s motor involvement when acting jointly remains to be clarified . moreover , in joint action , different aspects of an observed action ( such as the timing ) might serve as relevant cues for adapting their own action . the question arises whether in an individual action context similar nonsocial cues would subserve the same purpose and result in the same neural response . since this question can not be answered by the current experimental design , further investigations contrasting social and nonsocial situations are needed . how do the current findings of 3-year - olds brain activity relate to the development of young children s joint action performance ? a recent developmental study by grfenhain and colleagues revealed that it is around the same age that children understand the obligations and commitments they have toward a joint action partner ( grfenhain et al . at this age , children were also found to interact successfully when the joint action requires more complex interactions of the action partners ( ashley and tomasello 1998 ) . this indicates changes in children s responsiveness to the joint action partner occurring around the age of 3 years . we have previously investigated 3-year - olds joint action coordination in a behavioral study with a comparable joint task as used in the current experiment ( meyer et al . , we found that 3-year - old children made less errors when acting with an adult action partner than two - and - a - half - year - olds , while both age groups performed on a similar level when playing bimanually on their own ( meyer et al . 2010 ) . assessing children s brain activity during a similar task and at the age when children begin to establish well - coordinated joint actions revealed that their motor system involvement during action observation was related to their joint action performance . more activation in the motor system during action observation was thereby associated with fewer errors when playing jointly . this suggests that involvement of the motor system in observing the joint action partner might play a crucial role for the development of successful joint action performance . together , the results show an enhanced motor activation as indicated by decreased mu- and beta - power during action observation when the 3-year - olds were involved in a joint action game with the observed actor . while power differences in the beta - range show time - locked motor activation for the partner s actions , differences in mu - power rather indicate a more general involvement of the motor system in a joint action task . furthermore , the results show that the stronger the time - locked effect in beta - power , the fewer errors children made when acting jointly . this study is one of the first to investigate the neurocognitive mechanisms underlying joint action in young children . the present findings suggest that already in early childhood , others actions are integrated differentially in the motor system depending on whether or not children are engaged in a joint action . this context - specific involvement of the motor system might have important consequences for developing success in joint action .

when we are engaged in a joint action , we need to integrate our partner s actions with our own actions . previous research has shown that in adults the involvement of one s own motor system is enhanced during observation of an action partner as compared to during observation of an individual actor . the aim of this study was to investigate whether similar motor system involvement is present at early stages of joint action development and whether it is related to joint action performance . in an eeg experiment with 3-year - old children , we assessed the children s brain activity and performance during a joint game with an adult experimenter . we used a simple button - pressing game in which the two players acted in turns . power in the mu- and beta - frequency bands was compared when children were not actively moving but observing the experimenter s actions when ( 1 ) they were engaged in the joint action game and ( 2 ) when they were not engaged . enhanced motor involvement during action observation as indicated by attenuated sensorimotor mu- and beta - power was found when the 3-year - olds were engaged in the joint action . this enhanced motor activation during action observation was associated with better joint action performance . the findings suggest that already in early childhood the motor system is differentially activated during action observation depending on the involvement in a joint action . this motor system involvement might play an important role for children s joint action performance .

we have demonstrated that , melatonin is a bioprecursor of hypnotic acetyl metabolites produced by enzymatic acetylation of melatonin and 2-oxomelatonin under the control of acetyltransferases , most probably the nat enzymes . in 1994 , in our laboratory , we developed a specific and highly sensitive gas chromatography - mass spectrometry ( gc - ms ) method to assay , simultaneously and distinct ! ) , plasma concentrations of endogenous melatonin ( d0melatonin ) and exogenous melatonin ( d7-melatonin ) , in which 7 atoms of h have been substituted by 7 atoms of deuterium . using the same human volunteers ( 12 young subjects in june 1994 and 12 elderly subjects in october 1994 ) , we determined the pharmacokinetics of exogenous d7-melatonin , when given orally and intravenously , and the kinetics of the pineal secretion of endogenous d0-melatonin . , the results shown in figure 2 led to the following conclusions : secretion of melatonin by the pineal gland occurs only during the night . pharmacokinetic analysis shows that the rate of melatonin secretion by the pineal gland is constant throughout the whole nocturnal pineal melatonin production , for the same subject . the beginning and end of melatonin secretion from the pineal gland are the same for each subject , whatever the season and night , length . duration of melatonin pineal secretion is between 7.5 and 8 h. therefore , melatonin secretion and sleep are contemporaneous . there is a large interindividual variability in the amount , of melatonin released in plasma by the pineal gland during the night in young and old subjects alike . results of previous related studies show that melatonin secretion , and therefore the presence of melatonin in the central nervous system ( cns ) , is necessary for the induction and maintenance of nocturnal sleep . however , the presence of melatonin in the cns is insufficient for the induction and maintenance of sleep . indeed , figure 3 and table i show results of observations in chicks in an alternate light ( l)-dark ( d ) program ( l / d , 12 h:12 h ) , in which the light phase lasted from 8.00 am until 8.00 pm . when melatonin was administered intramuscularly ( pectoralis major muscle ) during the light phase from 2.00 pm to 8.00 pm , the chicks did not . the absence of a hypnotic effect during the light phase correlated with the very low level of nat activity in the pineal glands of chicks measured at the same times . in contrast , when chicks were observed in a 7-day permanent light , program ( l / l , 12 h:12 h ) , during which nat activity level was constantly higher , the administration of melatonin induced a significant , hypnotic effect . the duration of sleep ( between 4 and 5 h ) was much greater than that , observed with diazepam ( between 1 and 2 h ) when it . was administered intramuscularly at the same dose ( 1 m per 100 g body weight , at 2.00 pm ) . these results lead to the following conclusions : the simultaneous presence of melatonin and nat in the cns ( pineal gland ) is a necessary and sufficient condition for the induction and maintenance of sleep . in contrast to the classic so - called hypnotic drugs ( eg , benzodiazepines , barbiturates , zopiclone , and zolpidem ) , melatonin does not have direct , hypnotic properties related to its chemical structure . during the development of the gc - ms method for the assay of melatonin in plasma , our attention was focussed on the chemical reactivity of melatonin at position 3 , which allows cyciization of the side chain after acylation . this proceeds by nucleophilic attack and leads to a fluoroacyl--carboline ( figure 4 ) . considering our previous observations , we assumed that melatonin undergoes enzymatic acetylation during the night , under the control of nat , and that this leads to an n - acetyl--carboline , which we call carbo2 . we conclude that melatonin is a bioprecursor of hypnotic acetyl metabolites , such as carbo2 . chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase , they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min . figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) . gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) . we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low . the results are presented in table iii , together with some reference compounds . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect . in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep . chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase , they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min . figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) . gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) . we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect . in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep . we have evidenced the role played by melatonin in both inducing and maintaining nocturnal sleep . melatonin is the bioprecursor of hypnotic acetyl metabolites , such as carbo2 , which result from the enzymatic acetylation of melatonin ( and 2-oxomelatonin ) by nat . since insomnia and sleep disorders may be due to a lack of nat enzymes in the pineal gland , a therapeutic approach to sleep disorders could be suggested . patients with insomnia may be treated by administering hypnotic acetyl metabolites of melatonin or their synthetic analogs .

pharmacokinetic studies of melatonin in young and elderly human volunteers , and the measurement of hypnotic effects in chicks under alternate light - dark or permanent light conditions , show that melatonin is a bioprecursor of hypnotic acetyl metabolites produced by the enzymatic acetylation of both melatonin and 2-oxomelatonin under the control of serotonin n - acetyltransferases ( nats ) , which are present in the pineal gland . the acetyl metabolite of melatonin , which we call carbo2 , is an n - acetyl--carboline . the electroencephalographs ( eeg ) architecture of the sleep produced by this compound is similar to thai of physiological sleep , and is characterized by the significant proportion of slow - wave deep sleep and rapid eye movement sleep . this is in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds . since insomnia and sleep disorders are believed to be due to a lack of nat enzymes in the pineal gland , a new therapeutic approach of sleep disorders by administration of such hypnotic acetyl metabolites of melatonin , or synthetic analogs thereof , can be en visaged .

previously we reported that deletion of neutral endopeptidase ( nep ) provides protection from obesity- and diabetes - induced neural complications . we have also shown that treating obese and streptozotocin - diabetic mice with the vasopeptidase inhibitor ilepatril prevented neural complications including slowing of nerve conduction velocity , thermal hypoalgesia , and decreased intraepidermal nerve fiber density . vasopeptidase inhibitors are drugs that simultaneously inhibit nep and angiotensin - converting enzyme ( ace ) activity . recent studies have shown increased expression of angiotensin - ii - forming enzymes in adipose tissue and increased activity of the renin - angiotensin system being implicated in the development of insulin resistance and type 2 diabetes . nep is found in many tissues including vascular and renal tissue and its activity is increased by fatty acids and glucose in human microvascular cells [ 59 ] . in the peripheral nervous system nep is located in schwann cell membranes surrounding dorsal root ganglion cells and nerve fibers [ 10 , 11 ] . nep degrades many vaso- and neuroactive peptides including natriuretic peptides , adrenomedullin , bradykinin , and calcitonin - gene - related peptide [ 12 , 13 ] . therefore , inhibition of ace and nep activity would be expected to improve vascular and neural function . in this regard we have demonstrated that treating type 1 and type 2 diabetic rats as well as a genetic rat model of obesity with ilepatril improves vascular and neural dysfunction [ 1416 ] . however , little information is available about the effect of vasopeptidase inhibitors in animal models of diet - induced obesity . in order to further elucidate the effects of vasopeptidase inhibitors in peripheral nerve dysfunction associated with obesity we examined the effect of diet - induced obesity on nerve conduction velocity and thermal response latency in the hindpaw of c57bl/6j mice and mice deficient in nep treated with ilepatril , enalapril , ace inhibitor , or candoxatril , nep inhibitor [ 1 , 2 ] . unless stated otherwise all chemicals used in these studies were obtained from sigma chemical co. ( st . louis , mo ) . c57bl/6jj breeding pairs of neutral endopeptidase - deficient ( nep ) mice were provided by drs . these mice have been bred and a colony created at the veterans affairs medical center , iowa city , ia . deficiency of nep activity was confirmed in nep mice by measuring the specific activity of nep in kidney homogenates using the method described by ayoub and melzig with modification . activity of nep in kidney from c57bl/6j and nep mice was 0.35 0.02 and 0.02 0.02 mm 7-amido-3-methylcoumarin ( amc)/min / mg protein , respectively ( p < 0.001 versus c57bl/6j by unpaired t - test ) . this test was performed on all mice used in these studies in order to confirm that nep was functionally knocked out in the nep mice . mice were housed in a certified animal care facility and food ( harlan teklad , no . adequate measures were taken to minimize pain or discomfort and all of the experiments were conducted in accordance with international standards on animal welfare and were compliant with all institutional and national institutes of health guidelines for use of animals ( acurf protocol 1101009 ) . for the prevention protocol c57bl/6j and nep mice at 12 weeks of age were divided into five groups . a second group was fed a high - fat diet containing 24 gm% fat , 24 gm% protein , and 41 gm% carbohydrate ( d12451 ; research diets , new brunswick , nj ) . the primary source of the increased fat content in the diet was soybean oil and lard . the control and high - fat diet contained 0.4 and 0.3% sodium chloride , respectively . the average fat content of the control diet was 4.25 gm% ( harlan teklad , no . the third through fifth groups were fed the high - fat diet containing ilepatril ( 500 mg / kg in the diet ) , candoxatril ( 30 mg / kg in the diet ) , or enalapril ( 500 mg / kg in the diet ) . we have found that these doses provide maximal inhibition of nep and/or ace activity in vivo [ 2 , 19 ] . for the intervention study the same five groups of c57bl/6j and nep mice at 12 weeks of age were fed the control diet ( group 1 ) or high - fat diet ( groups 25 ) for 12 weeks . afterwards , the four groups of high - fat - fed mice were fed a high - fat diet with no additions ( group 2 ) or high - fat diet containing ilepatril , candoxatril , or enalapril ( groups 35 ) for 12 weeks . after an overnight fast mice were injected with a saline solution containing 2 g / kg glucose , i.p . immediately prior to the glucose injection and for 120 minutes afterwards blood samples were taken to measure circulating glucose levels with the use of glucose - dehydrogenase - based reagent strips ( aviva accu - chek , roche , mannheim , germany ) . blood samples ( 0.6 l ) were taken from a tail vein that was lanced once . thermal nociceptive response in the hindpaw was measured using the hargreaves method with instrumentation provided by iitc life science , woodland hills , ca ( model 390 g ) as previously described . the test was performed in a blind manner . thermal nociceptive responses were measured by placing the mouse in the observation chamber on top of the thermal testing apparatus and allowing it to acclimate to the warmed glass surface ( 33c ) and surroundings for a period of 15 min . the mobile heat source was maneuvered so that it was under the heal of the hindpaw and then activated , a process that starts a timer and locally warms the glass surface , when the mouse withdrew its paw , the timer , and the heat source was turned off . following an initial recording , which was discarded , two measurements were made for each hindpaw , with a rest period of 5 min between each set of measurements . the mean of the measurements , reported in seconds , was used as a measure of the thermal nociceptive response latency . , abbott laboratories , north chicago , il ) and sensory nerve conduction velocities were determined as previously described [ 1 , 2 ] . briefly , sensory nerve conduction velocity was recorded in the digital nerve to the second toe by stimulating with a square - wave pulse of 0.05 ms duration using the smallest intensity current that resulted in a maximal amplitude response ( grass s44 stimulator ; grass medical instruments , quincy , ma ) . the maximal sensory nerve conduction velocity was calculated by measuring the latency to the onset / peak of the initial negative deflection and the distance between stimulating and recording electrodes ( measured in millimeters using a vernier caliper ) . biopsies of skin of the right hindpaw were fixed , dehydrated , and embedded in paraffin . sections ( 7 m ) were collected and immunostained with anti - pgp9.5 antibody ( rabbit anti - human no . 7863 - 0504 ( this antibody cross - reacts with rat , mouse guinea pig , and other species ) , abd serotic , morpho sys us inc . , raleigh , nc ) overnight followed by treatment with secondary antibody alexa fluor 546 goat anti - rabbit ( invitrogen , eugene , or ) . profiles were imaged using a zeiss 710 confocal microscope with a 40x objective and were counted by two individual investigators that were blinded to the sample identity . length of the epidermis was determined by drawing a polyline along the contour of the epidermis and recording its length in mm . comparisons between the groups for body weight , blood glucose , sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber profiles were conducted using a one - way anova and bonferroni 's test for multiple comparisons ( prism software ; graphpad , san diego , ca ) . presented in table 1 are weight and blood glucose changes for c57/bl6j and nep mice used in the prevention study . at 12 weeks of age when fed a high - fat diet c57bl/6j and nep mice both gained a similar amount of weight . treating the high - fat diet with ilepatril or enalapril but not candoxatril completely prevented the gain in weight . the mass of the epididymal fat pad was significantly increased in high - fat - fed mice and mice fed the high - fat diet treated with candoxatril compared to control mice or mice fed the high - fat diet containing ilepatril or enalapril . when calculating the epididymal fat pad mass as a percent of total body weight epididymal fat pad mass was significantly increased in high - fat - fed c57bl/6j mice and mice fed a high - fat diet containing candoxatril compared to control . treating the high - fat diet with ilepatril and to a greater extent enalapril prevented the increase in epididymal fat pad mass when presented as percent of final body weight . nonfasting blood glucose levels were not significantly different between c57bl/6j and nep mice fed the control or high - fat diets and not affected by ilepatril , candoxatril , or enalapril treatment . in the intervention study all mice fed the high - fat diet for the initial 12 weeks of the experimental design gained a significant amount of weight ( table 2 ) . when transferred to a high - fat diet containing ilepatril or enalapril for an additional 12 weeks c57bl/6j and nep mice lost weight . mice remaining on the high diet or high - fat diet containing candoxatril continued to gain weight . the epididymal fat mass decreased after c57bl/6j or nep mice , fed a diet containing high fat for 12 weeks , were given a high - fat diet containing enalapril . the epididymal fat mass also was decreased after nep mice but not c57bl/6j mice were fed a high - fat diet treated with ilepatril . treating high - fat - fed c57bl/6j or nep mice with candoxatril did not reduce epididymal fat mass . a similar trend was observed when the epididymal fat mass data was presented as percentage of final body weight . nonfasting blood glucose levels were not changed in c57bl/6j or nep mice fed control or high - fat diets with or without ilepatril , candoxatril , or enalapril . in the prevention study c57bl/6j ( figure 1 ) and nep ( figure 2 ) mice fed the high - fat diet or high - fat diet containing candoxatril had an impaired glucose clearance curve compared to control mice . in contrast , the glucose clearance curve was near normal in c57bl/6j and nep mice fed a high - fat diet containing ilepatril or enalapril . in the intervention study feeding c57bl/6j ( figure 3 ) or nep ( figure 4 ) mice for 24 weeks a high - fat diet caused an impaired glucose clearance curve . treating high - fat - fed c57bl/6j or nep mice with enalapril for the last 12 weeks of the 24-week period partially corrected glucose utilization . treating nep mice with ilepatril treating c57bl/6j mice with ilepatril did not improve glucose utilization nor did treating c57bl/6j or nep with candoxatril . data in figure 5 ( left ) demonstrate that sensory nerve conduction velocity was significantly decreased in high - fat - fed c57bl/6j mice compared to control mice and that this was prevented by treating high - fat - fed mice with ilepatril or candoxatril using a prevention protocol . data in figure 5 ( center ) demonstrate that thermal nociception was significantly decreased in c57bl/6j mice after 12 weeks of a high - fat diet compared to control mice as indicated by an increase in paw withdrawal latency . treatment with ilepatril or candoxatril and to a lesser extent enalapril for the 12-week period prevented thermal hypoalgesia in high - fat - fed c57bl/6j mice . data in figure 5 ( right ) demonstrate that the number of intraepidermal nerve fiber profiles was significantly decreased in high - fat - fed c57bl/6j mice and that treatment of these mice with ilepatril or candoxatril but not enalapril for 12 weeks prevented the significant decrease . sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber density were not impaired in nep mice fed a high - fat diet ( figure 6 , left , center , and right , resp . ) . we also determined the effect of treating high - fat - fed c57bl/6j and nep mice using an intervention protocol ( figures 7 and 8 , resp . ) . in this study design high - fat - fed mice were untreated for 12 weeks followed by 12 weeks of a high - fat diet with or without treatment . feeding c57bl/6j mice a high - fat diet for 24 weeks caused a significant decrease in sensory nerve conduction velocity ( figure 7 , left ) . treating these mice for the last 12 weeks of the 24-week period with ilepatril , candoxatril , or enalapril the decrease in thermal nociception in high - fat - fed c57bl/6j mice was reversed by treating mice with ilepatril or candoxatril but not enalapril ( figure 7 , center ) . treating high - fat - fed c57bl/6j mice with ilepatril , candoxatril , or enalapril reversed the decrease in intraepidermal nerve fiber profiles ( figure 7 , right ) . feeding nep mice a high - fat diet for 24 weeks with or without treatment had no effect on sensory nerve conduction velocity , thermal nociception , or intraepidermal nerve fiber density ( figure 8 , left , center , and right , resp . ) . the main findings from these studies were that diet - induced obesity caused significant weight gain and impaired glucose utilization in c57bl/6j and nep mice that was improved when the mice were treated with ilepatril or enalapril using a prevention protocol . using the intervention protocol enalapril but not ilepatril treatment was effective improving glucose utilization by c57bl/6j mice . treating c57bl/6j or nep mice with candoxatril did not improve weight gain or glucose utilization . these data suggest that inhibition of ace and not nep activity was responsible for improving weight gain and glucose utilization in high - fat - fed mice . diet - induced obesity also caused slowing of sensory nerve conduction velocity , thermal hypoalgesia , and decrease in epidermal nerve fiber density in the paw of the hindlimb in c57bl/6j mice but not nep mice . the impairment in sensory nerve function endpoints in c57bl/6j mice fed a high - fat diet was prevented / improved when the mice were treated with ilepatril or candoxatril and to a much lesser extent enalapril using either the prevention or intervention protocol . these data suggest that increased nep activity / expression but not ace activity contributes to diet - induced obesity - related deficits in sensory nerve function . previous studies have shown that diet - induced obesity in rodent models can be prevented by ace inhibitors and angiotensin ii receptor blockers [ 2124 ] . in addition , diet - induced weight gain and fat mass are reduced , energy expenditure increased , and glucose tolerance improved in mice lacking ace or the angiotensin ii type 1a receptor [ 25 , 26 ] . the mechanisms proposed for the improvement in obesity and glucose tolerance with treatment of rodent models with ace inhibitors are increased energy expenditure , liver and adipose tissue metabolic modulation , lower concentration of leptin , improved insulin signaling , and increased glucose and fatty acid utilization by muscle [ 2130 ] . in a study comparing the effects of ramipril , an ace inhibitor , to ilepatril in jcr : la - cp rats , an obese , insulin - resistant , hyperinsulinemic , normoglycemic model , it was found that both compounds reduced the surge of plasma insulin in a meal tolerance test by about 50% but ilepatril was more beneficial in improving vascular reactivity . in our study we found that enalapril trended to be more effective than ilepatril preventing / reducing fat pad mass . this could be because enalapril at the dosage used was a more effective ace inhibitor than ilepatril in target tissues . in another study using obese zucker rats it was found that dual inhibition of ace and nep improved insulin - mediated glucose disposal more effectively than monotherapy and this effect was linked to increased activation of the kinin - nitric oxide pathway . in a similar independent study it was found that omapatrilat , a vasopeptidase inhibitor , induced insulin sensitization and increased myocardial glucose uptake in obese zucker rats and that the effect of omapatrilat was greater than ramipril in part due to stimulation of the b2 receptor . later this group reported that treatment of obese zucker rats with a vasopeptidase inhibitor increased muscle glucose uptake independent of insulin signaling . in two of these studies protection of bradykinin from degradation by nep interestingly , it has been shown that natriuretic peptides promote muscle mitochondrial biogenesis and fat oxidation as to prevent obesity and glucose intolerance . the natriuretic peptides are also degraded by nep . because nep is expressed in skeletal muscle in relatively large amounts and being located on the cell surface , nep is able to hydrolyze peptides in the vicinity of their receptors thereby neutralizing their bioactivity [ 34 , 36 ] . however , inhibition of ace may also lead to protection of bradykinin levels by inhibiting kininase - ii - mediated degradation of this nonapeptide . reported that ace inhibition by captopril improved glucose transport in insulin - resistant muscle of the obese zucker rat . they attributed the improvement to modulation of insulin action by bradykinin mediated through b2 receptors and by an increase in nitric oxide production . since bradykinin and natriuretic peptides may have a role in enhancing insulin action and regulating glucose and fatty acid metabolism by muscle protecting their bioactive function by preventing degradation through inhibition of ace and/or nep may be a therapeutic approach for treatment of obesity and insulin resistance [ 34 , 36 , 38 ] . we have previously shown that treating diabetic rats with enalapril improved vascular and nerve endpoints but enalapril was less effective in rats fed a high - fat diet [ 19 , 36 , 39 , 40 ] . in contrast , we found that treating obese or diabetic rats with ilepatril was more effective than enalapril in improving peripheral nerve dysfunction [ 19 , 36 , 40 ] . we have shown that treating diabetic or diet - induced obese rodents with ilepatril improves vasodilation of blood vessels that provide circulation to the sciatic nerve by reducing oxidative stress and preventing degradation of vasoactive peptides by nep including calcitonin gene - related peptide and c - type natriuretic peptide thereby maintaining endoneurial blood flow and preventing ischemia . since nep degrades both calcitonin gene - related peptide and substance p , peptides important in pain signaling pathways , it is likely that blocking nep activity or mice deficient in nep would maintain higher levels of both of these neuroactive peptides and perhaps be more sensitive to painful stimuli . data from clinical trials clearly suggest that activation of the renin - angiotensin - aldosterone system plays an important role in the pathophysiology of the metabolic syndrome through interaction of a wide range of physiological and molecular mechanisms . more recently in humans activity of nep has been shown to correlate with body mass index and measures of insulin resistance with increasing activity found in subjects with multiple cardiovascular risk factors . in these studies we found that feeding mice a high - fat diet causes weight gain , impaired glucose tolerance , and sensory nerve dysfunction . inhibition of ace was effective in reducing weight gain and improving glucose utilization but generally noneffective in improving sensory nerve dysfunction . in contrast , inhibition of nep improved sensory nerve dysfunction and had no effect in improving weight gain or glucose utilization . treating high - fat - fed mice with a vasopeptidase inhibitor improved weight gain , glucose utilization , and sensory nerve function . thus , we conclude that increased activity of nep is associated with sensory nerve dysfunction in an animal model of diet - induced obesity and that dual inhibition of ace and nep may be more effective than monotherapy in reducing insulin resistance and the sensory nerve complications associated with metabolic syndrome .

we have demonstrated that treating diet - induced obese ( dio ) mice with the vasopeptidase inhibitor ilepatril improved neural function . vasopeptidase inhibitors block angiotensin - converting enzyme ( ace ) and neutral endopeptidase ( nep ) activity . we propose that increased activity of ace and nep contributes to pathophysiology of dio . to address this issue c57bl/6j mice or mice deficient in nep were fed a high - fat diet and treated with ilepatril , enalapril , ace inhibitor , or candoxatril , nep inhibitor , using both prevention and intervention protocols . endpoints included glucose utilization and neural function determination . in the prevention study glucose tolerance was impaired in dio c57bl/6j mice and improved with ilepatril or enalapril . sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber density were impaired in dio c57bl/6j mice and improved with ilepatril or candoxatril . in the intervention study only enalapril improved glucose tolerance . sensory nerve conduction velocity and intraepidermal nerve fiber density were improved by all three treatments , whereas thermal nociception was improved by ilepatril or candoxatril . in nep - deficient mice dio impaired glucose utilization and this was improved with enalapril . nerve function was not impaired by dio in nep - deficient mice . these studies suggest that ace and nep play a role in pathophysiology associated with dio .

rickettsia are obligatory , intracellular , small gram - negative bacteria associated with eukaryotic hosts . they are traditionally divided into three groups : the spotted fever group , the typhus group and the scrub typhus group . the spotted fever results from a large group of tick- , mite- and flea - borne zoonotic infections that are caused by closely related rickettsiae . rickettsia conorii has been identified as the agent causing mediterranean spotted fever ( msf ) . a variety of geographically distinct types of r. conorii have been described that cause variable clinical presentations different from typical msf . a 27-year - old primigravida with a 16week history of amenorrhea and respiratory failure was transfered to a tertiary care center . she had high - grade fever for 8 days prior to the transfer and had developed an erythymatous maculopapular rash on day 4 of the illness , involving the trunk initially , later extending to the palms and soles [ figure 1 ] . erythematous maculopapular rash in a hand a persistant dry cough , not associated with hemoptysis , was noticed on day 5 . she developed a progressively worsening dyspnea with bilateral ankle edema not associated with oliguria or orthopnea . her condition deteriorated rapidly , warranting intubation for respiratory failure and transfer to a tertiary care center . on admission to the tertiary care center icu , the patient also had tachycardia ( 128 beats / min ) without any significant st segment changes on the ecg . the blood urea measured 8.7 mmol / dl with a serum sodium of 140 meq / l and a pottasium of 5.0 meq / l . full blood count showed neutrophilia of 78% in a total of 14.4 10 l. hemoglobin was 7.4 g / dl with a mild thrombocytopenia of 160,000 l . the blood picture revealed normochromic normocytic anemia with no evidence of disseminated intravascular coagulation ( dic ) . the international normalized ratio ( inr ) was 0.96 . the serum glutamate oxaloacetate transaminase ( sgot ) activity was 42 u / l and serum glutamate pyruvate transaminase ( sgpt ) activity was 28 the c - reactive protein ( crp ) level was 6 mg / dl and the antinuclear antibody ( ana ) was negative . chest x - ray on day 1 at icu , showing diffuse bilateral opacities the patient was started on oral azithromycin , iv cefotaxime , iv hydrocortisone 50 mg 6 hourly . the patient 's condition deteriorated rapidly on day 3 of icu stay and needed adrenaline , noradrenaline , dobutamine and vasopressin to maintain a blood pressure of 70/50 mmhg . the urine output dropped and the urine microscopic examination revealed granular casts and dysmorphic red blood cells . blood urea rose to 12.9 mmol / dl and the electrolytes showed pottasium level of 5.6 meq / l . on the 4 day at the icu and 12 day of the disease , she devoloped anuria with further increase in blood urea and serum creatinine . she was hemodynamically unstable with a blood pressure of 60 mmhg even with total inotrope support . endocardium of the heart showed vegitations on a narrowed mitral valve [ figure 4 ] . the kidneys were of normal size , but the cortico - medullary demarcations were less clear . the grossly hemorrhagic lung the heart showing narrowing and vegetations at the mitral valve the histology of the lung showed gross hemorrhage and foci of pneumonia . the vegetations of the heart were reported as fibrinous with entrapped white cells , but with no bacteria or fungi . histology of the liver revealed centrilobular necrosis with a tendency to confluence , and a mild fatty change . a final diagnosis of disseminated r. conorii infection with pulmonary hemorrhage , endocarditis and tubulointerstitial nephritis was made . r. conorii is an obligate , intracellular , slow - growing , gram - negative bacterium belonging to the spotted fever group of rickettsiae . unusual rickettsial strains related to r. conorii have been described as belonging to an r. conorii complex which includes the indian tick typhus rickettsia ( atcc vr-597 ) with r. conorii subsp . it is transmitted to humans through the bite of dog ticks ( rhipicephalus sanguineus ) widely prevalent among the old world . it is also known to be transmitted by haemaphysalis ticks , especially in pakistan and kenya . the infection is transmitted via larvae and nymphs , and the tick bite is usually not felt . the incubation period ranges from 3 to 15 days depending upon the route of rickettsial entry and the rickettsial load . after introduction into the skin at the site of the tick bite or through the conjunctiva contaminated by blood or excretions from an infective tick , the primary multiplication occurs . in the skin , the localized multiplication of the rickettsiae in the endothelial cells of the capillaries leads to the formation of a raised red papule . the inflammation and thrombosis of the affected capillaries lead to necrosis of the center of the papule and the formation of the typical red lesion with a black center , the tache noire . this is followed by the spread of infection through lympho - hematogenous routes throughout the body , causing disseminated vascular lesions in multiple organs . about 6% of the cases are severe , and fatal cases occur even in young , healthy adults , with a reported death rate of about 2.5% . old age , alcoholism and glucose-6-phosphate dehydrogenase ( g6pd ) deficiency are known risk factors for severe disease . the patient described here is from kotagla , a tea estate village in central sri lanka . the patient reported a history of significant exposure to stray dogs , abundant in any sri lankan village , but not of any tick bites . the absence of the tachy noire is significant in this case as the clinical diagnosis of msf ( caused by r. conorii subsp . however , the absence of tache noire has been noted in israeli spotted fever caused by r. conorii subsp . the fever starts after an incubation period of 7 days followed by a febrile period ( up to 40c , usually continuous ) associated with a maculopapular rash . the papules of the rash are first noticed on the 3 to 5 day of illness , and they come out in crops and are palpable as small nodules in the skin . in severe cases , characteristically , the rash involves the palms of the hands , the soles of the feet and , to a lesser extent , the face . in this patient , the rash involved the palms and soles , sparing the face , and was of dusky cyanotic appearence with prominant macules . the cough and the dyspnea reported in our patient were relatively rare in a case series of msf , with a representation of 10% and 21% , respectively . renal insufficiency is a known complication in around 6% of the patients and is caused by tubulointerstitial nephritis . myocarditis had been obseved in 11% of patients and might explain the severe tachycardia with poor response to inotropes in this patient . the diagnosis of r. conorii infection in this patient is justfied as there is a compatible clinical picture along with a single titer of > 1/512 . endocarditis is not described as a complication of r. conorii infection . identifying r. conorii as the causative organism of endocarditis in this patient is supported by the absence of previous heart disease and lack of clinical features to suggest subacute bacterial endocarditis on admission and during prenatal clinics . the vegetations isolated from this patient as well as the blood cultures did not reveal bacteria or fungi . this patient was correctly started on oral azithromicin and intravenous chlorampenicol at the tertiary hospital . a diagnostic scoring system with microbiological , epidemiologic , and clinical parameters has been proposed for msf and it had shown good sensitivity and specificity . this case highlights the need of clinical suspicion of uncommon diseases based on the geographic and socioeconomic background of a patient . relatively benign diseases may have severe manifestations resulting in fatality ; therefore , physicians should consider there factors for early diagnosis and intervention .

rickettsial diseases are common in srilanka . the spotted fever group of rickettsiae presents in many ways , including very severe disease causing significant morbidity and mortality . a regional variation of the rickettsia conorii subspecies and differences in clinical presentations are reported . this case describes disseminated rickettsia conorii infection in a pregnant woman presenting with endocarditis .

despite many years of research efforts and impressive progress in knowledge of mechanisms of endometriosis development , the etiopathogenesis of the disease and exact cause of infertility in patients suffering from endometriosis still remain poorly understood . none of the theories and models of endometriosis pathogenesis provide definitive explanation of the disease development , considering its different manifestations and various localizations . recently published studies present new data on potential role of free radicals in endometriosis pathophysiology . although the origin of the oxidative stress occurring in the peritoneal cavity in endometriotic patients is unknown , accumulating data suggest that increased iron levels , together with apoptotic endometrial fragments and activated macrophages , may promote prooxidant environment . in addition , oxidative stress in endometriotic patients may potentially be induced by environmental factors , including dioxins or heavy metals [ 1 , 2 ] . in our preliminary work we found significantly increased levels of ox - ldl in peritoneal fluid of women with stage iii / iv endometriosis compared to patients with follicle ovarian cysts . however , peritoneal fluid oxldl concentrations did not differ significantly between patients with minimal / mild endometriosis and women from the reference group . murphy et al . showed increased low - density lipoprotein ( ldl ) oxidation in peritoneal fluid of patients with endometriosis , which may be a result of peritoneal cavity macrophages hyperactivity . it was also proved that oxidized ldls stimulate monocyte chemotactic protein-1 ( mcp-1 ) expression in mesothelial and endometrial cells which provides direct evidence of oxidative stress role in etiopathogenesis of the disease . increased concentrations of lipid peroxidation end products , malondialdehyde ( mda ) , 8-isoprostane , and 25-hydroxycholesterol , were found in peritoneal fluid of infertile women with endometriosis [ 610 ] . serum of patients with endometriosis , compared to healthy women , contains also significantly higher 8-isoprostane levels . murphy et al . showed that peritoneal fluid of patients with endometriosis contains increased concentration of lysophosphatidylcholine , another lipid peroxidation product with confirmed chemotactic properties for monocytes . mda and 7-hydroxynonenal ( hne-7 ) expression were increased in endometriosis implants tissue ; however , both lipid proteins are also expressed in eutopic endometrium . concentrations of antibodies against lipid peroxidation products were found to be increased in serum of women with endometriosis , with no immunoglobulins detected in their peritoneal fluid . serum of women with endometriosis contains also elevated concentration of lipid hydroperoxide ( looh ) , and its levels correlate positively with the stage of the disease according to revised american fertility society classification . peritoneal fluid of endometriotic patients contains oxidatively modified protein - lipid complexes , showing both chemotactic properties and ability to stimulate selected cytokines production . however , there are relevant data published in the literature according to which the concentrations of mda and mda - cu complexes demonstrate no significant differences , being comparable in peritoneal fluid of patients with and without endometriosis , showing also no significant correlation with the stage of the disease [ 1618 ] . no differences were also found in peritoneal fluid concentration of another lipid peroxidation product , cholest-3,5-dien-7-one . the objective of the study was to assess concentrations of oxidized low - density lipoproteins ( oxldl ) in peritoneal fluid ( pf ) of women with endometriosis . clinically and histologically confirmed diagnosis established the following groups : women with endometriosis ( e , n = 110 ) and as the reference groups : patients with simple serous ( r1 , n = 78 ) and dermoid ( r2 , n = 41 ) ovarian cysts . in each case the disease was found to be minimal ( e1 ) in 23 cases , mild ( e2 ) in 25 patients , moderate ( e3 ) in 39 women , and severe ( e4 ) in 23 cases . subjects were not given hormonal therapy and/or anti - inflammatory medications for at least 3 months before laparoscopy . medical history of the patients and basic clinical examination showed no general chronic diseases , except for the condition , which was the indication for laparoscopy . similarly , no significant difference was found in the phase of menstrual cycle of the time of laparoscopic procedures between women in all study groups . all patients signed an informed consent , and the lublin medical university ethics committee approval was obtained for the study . all visible pfs were aspirated during laparoscopy from the anterior and posterior cul - de - sacs , under direct vision to avoid blood contamination . samples were immediately centrifuged at 500 g for 5 minutes , and the supernatants were aspirated and stored at 70c until analysis . oxldl concentration in the pf was measured in duplicate using a commercially available enzyme - linked immunoassay kit ( immundiagnostik ag , cat . all data were tested with the shapiro - wilk test for normality . because data were not normally distributed , statistical significance between e and r groups was determined with the mann - whitney u test . data are presented as medians ( me ) , minima ( min ) , maxima ( max ) , and lower and upper quartiles . concentrations of oxldl in pf of patients with endometriosis were significantly higher compared to women with serous ovarian cysts ( p = 0.03 ) . however , no significant difference in the pf oxldl levels was found between patients with endometriosis and women with dermoid ovarian cysts ( p = 0.4 ) . levels of oxldl in pf of women with serous ovarian cysts were similar to those noted in patients with dermoid cysts ( figure 1 , table 1 ) . by analyzing concentrations of oxldl in pf of women with different stages of the disease , it was noted that they were higher only in the subgroup of patients with stage iv endometriosis as compared to women with ovarian serous cysts . no significant differences were found between concentrations of oxldl in pf of women with different stages of the disease ( table 2 ) . pf oxldl concentration did not differ significantly between the subgroups of women in the follicular and the luteal phase of the menstrual cycle ( me , range : 71.5 , 1.21470 ng / ml versus 80.3 , 16.71870 ng / ml , p = 0.5 ) . in our work , we demonstrated that peritoneal fluid oxldl concentration was significantly higher in patients with endometriosis than in women with serous ovarian cysts ; however , it did not differ significantly as compared to subjects with dermoid cysts . after analysis of data obtained in women with different stages of the disease , it was noted that these results are found only in patients with severe endometriosis . in case of minimal , mild , and moderate disease , to our knowledge , only murphy and colleagues investigated the possible role of oxldl in the pathogenesis of endometriosis . based on a small number of cases , they found increased oxidation of low - density lipoprotein in women with pelvic endometriosis and increased levels of oxldl in the pf of patients with this disease . our results agree with these findings . however , our data suggest that only the severe stage of endometriosis is associated with increased oxidation of low - density lipoprotein in the peritoneal cavity , probably as the result of an imbalance in prooxidant / antioxidant pf systems . shanti et al . demonstrated that women with endometriosis had increased serum concentrations of autoantibodies to markers of oxidative stress including oxldl . data from our work indirectly confirm these results . based on many findings , there is an emerging concept of treating endometriosis as an autoimmune disease . an increased incidence of endometriosis was observed in the group of women with autoimmune diseases such as multiple sclerosis , lupus erythematosus , psoriasis , crohn 's disease , hypothyroidism , hyperthyroidism , and rheumatoid arthritis . endometriosis shares many similarities with other autoimmune diseases including elevated levels of cytokines , decreased cell apoptosis , and t- and b - cell abnormalities . a variety of autoantibodies have been detected in patients with endometriosis patients , which suggests a polyclonal activation of b cells . the most commonly reported types are antiendometrial , antiovarian antibodies , and autoantibodies against phospholipids , histones , and nucleotides . other similarities between endometriosis and autoimmune diseases include familial occurrence , tissue damage , preponderance of females , and multiorgan involvement [ 21 , 22 ] . lipid peroxidation processes are closely associated with the pathophysiology of autoimmune diseases . therefore , increased levels of oxidized ldl in pf of women with endometriosis support the theory of treating endometriosis as an autoimmune disease . oxidized ldl induces secretion of numerous proinflammatory cytokines including macrophage colony - stimulating factor ( m - csf ) , interleukin-6 ( il-6 ) , and tumor necrosis factor ( tnf- ) . concentrations of these cytokines were found to be elevated in the pf of patients with endometriosis . we can speculate that increased levels of oxidized ldl in the peritoneal cavity of women with severe endometriosis may be one of the factors responsible for increased levels of m - csf , il-6 , and tnf- in pf . elevated pf concentrations of these cytokines promote adhesion , invasion , proliferation , and angiogenesis of ectopic endometrium and create an inflammatory environment in the peritoneal cavity in women with endometriosis . . demonstrated that oxldl caused an increase in accumulation of monocyte chemotactic factor-1 ( mcp-1 ) in the medium of cultured mesothelial and endometrial cells . they also found that cells cultured in the presence of pf from endometriosis patients secreted more mcp-1 than those cultured with pf from subjects without the disease . therefore , we hypothesize that increased concentration of oxldl may be responsible for , as demonstrated in other studies , higher concentrations of mcp-1 in the pf of women with endometriosis . stimulation of mcp-1 production by increased pf levels of oxidized ldl may hypothetically be another factor responsible for the creation of proinflammatory environment in the peritoneal cavity of patients with endometriosis . unfavorable changes in lipid profile are present not only in the pf of women with endometriosis . it has been recently shown that plasma of patients with this disease contains higher concentrations of total cholesterol , low - density lipoproteins , high - density lipoproteins ( hdl ) and triglycerides as compared to healthy women . although all lipoproteins were significantly elevated in endometriosis patients , the difference was most substantial for ldl levels , which were 38% higher in women with endometriosis . verit et al . found that patients with endometriosis displayed significantly lower serum levels of hdl and higher levels of triglycerides , total cholesterol , and ldl than women without the disease . they also demonstrated that serum of women with endometriosis is characterized by significantly lower activity of paraoxonase-1 ( pon-1 ) , which negatively correlated with the progression of the disease . this hdl - associated antioxidant enzyme with paraoxonase activity prevents ldl and hdl oxidation and is also responsible for the antioxidant effect of hdl . therefore , authors speculated that unfavorable lipid profile combined with lower pon-1 activity in women with endometriosis may contribute to the increased susceptibility for the development of atherosclerosis . in conclusion , oxldl levels were significantly higher in the peritoneal fluid of patients with severe endometriosis than in women with serous ovarian cysts . this suggests that disrupted oxidative status in the peritoneal cavity of women with endometriosis may play a role in the etiopathogenesis of the more advanced stages of the disease . however , it can not be excluded that high oxldl levels are the result of more oxidant environment in the peritoneal cavity of patients with severe endometriosis than with less advanced disease .

the etiopathogenesis of endometriosis still remains unknown . recent data provide new valuable information concerning the role of oxidative stress in the pathophysiology of the disease . it has been proved that levels of different lipid peroxidation end products are increased in both peritoneal fluid ( pf ) and serum of endometriotic patients . we assessed the concentration of oxidized low - density lipoproteins ( oxldl ) in pf of 110 women with different stages of endometriosis and 119 women with serous ( n = 78 ) or dermoid ( n = 41 ) ovarian cysts , as the reference groups . pf oxldl levels were evaluated by elisa . we found that concentrations of oxldl in pf of endometriotic women were significantly higher compared to women with serous but not dermoid ovarian cysts . interestingly , by analyzing concentrations of oxldl in women with different stages of the disease , it was noted that they are significantly higher only in the subgroup of patients with stage iv endometriosis as compared to women with ovarian serous cysts . in case of minimal , mild , and moderate disease , pf oxldl levels were similar to those noted in reference groups . our results indicate that disrupted oxidative status in the peritoneal cavity of women with endometriosis may play a role in the pathogenesis of advanced stages of the disease .

posttraumatic stress disorder ( ptsd ) is a complex mental disorder that can develop in response to a traumatic event such as a motor vehicle accident , rape , combat exposure or natural disaster . the national vietnam veterans readjustment study estimated that 53.4% of us male veterans of the vietnam war developed full or partial ptsd and 15.2% still suffered from the disorder in 1990 1 . the application of conservative procedures for analysis led dohrenwend et al to document that 18.7% of male veterans developed war - related ptsd during their life time and 9.1% were still suffering from ptsd 11 to 12 years after the war 2 . in the general population , the estimated lifetime prevalence of ptsd is 7.8% in adult americans , with women having a 2.3-fold higher prevalence than men 3 . ptsd symptoms include intrusive thoughts , hyperarousal , nightmares , flashbacks , emotional detachment or numbing of feelings , insomnia , fear , avoidance of reminders , distress when exposed to reminders , irritability , hypervigilance , and heightened startle response 4 . several lines of evidence support neurochemical , functional and structural alterations in the neuroendocrine system in patients with ptsd . the neurochemical alterations range from adrenergic hyperresponsiveness , increased thyroid activity , increased levels of corticotrophin - releasing factor , to low cortisol levels and increased negative feedback sensitivity of the hypothalamo - pituitary - adrenal - axis after the administration of low - dosage dexamethasone 5 . functional brain imaging studies in patients with ptsd demonstrate increased function in the amygdala and decreased activity in the hippocampus and prefrontal cortex ( pfc ) 6;7 . these functional alterations are consistent with the structural findings in animal models of ptsd that neurons in the hippocampus and pfc display atrophy while those in amygdala show growth in response to repeated stress 8 . dlpfc including ba46 is one of the three regions of pfc , which regulates working memory and execution of fear responses 9 . this brain region has been correlated with structural and functional alterations , and with the treatment response of patients with ptsd . in children with ptsd symptoms , decreased volume of gray matter in the dlpfc is correlated with increased functional impairment 10 . adult patients with ptsd core symptoms ( i.e. re - experiencing , avoidance ) were markedly improved by treatment with 10-hz repetitive transcranial magnetic stimulation over the right dlpfc 11 . furthermore , the indicator of neuronal viability , n - acetylaspartate , in dlpfc was correlated positively in healthy people with verbal intelligence 12 , and negatively in patients with a generalized anxiety disorder following a previous traumatic event 13 . although the prefrontal cortex , amygdala and hippocampus are the brain regions considered to be related to ptsd 6;7 , the underlying molecular mechanisms are unknown . one of the possibilities is that functional and structural changes in the brain may result from mitochondria - centered responses to repeated or chronic harmful stresses 14;15 . mitochondria provide the cell with energy in the form of atp and play fundamental roles in many metabolic pathways , such as -oxidation , the tricarboxylic acid cycle and urea cycle , the synthesis of steroid hormones and heme , and calcium signaling . in addition , mitochondrial biochemical and molecular pathways are a natural central target of diverse pharmacological agents 16 . however , reactive oxygen species ( ros ) , an inevitable by - product of mitochondrial oxidative phosphorylation , can impair molecules , contributing to various diseases including neurodegeneration 17 . thus , homeostasis of these mitochondrial functions is critical in human brain because of its high energy demand . mitochondrial dysfunctions are increasingly recognized as key components in stress related mental disorders 14;15 . although long or repetitive exposure to stress can induce a neurological change resulting in ptsd , the molecular pathology involving in such a neuronal damage remain elusive . overexpression of antioxidant enzymes ( glyoxalase and glutathione reductase 1 ) in mouse brain was also associated with increase in anxious behavior 19 . in addition , chronic stress of rats inhibits the activities of mitochondrial respiratory complexes ( inhibition of 69% in complex i - iii and of 67% in complex ii - iii ) 20 . cortisol has widespread functions including activation of glucocorticoid receptor which bind directly to mitochondrial membrane and regulate apoptosis 15 . the steroidogenic acute regulatory protein is one of the outer mitochondrial membrane protein required for stress responses 21 . although all these studies indicate mitochondrial dysfunction , they are all focusing on a specific candidate gene(s ) . the lack of a holistic study of all the mitochondria - related genes hinders the progress of the ptsd research . to date , methods employing a systems biology approach to identify the mitochondria - focused genes underlying the pathogenesis in ptsd - related brain regions have not been documented , probably due to the lack of human postmortem brain tissue from ptsd patients . the expression signatures in blood cells have been associated with changes in peripheral lymphocytes of subjects with ptsd 22;23 , but provide no information for molecular abnormalities in brain tissues . here we report the identification of expression signatures , canonical pathways , molecular networks and drug targets of dysregulated neuropsychiatric disease - related genes in ba46 of postmortem brain tissue from patients with ptsd using a human mitochondria - focused cdna microarray ( hmitchip3 ) 24 . our results indicate mitochondrial dysfunction in the dlpfc ba46 and may prove useful for developing methods for diagnosis and treatment of ptsd . postmortem brain tissue : postmortem brain tissues were collected by the traumatic stress brain study group who diagnosed , collated , planned , coordinated , made decisions on inclusion and exclusion , established procedures of diagnosis and interrogated reliability , and obtained normal brains for controls as well . medical records were meticulously reviewed and clinical diagnoses were confirmed using dsm - iv criteria 4 . table 1 lists all available brain tissues employed for this study , with some clinical data including patient 's gender and age ( 4-year difference ) , brain ph ( 0.65 difference ) and reasons of death . eight subjects died from myocardial infarction / heart attack , two from suicide by overdose , one from alcoholism complications and another from pulmonary embolism . tissue ( 0.4 - 0.8 g ) was thawed and homogenized in 10 times the tissue volume of deionized distilled h2o and the homogenate ph was measured with the perphect ph 370 model meter with a compatible perphect sure - flow type probe ( ati orion , boston , ma , usa ) . the ph electrode was washed thoroughly in deionized distilled h2o after each sample and recalibrated at the standard 3 points of ph ( 4 , 7 , and 10 ) . rna preparation : dlpfc ba46 tissues were dissected from postmortem brains and the samples contained primarily gray matter with small but random amount of white matter because the dissections were done from frozen tissue blocks but not from cryostat sections . carlsbad , ca , usa ) and then purified with rneasy kit ( qiagen , valencia , ca ) , following the manufacture 's instructions . gene chip and microarray data analysis : a third - generation human mitochondria - focused cdna microarray ( hmitchip3 ) containing 37 mitochondrial dna - encoded genes , 1,098 nuclear dna - encoded and mitochondria - related genes and 225 controls was printed as described previously 24 . a total of 1135 mitochondria - related genes include 946 genes associated with 645 molecular functions , 930 genes with 612 biological processes , 476 genes with biological chemistry pathways , 227 genes with 23 reactome events , 237 genes with 320 genetic disorders , and 55 genes with 87 drugs targets24 . for examples , 89 genes are related to oxidative phosphorylation , 20 genes to apoptosis , and 14 genes to neurotransmitter ( tyrosine , l - dopa and dopamine ) metabolism . five g of rna per sample were used for microarray labeling and hybridization as previously described 24 . slides were scanned using the scanarray express microarray scanner ( perkinelmer , boston , ma , usa ) as described previously 24 . microarray database construction , data filtering and normalization were performed as described previously 24;25 . the normalized data were used to cluster and visualize expression levels of genes in these brain tissue specimens by using eisen 's cluster software 26 . heat map was visualized by using eisen 's maple tree software ( http://rana.lbl.gov/eisensoftware.htm ) . gene information analysis : gene i d , symbols and names were derived from human unigene build 204 ( ftp://ftp.ncbi.nih.gov/repository/unigene/ ) based on human cdna i.m.a.g.e . ontology , pathways and phenotypes of genes were compiled from entrez ( ftp://ftp.ncbi.nlm.nih.gov/gene ) and david bioinformatics resources 2008 ( http://david.abcc.ncifcrf.gov/ ) . ingenuity pathway analysis software ipa version 6.0 ( ingenuity systems inc . , redwood city , ca , usa ) was used to map canonical pathways , diseases , disorders , molecular networks and drug targets and to calculate the percentages and p - values ( fisher exact test ) . quantitative rt - pcr ( qrt - pcr ) : two g of total rna was reverse - transcribed into cdna by using superscript first - strand synthesis system ( invitrogen ) . 30 ng cdna was used for qpcr reactions with the universal pcr master mix ( no amperase ung ) on an applied biosystems 7300 real time pcr system ( foster city , ca , usa ) , following the manufacturer 's instructions . after 40 cycles , taqman probes and primers for qpcr were purchased from applied biosystems and include those of glyceraldehyde-3-phosphate dehydrogenase ( gapdh , 4352934e ) , dnaj ( hsp40 ) homolog subfamily c member 19 ( dnajc19 , hs00829488_s1 ) , solute carrier family 1 ( high affinity aspartate / glutamate transporter ) member 6 ( slc1a6 , hs00192604_m1 ) , solute carrier family 9 ( sodium / hydrogen exchanger ) member 6 ( slc9a6 , hs00234723_m1 ) , amyloid beta ( a4 ) precursor protein ( app , hs00169098_m1 ) , and estrogen receptor 2 ( er beta ) ( esr2 , hs00230957_m1 ) . statistics : statistical calculations were performed on triplicate array experiments using xlstat 2006 ( xlstat , new york , ny , usa ) . differentially expressed genes were identified arbitrarily by 1.25-fold change in the average expression of the background - subtracted mean intensity ratios of a gene between ptsd and control , with p - value < 0.05 . student t - test was used to calculate p - values for gene expression , while fisher exact test in ingenuity pathway analysis software was used to calculate p - values for pathways and diseases . the level of statistical significance was set at a p - value < 0.05 . total rna samples were extracted from gray matter that was dissected from postmortem brain dlpfc ba46 of ptsd brains ( n=6 ) and unaffected controls ( n=6 ) and labeled for triplicate microarray experiments using our recently developed third generation human mitochondria - focused cdna microarray hmitchip3 . because use of 3 replicates in cdna microarrays analysis of even a single specimen greatly reduces misclassification rates 29 , the hmitchip3 genes were all measured 9 times ( 3 identical probes per microarray and 3 microarray experiments per specimen ) , which generated reliable expression data for further analysis . the microarray data of 4,080 spots across all 36 gene chips used for 12 rna samples were filtered by uniform statistic and bioinformatic criteria as described previously 24;30 , which generated 800 genes with informative expression profiles . the supplemental table 1 reports the expression data of the selected 800 genes in each of the triplicate experiments before and after normalization as the raw and processed data , respectively ; while figure 1 shows the box plots of mrna levels of 800 genes before and after the data normalization . the resultant dendrograms for all of these 800 genes and 12 brain dlpfc ba46 specimens classified p1 , p2 , p3 , p5 , p6 and n2 in one group and n1 , n3 , n4 n5 , n6 and p4 in another ( fig . 2a and supplemental figure 1 ) ; these analysis separated the ptsd brain from the control except for n2 and p4 ( opposite from their clinical diagnosis ) . based on the unsupervised cluster results , we calculated an average expression level of each gene in the ptsd group ( p1 , p2 , p3 , p5 , p6 and n2 ) and in the non - ptsd control group ( n1 , n3 , n4 n5 , n6 and p4 ) . the ratios and p - values ( t - test ) were calculated between the ptsd and control specimens , resulting in the identification of 119 genes ( p<0.05 , 1.25 ) and 42 genes ( p<0.05 , 1.60 ) whose expression were dysregulated in the ptsd dlpfc ba46 as compared to the controls ( supplemental table 2 ) . clustering analysis of these 119 and 42 genes across all 12 brain samples generated double dendrograms and heat maps that clearly distinguish the ptsd dlpfc ba46 specimens from the controls ( fig . the identification of these dysregulated genes provides candidates with biological clues for validation of mrna expression and future functional studies . qrt - pcr confirmation of microarray results . to validate the microarray results , we conducted qrt - pcr analysis on 5 genes including app , dnajc19 , esr2 , slc1a6 and slc9a6 because of the highest ( slc9a6 ) and lowest ( danjc19 ) expression levels in ptsd ba46 or because of their known relevance to neuropsychiatric disorders ( app , est2 and slc1a6 ) . the results showed that the relative mrna levels of these genes measured by qrt - pcr were essentially in agreement with the data detected by microarray experiments ( fig . 3 ) . specifically , 11 of 12 ( 92% ) of expression changes were consistent between these two methods for app , 9 of 12 ( 75% ) for slc9a6 , and 8 of 12 ( 67% ) for dnajc19 , slc1a6 and esr2 . the discrepant data were randomly distributed among the genes and samples tested ( fig . thus , we subjected all the 119 dysregulated genes to analysis of a systems biology including the pathway , ontology and network . the mitochondrial dysfunction and oxidative phosphorylation pathways contained the highest number of dysregulated genes . to see functional relationships the results showed that out of 94 canonical pathways , 16 ( 17.0% ) contained a significant number of dysregulated genes per pathway , of which 10 were listed in figure 4a . the mitochondrial dysfunction pathway contained 8 ( 4.8% ) dysregulated genes ( p=6.61x10 ) including upregulated app , cat , ndufa10 and ucp2 and downregulated cox8a , ndufb5 , ndufs2 and pdha1 . the oxidative phosphorylation pathway had 6 ( 3.8% ) dysregulated genes ( p=9.04x10 ) including upregulated atp5c1 and ndufa10 and downregulated atp5e , cox8a , ndufb5 and ndufs2 . the citrate cycle pathway had 3 ( 5.1% ) dysregulated genes ( p=1.71x10 ) including upregulated idh3b and downregulated cs and suclg1 . the methane metabolism pathway contained 2 ( 3.1% ) dysregulated genes ( p=5.38x10 ) including upregulated cat and downregulated mpo . the ubiquinone biosynthesis pathway had 3 ( 2.9% ) dysregulated genes ( p=1.11x10 ) including upregulated ndufa10 and downregulated ndufb5 and ndufs2 . the erk / mark signaling pathway included upregulated h3f3a , ppp2r1b , prkcd and tln2 , and downregulated prkar2b ( n=5 [ 2.8% ] , p=1.18x10 ) . the propanoate metabolism pathway had upregulated acad8 and acadl and downregulated suclg1 ( n=3 [ 2.4% ] , p=1.21x10 ) . the xenobiotic metabolism pathway contained upregulated cat , hsp90ab1 , ppp2r1b and prkcd and downregulated aldh18a1 and mgst1 ( n=6 [ 2.4% ] , p=1.28x10 ) . the valine , leucine and isoleucine degradation pathway contained upregulated acad8 , acadl and hmgcl ( n=3 [ 2.8% ] , p=1.38x10 ) . the pyruvate metabolism pathway had downregulated akr1b1 , hagh and pdha1 ( n=3 [ 2.1% ] , p=2.05x10 ) ( fig . 46 dysregulated genes were mapped to neurological diseases and other systemic disorders . to see phenotypes involved , we performed mapping of all 119 dysregulated genes to known human diseases and disorders . the results revealed that 35 dysregulated genes had known roles in a variety of neurological diseases ( 28 genes ) and other systematic disorders including endocrine ( 14 genes ) , genetics ( 11 genes ) , metabolism ( 12 genes ) and psychology ( 6 genes ) . the number of these dysregulated genes were all statistically significant ( p<0.05 ) . in addition , 17 ( 61% ) of the 28 genes that were mapped to neurological diseases were known to play a role in survival and apoptosis of a cell . moreover , a total of 30 dysregulated genes were mapped to the cell survival - apoptosis category ( fig . many of these genes are involved in multiple abnormalities , suggesting that ptsd is a neurological and systemic disorder . table 2 lists 30 ptsd - dysregulated genes that have been described to play a pathogenic role in various neurological diseases and psychiatric disorders . upregulated app induces changes in nitric oxide production and mitochondrial activity , leading to apoptosis 31 , which have been implicated in amyloidosis 32;33 , neurodegeneration 34 , alzheimer 's disease 35;36 and down syndrome 37 . considering the role of myeloperoxidase ( mpo ) in modulating vascular inflammatory responses and transporting molecules as well as its incapability of clearing up amyloid when mutated 38 - 40 , mpo downregulation may lead to regional accumulation of amyloid , resulting in mitochondrial dysfunction and cell death . peptidylprolyl isomerase d ( ppid ) suppresses apoptosis via a mitochondrial hexokinase ii - dependent mechanism and its decreased expression has been associated with corticobasal degeneration characterized by nerve cell loss and atrophy 41 . dysregulation of genes with known functions as neurotransmitters in synaptic vesicles ( slc1a6 and vamp1 ) and cell adhesion ( pecam1 , cdh13 and spg7 ) are related to diseases in the central nervous system . dysregulation of these genes might reduce synaptic communication , and jeopardize cell - to - cell interactions and/or cell - to - matrix adhesion . these are but a few examples ( table 2 ) . networks of 54 dysregulated genes involved in neuron function and survival and 7 protein targets for neuropsychiatric drugs : to reveal complex interactions among dysregulated genes and to identify gene targets for the central nervous system drugs , we mapped 54 dysregulated genes to molecular networks . these genes were selected for analysis because of their obvious relevance to neurological diseases , psychological disorders , psychiatric disorders , and stress response in the central nervous system . the results revealed interactive networks with a total of 75 molecular elements that maintain neuronal function and survival . within these networks , 27 ( 36% ) genes were upregulated ; 27 ( 36% ) genes were downregulated ; and 21 ( 28% ) elements appear to be unchanged ( fig . these data strongly suggest molecular and cellular abnormalities in neuronal function and survival in ptsd dlpfc ba46 . importantly , 7 gene products within the neuronal function - survival networks were identified as known targets of drugs that are used for neurological diseases , psychological and/or behavioral disorders , including app as a target for bapineuzumab ( aab-001 ) , vamp1 for dysport , slc1a6 for riluzole , esr2 for 17-estradiol ( agonist ) and tamoxifen ( antagonist ) , prkcd for tamoxifen and rottlerin , pp2a for okadaic acid and fostriecin , and ucp2 for mptp ( 1,2,3,6-methyl - phenyl - tetrahydropyridine ) . human brain dlpfc including ba46 is involved in regulation of working memory and preparation and selection of fear responses and has been correlated with structural and functional alterations and treatment response of patients with ptsd 9 - 13 . however , the underlying cellular and molecular mechanisms are unknown . in this study , we applied human mitochondria - focused cdna microarrays ( hmitchip3 ) to ptsd brain samples and have successfully identified expression signatures , canonical pathways , molecular networks and drug targets of neurological disease- and psychological / psychiatric disorder - related genes that are dysregulated in the dlpfc ba46 . our results indicate mitochondrial dysfunction involved in neuronal function and survival in the dlpfc ba46 and may prove useful for development of methods for diagnosis , prevention and treatment of ptsd . unsupervised clusters of 12 dlpfc ba46 rna samples based solely on expression similarities of informative 800 mitochondria - focused genes clearly distinguish the ptsd brains from the controls . the identification of 119 ( p<0.05 , 1.25 ) and 42 ( p<0.05 , 1.60 ) dysregulated genes provides candidates for qrt - pcr validation and for subsequent functional studies . the clustering signatures of these genes may facilitate further development of methods and tools for forensic diagnosis of ptsd . our approach has generated informative results partially because of our accurate dissection of brain dlpfc ba46 gray matter , and partially because of the utilization of our hypothesis - driven mitochondria - focused cdna microarrays . however , we can not explain why the samples p4 and n2 had expression profiles different from their clinical classification . due to a small sample size , p4 and n2 were kept in the groups based on their gene expression rather than a clinical diagnosis because the former might be more objective than the latter . we would like to repeat the study when more ptsd and control specimens become available in order to improve the molecular classification of ptsd . the subsequent experiments should examine both mrna and protein levels in the dlpfc ba46 , as well as other ptsd - related brain regions such as medial and ventral pfc , hippocampus , and amygdala 6;7;42 . the mitochondria - mediated cellular and molecular response to stress in the central nervous system of patients with ptsd is the key part of our hypothesis because mitochondria are at the center of a cellular response to stress 14;15 . study of mitochondrial function is inevitable and our application of the mitochondria - focused cdna microarrays to ptsd is just the beginning . mitochondrial function in the brain is of particular importance because of its high energy demand . although human brain represents only 2% of body weight , it receives 15% of the body 's cardiac output , and uses 20% of total body oxygen . these high level oxygen and energy requirements are continuous and imply that even brief periods of oxygen or glucose deprivation may impair neuron function and even result in neuron death . our demonstration that a highly significant number of oxidative phosphorylation genes were dysregulated in the ptsd brain ba46 strongly suggests the presence of at least energy deficiency in this brain region . our findings support the postulation that mitochondria - focused and stress - responsive genes may play a key role in the pathogenesis of ptsd , although the specific impact of these dysregulated genes and pathways on brain function remains to be determined . the identification and validation of the dysregulated genes enhances our understanding of the cellular and molecular mechanisms underlying the pathophysiology of ptsd . the genes listed in table 2 are critical to the ba46 neuronal functions including survival and apoptosis , carbohydrate and lipid metabolism , atp and ros production , synthesis and transport of neurotransmitter , mitochondrial and nuclear dna expression , and protein phosphorylation and dephosphorylation as well as folding and degradation . because homeostasis of these mitochondrial functions is vital for neuronal function and survival , the presence of a hundred consistently dysregulated genes in the ptsd dlpfc ba46 implies a pathological role . in addition , more genes were downregulated than those upregulated ( 63 vs. 56 in supplemental table 1 and 18 vs. 12 in table 2 ) . moreover , some upregulated genes play a degradative or negative role such as proapoptosis ( e.g. , app ) , reduction of atp synthesis ( e.g. , ucp2 ) and protein breakdown ( e.g. , hspa1a ) . taken together , functional impairment and atrophy in the ptsd dlpfc ba46 might be a consequence of these dysregulated genes . thus , the neurons in the ptsd dlpfc ba46 may be unable to regulate working memory and preparation and selection of healthy responses to fear or may even send abnormal signals to their downstream executive neurons such as those in the hippocampus and/or amygdala . our molecular insight in the ptsd dlpfc ba46 pathogenesis is reminiscent of the neurocircuitry model by bremner 43 and rauch et al 42 that the fear extinction abnormalities in ptsd are characterized by exaggerated amygdala responses and deficiency in ventral / medial pfc and hippocampus . therefore , our systems biology results appear not only to extend the neurocircuitry model to brain dlpfc ba46 , but they also provide genes and proteins that should be further investigated for better understanding of the molecular pathogenesis of ptsd . seven protein targets for neuropsychiatric drugs are present in the neuronal function - survival networks in human brain dlpfc ba46 . in addition of dysregulation of 72% ( 54/75 ) genes in these networks , many dysregulated genes are associated with multiple neuropsychiatric conditions such as alzheimer 's disease , parkinson 's disease , schizophrenia , and major depressive disorder ( mdd ) , down syndrome , attention disorders and mood disorders ( table 2 ) . obviously , modulations of these genes with drugs would affect neuropsychiatric functions of the neurons in human dlpfc ba46 . for example , the app is a target for the immunotherapeutic drug bapineuzumab that is currently used for the phase 3 clinical trials for the treatment of patients with alzheimer 's disease 44 . the esr2 is a target for 17-estradiol ( agonist ) and tamoxifen ( antagonist ) . beside the treatment of estrogen - receptor positive breast cancer , tamoxifen is used to treat mania in patients with bipolar disorder , probably by blocking prkcd 45 . the prkcd is also a target for rottlerin used in preclinical study of parkinson 's disease 46 . the ucp2 is a target for protection of dopaminergic neurons from oxidative stress caused by 1,2,3,6-methyl - phenyl - tetrahydropyridine ( mptp ) toxicity 48 . however , whether these drugs and related targets might be useful for prevention and treatment of ptsd is interesting and deserves further studies . supplemental table 1a : microarray mean data before normalization click here for additional data file . supplemental table 1b : microarray mean data after normalization click here for additional data file . click here for additional data file . traumatic stress brain study group includes david benedek and harry holloway , dept of psychiatry & center for the study of traumatic stress , usuhs , school of medicine ; maree j. webster , stanley medical research institute ; christopher j. hough , fda ; ronald duman , abraham ribicoff research facilities laboratory of molecular psychiatry , yale university school of medicine ; matthew friedman , national center for ptsd , us dept of veterans affairs & dartmouth medical school ; john krystal , clinical neuroscience division , va national center for ptsd , va connecticut healthcare system , west haven , ct & dept of psychiatry , yale university school of medicine ; gregory leskin , national center for ptsd , va palo alto health care system ; james meyerhoff , georgetown university school of medicine & division of psychiatry & neurosciences , walter reed army institute of research ; elizabeth osuch , dept of psychiatry , university of western ontario , schulich school of medicine and dentistry .

posttraumatic stress disorder ( ptsd ) is associated with decreased activity in the dorsolateral prefrontal cortex ( dlpfc ) , the brain region that regulates working memory and preparation and selection of fear responses . we investigated gene expression profiles in dlpfc brodmann area ( ba ) 46 of postmortem patients with ( n=6 ) and without ptsd ( n=6 ) using human mitochondria - focused cdna microarrays . our study revealed ptsd - specific expression fingerprints of 800 informative mitochondria - focused genes across all of these 12 ba46 samples , and 119 ( >1.25 , p<0.05 ) and 42 ( >1.60 , p<0.05 ) dysregulated genes between the ptsd and control samples . quantitative rt - pcr validated the microarray results . these fingerprints can essentially distinguish the ptsd dlpfc ba46 brains from controls . of the 119 dysregulated genes ( 125% , p<0.05 ) , the highest percentages were associated with mitochondrial dysfunction ( 4.8% , p=6.61x10 - 6 ) , oxidative phosphorylation ( 3.8% , p=9.04x10 - 4 ) , cell survival - apoptosis ( 25.2% , p<0.05 ) and neurological diseases ( 23.5% , p<0.05 ) . fifty ( 50 ) dysregulated genes were present in the molecular networks that are known to be involved in neuronal function - survival and contain 7 targets for neuropsychiatric drugs . thirty ( 30 ) of the dysregulated genes are associated with a number of neuropsychiatric disorders . our results indicate mitochondrial dysfunction in the ptsd dlpfc ba46 and provide the expression fingerprints that may ultimately serve as biomarkers for ptsd diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of ptsd .

there is increasing evidence that higher levels of high sensitivity c - reactive protein ( hs - crp ) is a marker of inflammation and a potential independent predictor of cardiovascular disease , as it may play a role in the development of atherosclerosis , adversely affecting mortality.1)2 ) rate of obesity is increasing globally where studies have shown association of body weight as assessed by body mass index ( bmi ) with clinical outcomes after percutaneous coronary intervention ( pci ) in acute myocardial infarction ( ami ) patients.3 ) previous reports have also indicated that hs - crp level is correlated with bmi and fat distribution.4)5 ) the mechanism for elevation of plasma hs - crp in obese subjects could be through a high production of cytokines , e.g. , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) and tumor necrosis factor- ( tnf- ) , by excess adipose tissue which could induce high hs - crp production in liver.4 ) curently there is no data available evaluating the relationship between higher baseline hs - crp levels , body mass index ( bmi ) status , and adverse outcomes in patients with ami undergoing pci . therefore , in the present study we attempted to assess the association between baseline hs - crp level and 12-month clinical outcomes in patients with ami . this retrospective study was carried out at the heart center of chonnam national university hospital , korea . the permission to carry out the study had been sought from the hospital authorities ( irb no.05 - 49 ) and informed consent obtained from the patients . patient medical documents were used to obtain the demographic data , clinical characteristics and relevant laboratory results . patients were enrolled in the registry after admission to participating hospitals with a suspected diagnosis of ami . the korea acute myocardial infarction registry ( kamir ) is a prospective , multicenter , observational registry designed to examine current epidemiology in hospital management and outcome of patients with ami in korea . the registry included 52 community and university hospitals capable of primary pci with one - year clinical follow up . data was collected at each site by a well - trained study coordinator based on a standardized protocol.6 - 8 ) a total of 10974 consecutive patients with ami ( both st - segment elevation mi and non - st - segment elevation mi ) undergoing pci from november 2005 to september 2008 were assessed in this study . patients with unknown bmi ( n=395 ) and missing hs - crp ( n=2405 ) data were excluded from this study ; hence the total study population was 8174 patients . the aim was to evaluate the impact of baseline hs - crp on 12-month clinical outcomes according to bmi status . then , according to bmi categories suggested by the world health organization for asian population , the total study population was divided into three groups that included 1 ) overweight / obese ( bmi 23 kg / m , n=5647 ) , 2 ) normal - weight ( bmi 18.5 to 22.9 kg / m , n=2246 ) and 3 ) underweight ( bmi < 18.5 kg / m , n=281).9 ) each group was then stratified into quartiles based on their hs - crp levels . a final diagnosis of ami was made by the european society of cardiology / american college of cardiology ( esc / acc ) diagnostic criteria for ami.10 ) bmi was calculated as weight ( kg ) divided by height squared ( m ) . diabetes mellitus ( dm ) was defined as requiring the use of oral hypoglycaemic agent or insulin to lower blood glucose levels . hypertension was defined as patient systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at rest , over a series of repeated measurements , or after treatment with anti - hypertensive medications . for diabetics and patients with chronic renal disease , hypertension was defined as systolic blood pressure 130 mm hg and/or diastolic blood pressure 80 mm hg . hyperlipidemia was defined as patient total cholesterol level > 200 mg / dl or treatment with a lipid - lowering agent . coronary artery disease ( cad ) was defined as patient having a history of myocardial infarction ( mi ) revascularization procedure , or obstructive cad . blood samples were centrifuged and serum was removed and stored at a temperature of -700 until the assay for sugar and proteins was performed . absolute creatine kinase - mb levels were determined by radioimmunoassay ( dade behring , inc . , cardiac specific troponin i levels were measured using a paramagnetic particle , chemiluminescent immunoenzymatic assay ( beckham , coulter , inc . , twelve - hour fasting serum levels of total cholesterol , triglyceride , and low and high density lipoprotein - cholesterol were measured by standard enzymatic methods . blood sample for hs - crp was obtained on admission and was analyzed turbidimetrically with sheep antibodies against human crp ; this has been validated against the dade behring method . overweight / obese quartiles were : first quartile < 0.2 mg / dl , n=1370 , second quartile 0.2 to < 0.82 mg / dl , n=1451 , third quartile 0.82 to < 4.08 mg / dl , n=1413 and fourth quartile 4.08 mg / dl , n=1413 . normal - weight quartiles were : first quartile < 0.2 mg / dl , n=548 , second quartile 0.2 to < 0.92 mg / dl , n=575 , third quartile 0.92 to < 5.23 , n=563 mg / dl and fourth quartile 5.23 mg / dl , n=563 . underweight quartiles were : first quartile < 0.31 mg / dl , n=70 , second quartile 0.31 to < 1.54 mg / dl , n=70 , third quartile 1.54 to < 8.76 mg / dl , n=71 and fourth quartile 8.76 mg / dl , n=70 . two - dimensional echocardiography was performed in all patients and left ventricular ejection fraction ( lvef ) was assessed using a modified simpson 's biplane method . the morphology in coronary angiography was classified by criteria from the american college of cardiology / american heart association ( acc / aha).11 ) the degree of coronary flow was classified by thrombolysis in myocardial infarction ( timi ) score . the presence of left main coronary artery stenosis was defined as a luminal stenosis 50% . multivessel disease was defined as the presence of a lesion with > 50% diameter stenosis in a non - infarct related coronary artery . successful pci was defined as timi flow 3 with residual stenosis 50% in the infarct related artery . in - hospital complications included atrio - ventricular block , bradycardia , ventricular tachycardia / ventricular fibrillation , atrial fibrillation , no reflow , dissection , cardiogenic shock , acute renal failure , metabolic acidosis / lactic acidosis . major advense cardiac event ( mace ) were defined as all - cause death , recurrent mi , or repeat revascularization . the primary endpoint of this study was all - cause mortality at 12-month clinical follow up . all data were recorded on a standardized , electronic , web based registry at http://www.kamir.or.kr . continuous variables were presented as the meansd ; comparisons were conducted by one - way anova test . discrete variables were presented as percentages and frequencies ; comparisons were conducted by chi - square statistics . cox regression analysis was performed to identify a model with independent predictive factors including determination of a hazard ratio and its 95% confidence interval ( ci ) for each variable in the model . cox analysis was performed to identify variables that most affected the hazard ratio of hs - crp , with a cut off at p<0.20 for entry into the model . the korea acute myocardial infarction registry ( kamir ) is a prospective , multicenter , observational registry designed to examine current epidemiology in hospital management and outcome of patients with ami in korea . the registry included 52 community and university hospitals capable of primary pci with one - year clinical follow up . data was collected at each site by a well - trained study coordinator based on a standardized protocol.6 - 8 ) a total of 10974 consecutive patients with ami ( both st - segment elevation mi and non - st - segment elevation mi ) undergoing pci from november 2005 to september 2008 were assessed in this study . patients with unknown bmi ( n=395 ) and missing hs - crp ( n=2405 ) data were excluded from this study ; hence the total study population was 8174 patients . the aim was to evaluate the impact of baseline hs - crp on 12-month clinical outcomes according to bmi status . then , according to bmi categories suggested by the world health organization for asian population , the total study population was divided into three groups that included 1 ) overweight / obese ( bmi 23 kg / m , n=5647 ) , 2 ) normal - weight ( bmi 18.5 to 22.9 kg / m , n=2246 ) and 3 ) underweight ( bmi < 18.5 kg / m , n=281).9 ) each group was then stratified into quartiles based on their hs - crp levels . a final diagnosis of ami was made by the european society of cardiology / american college of cardiology ( esc / acc ) diagnostic criteria for ami.10 ) bmi was calculated as weight ( kg ) divided by height squared ( m ) . diabetes mellitus ( dm ) was defined as requiring the use of oral hypoglycaemic agent or insulin to lower blood glucose levels . hypertension was defined as patient systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at rest , over a series of repeated measurements , or after treatment with anti - hypertensive medications . for diabetics and patients with chronic renal disease , hypertension was defined as systolic blood pressure 130 mm hg and/or diastolic blood pressure 80 mm hg . hyperlipidemia was defined as patient total cholesterol level > 200 mg / dl or treatment with a lipid - lowering agent . coronary artery disease ( cad ) was defined as patient having a history of myocardial infarction ( mi ) revascularization procedure , or obstructive cad . blood samples were centrifuged and serum was removed and stored at a temperature of -700 until the assay for sugar and proteins was performed . absolute creatine kinase - mb levels were determined by radioimmunoassay ( dade behring , inc . , cardiac specific troponin i levels were measured using a paramagnetic particle , chemiluminescent immunoenzymatic assay ( beckham , coulter , inc . , twelve - hour fasting serum levels of total cholesterol , triglyceride , and low and high density lipoprotein - cholesterol were measured by standard enzymatic methods . blood sample for hs - crp was obtained on admission and was analyzed turbidimetrically with sheep antibodies against human crp ; this has been validated against the dade behring method . overweight / obese quartiles were : first quartile < 0.2 mg / dl , n=1370 , second quartile 0.2 to < 0.82 mg / dl , n=1451 , third quartile 0.82 to < 4.08 mg / dl , n=1413 and fourth quartile 4.08 mg / dl , n=1413 . normal - weight quartiles were : first quartile < 0.2 mg / dl , n=548 , second quartile 0.2 to < 0.92 mg / dl , n=575 , third quartile 0.92 to < 5.23 , n=563 mg / dl and fourth quartile 5.23 mg / dl , n=563 . underweight quartiles were : first quartile < 0.31 mg / dl , n=70 , second quartile 0.31 to < 1.54 mg / dl , n=70 , third quartile 1.54 to < 8.76 mg / dl , n=71 and fourth quartile 8.76 mg / dl , n=70 . two - dimensional echocardiography was performed in all patients and left ventricular ejection fraction ( lvef ) was assessed using a modified simpson 's biplane method . the morphology in coronary angiography was classified by criteria from the american college of cardiology / american heart association ( acc / aha).11 ) the degree of coronary flow was classified by thrombolysis in myocardial infarction ( timi ) score . the presence of left main coronary artery stenosis was defined as a luminal stenosis 50% . multivessel disease was defined as the presence of a lesion with > 50% diameter stenosis in a non - infarct related coronary artery . successful pci was defined as timi flow 3 with residual stenosis 50% in the infarct related artery . in - hospital complications included atrio - ventricular block , bradycardia , ventricular tachycardia / ventricular fibrillation , atrial fibrillation , no reflow , dissection , cardiogenic shock , acute renal failure , metabolic acidosis / lactic acidosis . major advense cardiac event ( mace ) were defined as all - cause death , recurrent mi , or repeat revascularization . the primary endpoint of this study was all - cause mortality at 12-month clinical follow up . all data were recorded on a standardized , electronic , web based registry at http://www.kamir.or.kr . spss 17.0 for windows ( spss , inc . , chicago , il , usa ) was used for all analyses . continuous variables were presented as the meansd ; comparisons were conducted by one - way anova test . discrete variables were presented as percentages and frequencies ; comparisons were conducted by chi - square statistics . cox regression analysis was performed to identify a model with independent predictive factors including determination of a hazard ratio and its 95% confidence interval ( ci ) for each variable in the model . cox analysis was performed to identify variables that most affected the hazard ratio of hs - crp , with a cut off at p<0.20 for entry into the model . a total of 8,174 ami patients , mean age=62.712.4 years and 76% male gender ( n=6217 ) , were stratified into quartiles based on their hs - crp level to study their 12-month clinical outcomes . twelve - month all - cause death and 12-month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard analysis showed 12-month mortality to be highest in patients of 4th quartile { hr=2.80 ( 1.335 - 5.873 ) , p=0.006 } followed by 3rd quartile { hr=2.495 ( 1.186 - 5.248 ) , p=0.016}. it was statistically significant among hs - crp quartiles ( p=0.045 ) . independent factors for 12-month mortality were age greater than 60 years , in - hospital complications , killip class > 1 , use of statin , heart rate > 100 beats / min , n - terminal pro - b - type natriuretic peptide ( nt - probnp ) > 3000 pg / ml , left ventricular ejection fraction ( lvef ) < 55% and creatinine clearance < 60 min / ml ( data not provided ) . as the 12-month mortality was statistically borderline significant among hs - crp quartiles in the total study population , we sought to further evaluate this finding by dividing the total study population into three groups based on their bmi . overweight and obese patients were grouped together as there was no baseline significant difference in their 12-month all - cause mortality ( p=0.076 , data not provided ) . baseline clinical characteristics and laboratory findings of this group are presented in table 1 and 2 respectively . overweight / obese patients with higher hs - crp ( i.e. , 2nd , 3rd and 4th quartiles ) were older , more often female , had higher killip classes , heart rate and nt - probnp ; and had lower blood pressure , lvef and creatinine clearance . they were associated with higher incidence of dm , hypertension , cad , heart failure and cerebrovascular disease . as per past regular medication history , 7.15% of the patients at the time of admission were on statin ( n=404 ) . multivessel involvement , acc / aha lesion type c , and pre - pci timi flow grade 0 were observed to increase with an increase in hs - crp . pci success rate decreased and in - hospital complication rate increased with increasing hs - crp . table 4 shows the clinical outcomes during the 12-month follow - up . in - hospital death , composite mace at 1 month and 12 months , and mortality at 1 month and 12 months were more frequent with increasing hs - crp levels . twelve - month mortality increased as the value of hs - crp increased ; 2.0% ( n=27 ) in 1st , 2.5% ( n=37 ) in 2nd , 4.4% ( n=62 ) in 3rd and highest 6.6% ( n=94 ) in the 4th quartile . cox proportional hazard model was implemented to identify the independent predictors of mortality at the 12-month clinical follow - up . serum hs - crp quartiles showed significant association with 12-month mortality when adjusted for age and gender ( p<0.001 ) . with quartile 1 as a reference , the risk of 12-month mortality was higher in the 3rd quartile { 2.153 ( 1.369 - 3.384 ) , p=0.001 } and 4th quartile { 3.268 ( 2.129 - 5.016 ) , p<0.001}. after adjustment with multiple covariates , 12-month mortality was highest in patients within the 4th quartile compared to other patients { hazard ratio ( hr ) 2.382 ( 1.079 - 5.259 ) , p=0.032 } though statistical significance was not achieved ( p=0.172 ) . independent factors for 12-month mortality were old age ( > 60 years ) , heart rate > 100 beats / min , lvef < 55% , creatinine clearance < 60 min / ml and in - hospital complications . the variables included in cox model were age , male gender , heart rate , systolic blood pressure , smoking history , history of hypertension , diabetes mellitus , coronary artery disease , cerebrovascular disease and heart failure , lvef , killip classes > 1 , glucose , total cholesterol , triglyceride , creatinine clearance , nt - probnp , left main and multivessel involvement , pre - timi flow grade 0 , post - timi flow grade 3 , acc / aha lesion type c , pci success rate , in - hospital complications and use of statin . patients were also stratified into quartiles based on hs - crp level to evaluate their 12-month clinical outcomes . patients with higher hs - crp were found to be older ( p<0.001 ) , with increased heart rate ( p=0.015 ) and higher killip classes ( p=0.002 ) . they had more association with diabetes mellitus ( p=0.019 ) , hypertension ( p=0.049 ) , heart failure ( p=0.001 ) and peripheral vascular disease ( p=0.001 ) . creatinine clearance , lvef and high - density lipoprotein cholesterol decreased with higher hs - crp ( p<0.001 , p<0.001 and p=0.001 respectively ) . total cholesterol , triglyceride and low density lipoproteincholesterol ( ldl - c ) were also significant among quartiles ( p<0.001 , p=0.001 and p=0.001 respectively ) . they were more associated with acc / aha lesion type c , multivessel involvement , pre - timi flow grade 0 and in - hospital complications . twelve - month composite of mace ( p=0.009 ) and all - cause death ( p<0.001 ) increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group ( p=0.681 ) . the variables included in the model were age , dm , hypertension , hyperlipidemia , heart failure , peripheral vascular disease , smoking history , clinical presentation st - elevation mi , total cholesterol , triglyceride , creatinine clearance , nt - probnp , systolic blood pressure , heart rate , in - hospital complications , killip class > 1 , lvef < 55% , pre - procedure timi grade 0 flow , acc / aha lesion type c , multivessel involvement , and use of statin . patients from this study were additionally divided into quartiles based on hs - crp level to study their 12-month clinical outcomes . the variables included in the model were age , systolic blood pressure , lvef < 55% , glucose , total cholesterol , ldl - c , creatinine clearance , nt - probnp , smoking history , heart failure , post - procedure timi grade 3 , pci success rate , and in - hospital complications . age , nt - probnp , killip class > 1 and in - hospital complications increased with increasing levels of hs - crp ( p=0.006 , p<0.001 , p=0.009 and p=0.032 respectively ) , while creatinine clearance decreased with rising level of hs - crp ( p=0.001 ) . systolic blood pressure , lvef , total cholesterol and post - timi flow grade iii were statistically significant among the quartiles ( p=0.043 , p=0.023 , p=0.038 and p=0.002 respectively ) . twelve - month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile , but it was statistically insignificant ( p=0.382 ) , and twelve - month all - cause death showed no association among quartiles ( p=0.079 ) . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group as well ( p=0.760 ) . 1 shows adjusted survival curves of 12-month mortality in hs - crp quartiles in ( a ) total study population , ( b ) overweight / obese group , ( c ) normal - weight group and ( d ) underweight group . baseline clinical characteristics and laboratory findings of this group are presented in table 1 and 2 respectively . overweight / obese patients with higher hs - crp ( i.e. , 2nd , 3rd and 4th quartiles ) were older , more often female , had higher killip classes , heart rate and nt - probnp ; and had lower blood pressure , lvef and creatinine clearance . they were associated with higher incidence of dm , hypertension , cad , heart failure and cerebrovascular disease . as per past regular medication history , 7.15% of the patients at the time of admission were on statin ( n=404 ) . multivessel involvement , acc / aha lesion type c , and pre - pci timi flow grade 0 were observed to increase with an increase in hs - crp . pci success rate decreased and in - hospital complication rate increased with increasing hs - crp . table 4 shows the clinical outcomes during the 12-month follow - up . in - hospital death , composite mace at 1 month and 12 months , and mortality at 1 month and 12 months were more frequent with increasing hs - crp levels . twelve - month mortality increased as the value of hs - crp increased ; 2.0% ( n=27 ) in 1st , 2.5% ( n=37 ) in 2nd , 4.4% ( n=62 ) in 3rd and highest 6.6% ( n=94 ) in the 4th quartile . cox proportional hazard model was implemented to identify the independent predictors of mortality at the 12-month clinical follow - up . serum hs - crp quartiles showed significant association with 12-month mortality when adjusted for age and gender ( p<0.001 ) . with quartile 1 as a reference , the risk of 12-month mortality was higher in the 3rd quartile { 2.153 ( 1.369 - 3.384 ) , p=0.001 } and 4th quartile { 3.268 ( 2.129 - 5.016 ) , p<0.001}. after adjustment with multiple covariates , 12-month mortality was highest in patients within the 4th quartile compared to other patients { hazard ratio ( hr ) 2.382 ( 1.079 - 5.259 ) , p=0.032 } though statistical significance was not achieved ( p=0.172 ) . independent factors for 12-month mortality were old age ( > 60 years ) , heart rate > 100 beats / min , lvef < 55% , creatinine clearance < 60 min / ml and in - hospital complications . the variables included in cox model were age , male gender , heart rate , systolic blood pressure , smoking history , history of hypertension , diabetes mellitus , coronary artery disease , cerebrovascular disease and heart failure , lvef , killip classes > 1 , glucose , total cholesterol , triglyceride , creatinine clearance , nt - probnp , left main and multivessel involvement , pre - timi flow grade 0 , post - timi flow grade 3 , acc / aha lesion type c , pci success rate , in - hospital complications and use of statin . patients were also stratified into quartiles based on hs - crp level to evaluate their 12-month clinical outcomes . patients with higher hs - crp were found to be older ( p<0.001 ) , with increased heart rate ( p=0.015 ) and higher killip classes ( p=0.002 ) . they had more association with diabetes mellitus ( p=0.019 ) , hypertension ( p=0.049 ) , heart failure ( p=0.001 ) and peripheral vascular disease ( p=0.001 ) . creatinine clearance , lvef and high - density lipoprotein cholesterol decreased with higher hs - crp ( p<0.001 , p<0.001 and p=0.001 respectively ) . total cholesterol , triglyceride and low density lipoproteincholesterol ( ldl - c ) were also significant among quartiles ( p<0.001 , p=0.001 and p=0.001 respectively ) . they were more associated with acc / aha lesion type c , multivessel involvement , pre - timi flow grade 0 and in - hospital complications . twelve - month composite of mace ( p=0.009 ) and all - cause death ( p<0.001 ) increased as the value of hs - crp increased from 1st to 4th quartile . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group ( p=0.681 ) . the variables included in the model were age , dm , hypertension , hyperlipidemia , heart failure , peripheral vascular disease , smoking history , clinical presentation st - elevation mi , total cholesterol , triglyceride , creatinine clearance , nt - probnp , systolic blood pressure , heart rate , in - hospital complications , killip class > 1 , lvef < 55% , pre - procedure timi grade 0 flow , acc / aha lesion type c , multivessel involvement , and use of statin . patients from this study were additionally divided into quartiles based on hs - crp level to study their 12-month clinical outcomes . the variables included in the model were age , systolic blood pressure , lvef < 55% , glucose , total cholesterol , ldl - c , creatinine clearance , nt - probnp , smoking history , heart failure , post - procedure timi grade 3 , pci success rate , and in - hospital complications . age , nt - probnp , killip class > 1 and in - hospital complications increased with increasing levels of hs - crp ( p=0.006 , p<0.001 , p=0.009 and p=0.032 respectively ) , while creatinine clearance decreased with rising level of hs - crp ( p=0.001 ) . systolic blood pressure , lvef , total cholesterol and post - timi flow grade iii were statistically significant among the quartiles ( p=0.043 , p=0.023 , p=0.038 and p=0.002 respectively ) . twelve - month composite of mace increased as the value of hs - crp increased from 1st to 4th quartile , but it was statistically insignificant ( p=0.382 ) , and twelve - month all - cause death showed no association among quartiles ( p=0.079 ) . cox proportional hazard model showed 12-month mortality to be statistically insignificant among hs - crp quartiles in this group as well ( p=0.760 ) . 1 shows adjusted survival curves of 12-month mortality in hs - crp quartiles in ( a ) total study population , ( b ) overweight / obese group , ( c ) normal - weight group and ( d ) underweight group . this study demonstrated that higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . over the past few years , it has become increasingly clear that inflammation plays a pivotal role in cardiovascular disease , and increased circulating inflammation markers may be predictive of cardiovascular events.12)13 ) among the inflammation markers available , hs - crp is one of the independent predictors of cardiovascular events.14)15 ) adiposity is an inflammatory condition and il-6 , tnf- and hs - crp levels are positively correlated with adipocyte size.4)15)16 ) these cytokines induce insulin resistance in adipose cells by inhibiting insulin signaling.17)18 ) crp along with il-6 and other cytokines / adipokines could be deleterious on the arterial wall since it has been found to directly promote endothelial cell inflammation and atherosclerotic processes.19)20 ) thus , the link between adiposity , plaque accumulation / progression / rupture and atherothrombotic events , especially ami , might be explained by higher levels of systemic inflammation and their chronic effect on coronary atherosclerosis over time . marsik et al.2 ) report that patients with crp concentrations > 5 mg / l at the time of hospital admission had a 50% to 330% increase in risk of death from any cause . this increase in risk was present in both short - term and long - term follow - ups , and rose in magnitude as concentrations of crp increased to > another study by koenig et al.1 ) deals with the issue of hs - crp as a predictor of death in the monitoring of trends and determinants in cardiovascular disease . after adjustment for age , smoking , hypertension , hyperlipidemia , diabetes , obesity and socioeconomic markers , those with baseline hs - crp concentrations > 3 mg / l had a 2-fold increase in risk of total mortality . some studies have shown that higher bmi is associated with higher crp concentrations even among young adults 17 to 39 years of age . these findings suggest a state of low - grade systemic inflammation in obese patients.21 ) as previously reported by sutter et al.22 ) it is known that increased levels of hs - crp are greatly associated with extensive tissue destruction or infarct size . in such situations with more severe inflammation and extensive tissue damage , the relationship between adverse outcomes and crp level might be considered independent from bmi status . the present study shows that hs - crp concentrations in the obese rise with increasing age , diabetes mellitus , hypertension , coronary artery disease , heart failure and cerebrovascular disease reaffirming data from earlier studies.23 - 25 ) a prospective study by bruci et al.26 ) on 329 patients with myocardial infarction at the time of admission showed crp to be associated with a strong , positive graded increase in the risk of heart failure and death independently of known risk indicators . ninety - four patients ( 29% ) were in killip class greater than 1 at presentation , and greater killip class was associated with increased crp . interestingly , a study in cairo by fareed et al . noted that patients with hs - crp -17 mg / l were prone to heart failure ( at this value sensitivity=88% , specificity=51.2% ) . our findings are in agreement with a recently published report that showed a trend to decreasing values of lvef in relation to higher crp levels.27 ) the significant relationship between crp and lvef suggests the implication of inflammatory mechanisms in left ventricular dysfunction and in the pathogenesis of congestive heart failure , independently of the underlying atherosclerotic burden . angiographic findings of multivessel involvement , frequency of acc / aha lesion type c and subsequently higher pre - timi grade 0 flow , lower procedure success rate and higher complications support the previous study findings on acute coronary syndrome that demonstrated multiple complex stenoses with high plaque burden suggesting a pan - coronary involvement.28 ) this study showed a negative relation of smoking with hs - crp in the overweight / obese subset . the mechanism whereby smoking is related to crp concentration is unclear , but it may be multi - factorial , involving bronchial injury , endothelial activation and systemic inflammation.29 ) use of a statin is a negative predictor of 12-month mortality in total study population . however , there was no significant association of statin use and hs - crp levels in higher bmi patients though earlier studies have shown that statin reduces hs - crp levels.30)31 ) this could be due to relatively small number of patients on a statin at the time of admission ( 7.15% ) or unavailability of follow - up hs - crp data in our registry . moreover , in anglo - scandinavian cardiac outcomes trial : lipid lowering arm trial neither baseline nor on - treatment crp provided useful information about the efficacy of statin treatment to reduce cardiovascular events beyond ldl - c reduction.32 ) this study should be interpreted in the light of the following limitations . this is not a randomized trial , but a retrospective evaluation on a large - scale multi - center registry . blood samples for hs - crp were obtained at the time of admission , hence higher levels of hs - crp in our study may not be associated with myocardial necrosis.33 ) the time of onset and size of the myocardial infarction may also increase the hs - crp levels . however , we did not determine the serial changes in hs - crp concentration after ami . other inflammatory markers , such as il-6 , tnf- or lipoprotein - associated phospholipase a2 ( lp - pla2 ) that could have added more robust evidence of other inflammatory pathways associated with poor outcome were not a part of this study . extreme variations in hs - crp levels that could have made a difference in standard deviation in each group have not been excluded from this study . higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . we suggest further long term evaluation with serial changes in hs - crp concentration after ami and other inflammation markers to clearly elucidate its role in more diverse population . this is not a randomized trial , but a retrospective evaluation on a large - scale multi - center registry . blood samples for hs - crp were obtained at the time of admission , hence higher levels of hs - crp in our study may not be associated with myocardial necrosis.33 ) the time of onset and size of the myocardial infarction may also increase the hs - crp levels . however , we did not determine the serial changes in hs - crp concentration after ami . other inflammatory markers , such as il-6 , tnf- or lipoprotein - associated phospholipase a2 ( lp - pla2 ) that could have added more robust evidence of other inflammatory pathways associated with poor outcome were not a part of this study . extreme variations in hs - crp levels that could have made a difference in standard deviation in each group have not been excluded from this study . higher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers . we suggest further long term evaluation with serial changes in hs - crp concentration after ami and other inflammation markers to clearly elucidate its role in more diverse population .

background and objectivesserum high sensitivity c - reactive protein ( hs - crp ) is a marker of inflammation and may lead to the development of atherosclerosis , adversely affecting mortality . the aim of this study was to evaluate the relationship between baseline hs - crp level and 12-month clinical outcomes in patients with acute myocardial infarction ( ami ) undergoing percutaneous coronary intervention ( pci ) according to their body mass index ( bmi ) status.subjects and methodsusing data from the korea acute myocardial infarction registry from november 2005 to september 2008 , a total of 8174 consecutive ami patients were studied . cox proportional hazard model revealed that higher baseline levels of hs - crp was associated with 12-month all - cause mortality ( p=0.045 ) . to further understand this association , patients were divided into 3 groups based on their body mass index : 1 ) overweight / obese , 2 ) normal weight , and 3 ) underweight patients . then each group was stratified into quartiles based on their hs-crp.resultsin overweight / obese patients , cox model showed significant association of hs - crp with 12-month mortality when adjusted for age and gender ( p<0.001 ) , however , after adjustment with multiple covariates , mortality was highest in the 4th quartile { hr 2.382 , ( 1.079 - 5.259 ) , p=0.032 } though statistically insignificant ( p=0.172 ) . we observed no significant association of serum hs - crp with 12-month mortality in normal weight ( p=0.681 ) and underweight ( p=0.760 ) patients.conclusionhigher baseline hs - crp level ( 4.08 mg / dl ) in overweight / obese ami patients showed significant association with 12-month all - cause mortality independent of other prognostic markers .

macrolide , lincosamide and streptogramin b ( mlsb ) antibiotics are structurally different with a same mechanism of action . these drugs , especially clindamycin , are alternative drugs for some staphylococcus aureus infections such as skin and soft tissue infections particularly in penicillin - allergic patients ( 1 , 2 ) . clindamycin resistance in s. aureus strains may be either constitutive ( cmlsb ) or inducible ( imlsb ) . relapse of infection in a patient with s. aureus endocarditis was reported in 1976 ( 3 ) . detection of isolates with inducible clindamycin resistance is a problematic issue for the routine diagnosis , because they are erythromycin resistant and clindamycin susceptible in vitro and in the condition that they are not place adjacent to each other . in these cases , in vivo treatment with clindamycin may lead to clinical therapeutic failures ( 4 , 5 ) . the aim of this study was to determine the incidence of inducible clindamycin resistance in s. aureus isolates collected from clinical specimens in this geographic area . totally 162 s. aureus isolates were collected from clinical specimens of patients admitted to three university hospitals with 462 , 420 and 300 bed capacities in kerman , south - east of iran , from march 2011 to february 2012 . confirmation of s. aureus isolates and detection of methicillin resistant s. aureus ( mrsa ) were performed by phenotypic and genotypic methods as previously described ( 6 ) . only one isolate per patient this test was performed by placing an erythromycin ( 15 g ) disk ( mast , group ltd . , merseyside , uk ) at a distance of 15 to 20 mm from clindamycin ( 2 g ) disk ( mast , group ltd . , merseyside , uk ) on a mueller - hinton agar plate inoculated with 0.5 mcfarland standard bacterial suspensions . following overnight incubation at 37c , flattening of the clindamycin zone adjacent to the erythromycin disk ( d shaped ) indicated inducible clindamycin resistance ( 7 ) . from 162 s. aureus isolates tested for determination of inducible clindamycin resistance , 70 ( 43.2% ) were mssa and 92 ( 56.8% ) isolates were mrsa ( table 1 ) . sensitivity to both erythromycin and clindamycin was significantly higher in mrsa compared to mssa isolates . resistance to methicillin , erythromycin and clindamycin was observed in 92 ( 56.8% ) , 75 ( 46.29% ) and 46 ( 28.4% ) of the isolates , respectively . inducible resistance to clindamycin was determined in 14 ( 8.64% ) isolates ( d - test positive , figure 1 ) . table 2 shows the distribution of d - test positive s. aureus isolates in clinical specimens . among those , 11 ( 78.57% ) abbreviations : mssa , methicillin susceptible s. aureus ; mrsa , methicillin resistant s. aureus ; ery , erythromycin ; cli , clindamycin ; s , sensitive , r , resistant ; cmlsb , constitutive macrolide lincosamide and streptogramin b ; imlsb , inducible macrolide lincosamide and streptogramin b. data are presented as no . ( % ) . flattening of the clindamycin zone adjacent to the erythromycin disk produces a d shape . ( % ) . including : blood , skin and soft tissue , cerebrospinal fluid . clindamycin is an effective antibiotic agent used in the treatment of skin and soft - tissue infections caused by s. aureus isolates . this antibiotic has good tissue penetration , potential antitoxin effects , and also accumulates in abscesses ( 8) . however , resistance to clindamycin may develop in s. aureus isolates with inducible phenotype and these isolates have a high rate of spontaneous mutation which would enable these isolates to develop constitutive resistance to clindamycin during therapeutic process ( 4 ) . a positive d - test indicates the existence of inducible resistance to clindamycin . in this study 8.64% of 162 s. aureus isolates were d - test positive that is comparable with the findings of the studies by rahbar and hajia ( 9.7% ) ( 9 ) and sedighi et al . the prevalence of inducible and constitutive clindamycin resistance differs in different geographic areas ( 12 ) . in our study high number ( 46.29% ) of s. aureus isolates were resistant to erythromycin . among those , 14 ( 18.66% ) isolates had positive and 15 ( 20% ) had negative d - test results and 46 ( 61.33% ) were constitutive clindamycin resistance . in this study the prevalence of inducible and constitutive resistance to clindamycin in mrsa ( 11.95% and 47.8% respectively ) is higher than those in mssa ( 4.28% and 2.85% , respectively ) . the results of this study revealed that inducible resistance to clindamycin in s. aureus isolates is relatively high in this region . therefore , d - test should be performed to prevent treatment failures of s. aureus infections caused by the agents that are resistant to erythromycin and sensitive to clindamycin .

background : clindamycin is a frequently used antimicrobial therapeutic medicine used for the treatment of skin and soft tissue infections caused by staphylococcus aureus strains . resistance to this antibiotic is either constitutive or inducible . constitutive resistance to clindamycin could be detected by standard susceptibility testing methods . inducible clindamycin resistance could not be detected by in vitro routine tests . this type of resistance can be identified by d-test.objectives:the outbreak of inducible resistance to clindamycin in methicillin resistant and - susceptible s. aureus isolates were investigated in this study.materials and methods : totally 162 s. aureus isolates were evaluated for inducible clindamycin resistance by d - test in accordance with clinical and laboratory standards institute ( clsi ) guidelines.results:inducible clindamycin resistance was detected in 8.64% of s. aureus isolates . inducible and constitutive resistance to clindamycin was found to be higher in methicillin resistant s. aureus ( 11.95% and 47.8% respectively ) compared to methicillin susceptible s. aureus ( 4.28% and 2.85% respectively ) isolates.conclusions:our results showed that inducible resistance to clindamycin in s. aureus isolates is relatively higher in this region . therefore , d - test should be performed to prevent treatment failures against infections caused by s. aureus , which are resistant to erythromycin and the sensitive ones against clindamycin .

the lingual tonsils develop at 6.5 weeks between the second and third arches ventrally while palatine tonsils develop at 8 weeks from second pouch ( ventral and dorsal ) . tonsils are predominantly b - organs and b - lymphocytes comprise 5060% of tonsillar lymphocytes . ample evidence shows that tonsils are involved in inducing secretory immunoglobulin production [ 4 , 5 ] . both adenoids and tonsils are favourably located to mediate immunologic protection of the upper aerodigestive tract as they are exposed to air borne antigens . tonsils are particularly designed for direct transport of foreign material from the exterior to the lymphoid cells . involution of the tonsils begins after puberty , resulting in a decrease of the b cell population and a relative increase in the ratio of t to b cells . the commonest indication for tonsillectomy is recurrent tonsillitis , which results in shedding the immunologically active cells and decreasing antigen transport function with subsequent replacement by stratified squamous epithelium [ 6 , 7 ] . we investigated the common pathogens causing this condition in our hospital and report here our findings . patients presenting at our clinic with signs and symptoms of chronic tonsillitis were enrolled for the study . the study was explained to them and where children were involved , to their parents . before the operation began , the laboratory was informed and a technician stood by to collect the tonsil as soon as it was removed . sterile wide - mouthed container was provided and the excised tonsils were aseptically put into them and carried immediately to the laboratory for processing . as soon as the tonsil reaches the laboratory , it is cut into two with a sterile surgical blade ; the inner surfaces were swabbed with sterile cotton swab , and inoculated onto two blood agar plates , one macconkey agar and one chocolate agar plate . one blood agar plate was incubated anaerobically , the chocolate plate in 510% co2 while the rest of the plates were incubated aerobically . the aerobic plates and the co2 plate were examined after 24 hours ; if no growth , they were reincubated for a further 24 hours after which if still no growth , they were discarded as negative . the anaerobic cultures were examined at 72 hours and if no growth they were reincubated for a total of 7 days . there were 34 bacterial isolates from 52 patients , giving a percentage positivity of 65.38% . thirty isolates were gram - positive bacteria and only four were gram - negative , made up of two genera , klebsiella and pseudomonas . staphylococcus aureus was the predominant isolate ( 15/34 , 44.1% ) , followed by group b streptococcus ( 12/34 , 35.3% ) . others were , streptococcus pyogenes ( group a streptococcus ) , 1/34 , 2.94% ; and untypable streptococcus spp . 2/34 , 5.88% . the gram - negative bacteria consist of klebsiella pneumoniae 3/34 , 8.82% and pseudomonas aeruginosa 1/34 , 2.94% ( table 1 ) . there were no growths in 7 patients while 11 yielded growth of normal flora only . all the cases were chronic and most of them took antibiotics before presenting to us . the ratio of anaerobic to aerobic bacteria in saliva is approximately 10 : 1 because of variations in oxygen concentration throughout the oral cavity . invargsson et al . revealed that streptococcus pneumoniae was recovered in 19% of healthy children , hemophilus influenzae in 13% , group a streptococcus in 5% , and moraxella ( branhamella ) catarrhalis in 36% . the frequency of pathogens decreases with age , possibly because of increased immunity . because the oropharynx is colonized by many organisms , most infections of wadeyer 's ring are polymicrobial . in our study , but in contrast with other researchers who found beta - hemolytic streptococci to be the predominant isolate [ 14 , 15 ] . these authors also found that streptococcus pyogenes was isolated more frequently in recurrent tonsillitis while in the tonsillar hypertrophy , streptococci beta - hemolytic non a group predominated . it has been suggested that fine - needle aspiration can be used in identifying tonsil core bacteriology in clinical settings . methicillin resistant staphylococcus aureus ( mrsa ) , has been isolated from the surface and core tonsils in children . we did not encounter any mrsa in our study ; and all the staphylococcus aureus isolated were sensitive to augmentin and vancomycin . it would appear from our results , that augmentin should be our drug of choice in future treatment of tonsillitis from this centre . the findings of kuhn et al . supported the etiologic role of hemophilus influenzae and staphylococcus aureus in hypertrophic tonsils with or without inflammation [ 18 , 19 ] .

objectives . tonsillitis is a common infection in all age groups , especially under the age of five . organisms causing this condition vary from place to place . our aim is to find out the main causative agents of this condition in our hospital . patients and methods . fifty - two consenting patients who needed tonsillectomy in khamis civil hospital , kingdom of saudi arabia , between september 2006 and april 2007 , were enrolled for the study . swabs were taken from their inner surfaces and cultured for anaerobes and aerobes according to standard microbiological techniques . results . fifty - two patients , consisting of 30 males and 22 females were enrolled . their mean age was 9.81 6.47 . nearly 65% of patients had positive cultures while 35% were negative . the commonest bacteria isolated were staphylococcus aureus ( 44.1% ) ; and group b streptococcus ( 35.3 % ) . two gram - negative bacteria , klebsiella pneumoniae , ( 8.82% ) , and pseudomonas aeruginosa ( 2.94 % ) , were also isolated . no anaerobe was isolated . conclusion . gram - positive cocci , consisting of staphylococcus aureus and group b streptococcus ( streptococcus agalactiae ) , are the major causes of tonsillitis requiring surgery in our hospital . antibiotic treatment of this condition should be directed largely against these organisms .

the basic goal of therapy is to achieve a steady state blood level that is therapeutically effective and nontoxic for an extended period of time . the design of proper dosage regimens is an important element in accomplishing this goal . in conventional oral dosage forms drug dosage this drawback of conventional dosage form can be overcome by formulation of controlled release dosage forms , which provides drug release in an amount sufficient to maintain the therapeutic drug level over extended period of time , with release profiles controlled by the special technological construction and design of the system . the term controlled release has become associated with those systems from which therapeutic agents may be automatically delivered at predetermined rates over a long period of time . products of this type have been formulated for oral , injectable , and topical use and inserts for placement in body cavities . controlled release also denotes systems , which can provide some control whether this be of a temporal or spatial nature or both for drug release in the body . matrix systems offer several advantages relative to other extended release dosage forms like easy to manufacture , versatile , effective , low cost , and can be made to release high molecular weight compounds . since the drug is dispersed in the matrix system , accidental leakage of the total drug components is less likely to occur , although occasionally , cracking of the matrix material can cause unwanted release . hypertension is one of the chronic disorders affecting a large number of populations in the world . hypertension often is defined as a diastolic pressure of more than 90 mmhg because at this value the frequency of complication due to hypertension rises significantly . hypertension may also refer to increase the blood pressure in any blood vessel , such as pulmonary or portal hypertension . systemic hypertension may leads to cerebrovascular accidents , myocardial infarction , congestive heart failure , and renal damage . the incidence of morbidity and mortality significantly decreases when hypertension is diagnosed early and is properly treated . losartan potassium is the first orally active angiotensin - ii antagonist used in the treatment of hypertension either alone ( or ) in combination of hydrochlorothiazides . it undergoes extensive biotransformation and has an elimination half life 1.5 - 2.5 h and hence it is suitable for oral controlled release . the current study aims at developing oral controlled release tablets of losartan potassium , using matrices hpmc k100 m and carbopol 934p , xanthan gum with chitosan , and trisodium citrate as a cross - linking agent . the developed formulations were evaluated for weight variation , hardness , friability , and in vitro release studies . xanthan gum , trisodium citrate , di - basic calcium phosphate , magnesium stearate were procured s.d . table 1 enlists the composition of different formulations prepared using varying amounts of the polymers ( i.e. , carbopol 934p and hpmc k100 m , xanthan gum , chitosan , trisodium citrate ) and dicalcium phosphate as the diluent , along with the fixed quantity of magnesium stearate as lubricant . drug and the excipients were homogeneously blended and subsequently compressed into flat - faced tablets using multi - punch tablet compression machine ( india ) . composition of losartan potassium control release matrix tablet ( f1-f12 ) fourier transforms infra - red ( ftir ) study was carried out to check compatibility of drug with polymers . the spectrum of dried mixture of drug and potassium bromide was run followed by drug with various polymers by using ftir spectrophotometer ( thermo nicolet 380 , india ) . the prepared controlled release tablets were evaluated for uniformity of weight using 20 tablets , hardness using 6 tablets ( monsanto hardness tester ) , and friability using 20 tablets ( roche friabilator ) . five tablets were weighed and triturate , from that transfer an accurately weighed portion of the powder equivalent to about 100 mg of losartan potassium in a 100 ml volumetric flask containing buffer solution and then concentration is measured at max 205 nm . the in - vitro dissolution studies were performed using the usp- ii ( paddle ) dissolution apparatus at 50 rpm . the dissolution medium consisted of 900 ml of an aliquot ( 5 ml ) was withdrawn at specific time intervals and drug content was determined by uv - visible spectrometer ( shimadzu-1700 , japan ) at 205 nm . were analyzed as per zero order , first order , higuchi equation models to assess the drug release kinetics and mechanism of release from the tablets . the optimized formulation was subjected for 3 month stability study according to international conference on harmonization ( ich ) guidelines . the selected formulations were packed in aluminum foils , which were in wide mouth bottles closed tightly . they were then stored at 25c/60% rh , 30c/65% rh , 40c/75% rh for 3 months and evaluated for their permeation study . table 1 enlists the composition of different formulations prepared using varying amounts of the polymers ( i.e. , carbopol 934p and hpmc k100 m , xanthan gum , chitosan , trisodium citrate ) and dicalcium phosphate as the diluent , along with the fixed quantity of magnesium stearate as lubricant . drug and the excipients were homogeneously blended and subsequently compressed into flat - faced tablets using multi - punch tablet compression machine ( india ) . fourier transforms infra - red ( ftir ) study was carried out to check compatibility of drug with polymers . the spectrum of dried mixture of drug and potassium bromide was run followed by drug with various polymers by using ftir spectrophotometer ( thermo nicolet 380 , india ) . the prepared controlled release tablets were evaluated for uniformity of weight using 20 tablets , hardness using 6 tablets ( monsanto hardness tester ) , and friability using 20 tablets ( roche friabilator ) . five tablets were weighed and triturate , from that transfer an accurately weighed portion of the powder equivalent to about 100 mg of losartan potassium in a 100 ml volumetric flask containing buffer solution and then concentration is measured at max 205 nm . the in - vitro dissolution studies were performed using the usp- ii ( paddle ) dissolution apparatus at 50 rpm . the dissolution medium consisted of 900 ml of an aliquot ( 5 ml ) was withdrawn at specific time intervals and drug content was determined by uv - visible spectrometer ( shimadzu-1700 , japan ) at 205 nm . in - vitro drug release data were analyzed as per zero order , first order , higuchi equation models to assess the drug release kinetics and mechanism of release from the tablets . the optimized formulation was subjected for 3 month stability study according to international conference on harmonization ( ich ) guidelines . the selected formulations were packed in aluminum foils , which were in wide mouth bottles closed tightly . they were then stored at 25c/60% rh , 30c/65% rh , 40c/75% rh for 3 months and evaluated for their permeation study . losartan potassium was dissolved in both ph 1.2 and ph 6.8 , further diluted with the same and scanned for maximum absorbance in uv double beam spectrophotometer ( shimadzu uv1700 , japan ) in the range from 190 to 380 nm , using ph 1.2 and ph 6.8 as blank . as shown in figure 1 the ftir analysis of losartan potassium showed characteristic peaks at 3621/cm due to n oh stretching , 2956 due to ar ( h ) , at 1645 because of c - o stretching and 1377 due to c - n stretching . moreover , we can observe that , as shown in figures 24 the peaks at the same as above with slight variation was observed for the mixture of drug with different polymers like hpmc k 100 m , carbopol934p , xanthan gum , respectively . hence , it was found that all the polymers used in formulations were compatible with losartan potassium . flow properties of the granules were evaluated by determining the bulk density 0.244 0.124 to 0.427 0.136 , tapped density 0.275 0.091 to 0.526 0.116 , angle of repose 16.43 0.53 to 25.90 0.45 , and compressibility index 11.27 0.092 to 24.37 0.147 [ table 2 ] . the measured hardness of tablets of each batch ranged between 5.0 0.05 kg / cm and 5.7 0.07 kg / cm [ table 3 ] this ensures good handling characteristics of all batches . hence , less than 1% in all formulations ensuring that the tablets were mechanically stable . hence , all the tablets were found to be uniform with low standard deviation values [ table 3 ] . the percentage of drug content was found to be above 90% of losartan potassium , which was within acceptable limits . the in - vitro drug release was studied with usp - ii ( paddle type ) dissolution apparatus in both stimulated gastric fluid ( ph 1.2 ) and intestinal fluid ( ph 6.8 phosphate buffer ) for 24 h [ table 4 ] . ft - ir spectra of losartan potassium ft - ir spectra of losartan potassium with hpmck100 m ft - ir spectra of losartan potassium with carbopol 934p ft - ir spectra of losartan potassium with xanthan gum precompressional parameters of all formulations post compressional parameters of all formulations drug release studies of f1 to f12 the present study reveals to control the drug release by increasing the concentration of chitosan as a cross - linking agent with different polymers like hpmc k100 m , carbopol 934p , xanthan gum . the combination of different ratios of carbopol 934p and xanthan gum with chitosan ( f11 and f12 ) showed better release profile 99.72% and 98.70% , respectively , rather than other combinations like hpmc k100 m and carbopol 934p , hpmc k100 m and xanthan gum [ figures 56 ] . the values of release rate exponent ( n ) , calculated as per the algorithm proposed by higuchi and korsemeyer , ranged between 0.961 and 1.259 . the drug release from matrix system follows zero - order kinetics and the mechanism was found to be diffusion controlled and case ii transport . the selected formulation f11 and f12 were subjected to accelerated stability studies for 90 days at 25c/60% rh , 30c/65% rh , 40c/75% rh , in vitro permeation study was performed on every 30 days and showed negligible change in permeation profile . the formulation subjected for stability studies cumulative percentage drug release vs time profile of formulation f1 to f6 cumulative percentage drug release vs time profile of formulation f7 to f12 the objective of the present was to investigate the possibility of controlling of losartan potassium release from matrix tablet prepared by different polymers . the preformulation studies were carried out , which ruled out the interaction between the drug and polymers . the granules were punched into tablet and evaluated by post compression parameter like weight variation , hardness , friability , and drug content . all formulation showed acceptable flow properties and with required hardness , weight variation , friability , and drug content . the in - vitro drug release was studies with usp xxii dissolution apparatus in both simulated gastric fluid and intestine fluid for a period of 24 h. the results of dissolution studies indicated that formulations f11 and f12 produced controlled effect with 99.72% and 98.70% of drug release over a period of 24 h than compared with other formulations . it can be concluded that the polymer plays a major role in the design of controlled release matrix tablet . the study reveals that the release of drug was low when the matrix tablet contained polymers with increasing concentration of chitosan as a cross - linking agent and also shows anomalous diffusion kinetics . hence , it clearly manifests that , the necessity of combining different classes of polymers is required to to get an acceptable pharmacokinetic profile .

aim and background : the novelty of the present study was to control the release profile of matrix tablets of losartan potassium prepared by using different concentrations of chitosan and trisodium citrate as cross - linking agent with combination of various release retardant polymers.materials and methods : twelve formulations were prepared using hpmc k100 m , carbopol 934p , and xanthan gum as polymers . matrix tablets were prepared by wet granulation technique . the granules were subjected to precompression parameters such as angle of repose , loose bulk density , tapped bulk density , compressibility index . tablets were evaluated for weight variation , hardness , drug content , in - vitro dissolution , stability studies , respectively . drug -polymer compatibility studies were determined by ftir spectroscopy . further stability studies were carried out for 3months in accelerated conditions at 40c and 75%rh . the granules of all formulations exhibited good flow and compressibility . in - vitro dissolution studies were carried out for 24 h using 0.1 n hcl for the first 2 h and ph 6.8 phosphate buffers for the remaining 22h.results:it was found that among the 12 formulations f11 and f12 showed good dissolution profile to control the drug release . the release data was fitted to various mathematical models such as , higuchi , korsmeyer , first - order , and zero - order to evaluate the kinetics and the drug release . the drug release follows zero - order kinetics and the mechanism was found to be diffusion controlled and case ii transport . ft - ir spectroscopic studies revealed no interaction between drug and polymer . the stability studies indicated that f11 and f12 formulations were stable for 3months.conclusion:the above results concluded that by combining different classes of polymers an acceptable release profile can be obtained in the fluctuating in vivo environment .

adenomyosis is a common gynecological condition that is characterized by ingrowths of the endometrial cells into the myometrium . adenomyosis and leiomyoma are benign conditions that are often responsible for uterine enlargement , menorrhagia , anemia , and infertility . adenomyosis or internal endometriosis may occur as a result of increased overgrowth of the endometrium with invasion of the underlying myometrium or the displacement of the endometrium during pregnancy , delivery , endometrial curettage , cesarean section , myomectomy , or metroplasty . because of its similarities to leiomyoma , it can be difficult in some cases to accurately diagnose adenomyosis by ultrasound . most gynecologists will consider conservative medical treatment more often for adenomyosis only , while surgical options will be chosen frequently for leiomyoma or combined adenomyosis and leiomyoma , especially in large uteri . the three most common methods of clinical diagnosis of adenomyosis are magnetic resonance imaging ( mri ) , transabdominal ultrasonography ( tas ) , and transvaginal ultrasonography ( tvs ) . there is limited diagnostic capacity with tas , while tvs is a more feasible option . moreover , tvs is also much more cost effective than mri and is generally more readily available in the office to most practicing gynecologists . the aim of this retrospective study was to determine the accuracy , sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of tvs in the diagnosis of adenomyosis and leiomyoma confirmed by postsurgical histopathological findings . the importance of an accurate noninvasive diagnostic method like ultrasound for adenomyosis , leiomyoma , or combined is an ongoing need in the gynecological community . this was an institutional review board ( irb ) approved retrospective consecutive case series study . from november 2006 to may 2012 , patients underwent surgery for the treatment of adenomyosis , leiomyoma , or combined adenomyosis and leiomyoma . diagnostic criteria of uterine adenomyosis include two of the five sonographic features on tvs : ( 1 ) no distinction of the endometrial - myometrial junction ; ( 2 ) asymmetry of the anterior and posterior myometrium ; ( 3 ) subendometrial myometrial striations ; ( 4 ) myometrial cysts and fibrosis ; and ( 5 ) heterogeneous myometrial echotexture . diagnostic criteria of uterine leiomyoma include two of the five sonographic features on tvs : ( 1 ) clear demarcation of the tumor margin ; ( 2 ) whorly appearance of the tumor content ; ( 3 ) the presence of blood vessels ( by color doppler ) surrounding the tumor ; ( 4 ) irregularities of the uterine surface ( subserous and intramural tumors ) ; and ( 5 ) irregularities of the endometrial surface ( submucous tumors type 1 and 2 ) . symptomatic patients diagnosed with adenomyosis and leiomyoma via tvs underwent myomectomy with excision of the surrounding myometrium which presumably contained adenomyosis . following surgery , a histopathological examination was performed by the hospital pathologists . student 's t - tests were used for parametric continuous variables ; and the chi - square or fisher 's exact test , where suitable , was used for categorical variables . sensitivity , specificity , npv , ppv , positive and negative likelihood ratios , and accuracy were determined for ultrasound findings as they corresponded to the final histopathological diagnosis . from november 2006 to may 2012 , 163 patients underwent surgery for the treatment of adenomyosis , leiomyoma , or combined adenomyosis and leiomyoma . one hundred and twenty - three patients were diagnosed with adenomyosis , and 134 patients were diagnosed with leiomyoma . twenty - nine patients were diagnosed with adenomyosis only , 40 patients were diagnosed with leiomyoma only , and 94 patients were diagnosed with combined adenomyosis and leiomyoma , as illustrated in figure 1 . patient diagnosis groups one hundred and thirty patients diagnosed with adenomyosis or combined adenomyosis and leiomyoma via tvs underwent hysterectomy . the mean age was 43.7 years with a standard deviation ( sd ) of 6.7 years and median of 43 years . there was no significant difference in the mean age , weight , height , gravidity , and parity of the patient diagnosis groups . patient characteristics for 163 females who underwent hysterectomy or myomectomy between 2006 and 2012 of the 123 patients ( 75.46% ) who were positively diagnosed with adenomyosis , 93 of the patients ( 75.61% ppv ) diagnoses were confirmed by the histopathological findings . histopathological reports found 23 ( 57.50% npv ) confirmed negative diagnoses and 17 ( 42.50% ) positive findings . the sensitivity of tvs in the diagnosis of adenomyosis was 84.55% ( 95% ci 76.4 - 90.7 , p < 0.0001 ) and the specificity was 43.40% ( 95% ci 29.8 - 57.7 , p = 0.41 ) . table 2 shows the sensitivities , specificities , ppvs , npvs , and accuracy for each of the initial tvs diagnoses . this could be explained by the difficulty to diagnose adenomyosis in patients with other intrauterine abnormalities or conditions . patients positively diagnosed with adenomyosis via tvs are 1.49 ( 95% ci 1.16 - 1.92 , p < 0.002 ) times more likely to have the condition . conversely , patients negatively diagnosed with adenomyosis via tvs are 2.81 ( 95% ci 1.65 - 4.79 , p = 0.0002 ) times less likely to have the condition . number of patients , positive and negative predictive values , sensitivity , specificity , and accuracy of transvaginal ultrasound for initial diagnosis with histopathological correlation ( n=107 ) one hundred and thirty - four patients ( 82.21% ) were diagnosed with leiomyoma and 29 patients ( 17.79% ) were negative for leiomyoma upon the initial tvs . of the 134 patients diagnosed with leiomyoma , 133 ( 99.25% ppv ) had a confirmed diagnosis represented in their postsurgical pathology report and one patient 's ( 0.75% ) pathology report did not confirm the diagnosis . of the 29 patients whose tvs diagnosis was negative for leiomyoma , 24 ( 82.76% ) patients histopathological findings were also negative , and five patients ( 17.24% ) had a positive histopathological diagnosis . the corresponding sensitivity and specificity of tvs as a diagnostic test for leiomyoma was 96.38% ( 95% ci 91.75 - 98.81 ) and 96.00% ( 95% ci 79.65 - 99.90 ) , respectively . patients with a positive diagnosis of leiomyoma via tvs are 24.09 times ( 95% ci 35.30 - 164.45 , p = 0.001 ) more likely to have leiomyoma . patients with a negative diagnosis of leiomyoma via tvs are 26.53 times ( 95% ci 11.16 - 62.89 , p < 0.0001 ) less likely to have leiomyoma . for the statistical calculations of adenomyosis and leiomyoma as coexisting conditions , analyses were performed excluding the patients who were diagnosed with a singular condition . these patients were classified as negative solely for the purpose of calculations regarding the combined condition . of the 94 patients ( 57.67% ) that were positively diagnosed with combined adenomyosis and leiomyoma , 70 ( 74.47% ppv ) had histopathological confirmation of both conditions and 24 patients ( 25.53% ) did not have evidence of both conditions in their post - surgical pathology report . sixty - nine ( 42.33% ) patients had a negative tvs diagnosis for combined adenomyosis and leiomyoma , and 49 ( 71.01% npv ) had histopathological confirmation of the negative diagnosis . however , 20 patients ( 28.99% ) had a positive histopathological diagnosis for both adenomyosis and leiomyoma . the sensitivity and specificity of tvs in the diagnosis of combined adenomyosis and leiomyoma was 77.78 ( 95% ci 67.79 - 85.87 ) and 67.12% ( 95% ci 55.13 - 77.67 ) , respectively . patients who obtained a positive tvs diagnosis are 2.37 times ( 95% ci 1.67 - 3.34 , p < 0.0001 ) more likely to be diagnosed with combined adenomyosis and leiomyoma . alternatively , patients who are negatively diagnosed with adenomyosis and leiomyoma via tvs are 3.02 times ( 95% ci 1.99 - 4.59 , p < 0.0001 ) less likely to be diagnosed with combined adenomyosis and leiomyoma . adenomyosis is a gynecological disorder that is characterized by the overgrowth of the endometrium into the underlying myometrium . the difficulty in diagnosing adenomyosis clinically is due to the lack of strong positive pathognomonic signs and/or clinical findings . the explanation for this wide range of values , as described by azziz , is the result of differences in the histological criteria for the diagnosis of adenomyosis , the care of which the pathologic specimens are handled , and the number of blocks of sampling specimens taken . various measures of accuracy were calculated for the diagnosis of adenomyosis , leiomyoma , and combined adenomyosis and leiomyoma . table 3 shows a comparison of sensitivity , specificity , ppv , and npv of this study with several previous studies that investigated the diagnosis of adenomyosis . it is important to note that the mean number of patients included in the 13 previous studies in table 3 is 76.8 patients ; however , this study utilized 163 patients in the analyses . along with differences in inclusion criteria , ultrasound equipment , and/or the differences in the criteria to diagnose adenomyosis , the larger sample size could contribute to the difference in results . the sensitivity and specificity of tvs for the diagnosis of adenomyosis in this study was 84.55 and 43.40% , respectively . the associated p value for the specificity was not significant , suggesting tvs as a diagnostic tool is sensitive , but not specific in the diagnosis of adenomyosis . the sensitivity in this study is similar to those previously reported in table 3 , but the specificity is the lowest of those reported . this could be due to the difficulty in diagnosing adenomyosis in the presence of other uterine abnormalities and conditions , especially uterine leiomyomata that may distort the uterus . patients that were diagnosed with multiple intrauterine conditions were not excluded from this study , since the principle inclusion criteria was patients who underwent hysterectomy or myomectomy and a preoperative tvs . sensitivity , specificity , positive and negative predictive values of tvs for the diagnosis of adenomyosis from previous series compared with this series the sensitivity of tvs for the diagnosis of leiomyoma was 96.38% and the specificity was 96.00% , which is similar to that of a previous study . the sensitivity for the diagnosis of combined adenomyosis and leiomyoma was 77.78% , and the specificity was 67.12% . it was accurate , sensitive , and specific in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma . tvs was both accurate and sensitive in the diagnosis of adenomyosis , but not specific . moreover , tvs is cost effective and readily available in the office to the majority of practicing gynecologists . this study demonstrated that tvs is a valuable noninvasive method that should be utilized in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma .

objective : to evaluate the accuracy , sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of the diagnosis of adenomyosis , leiomyoma , or combined adenomyosis and leiomyoma by the use of transvaginal ultrasonography ( tvs ) compared to the histopathological findings.subjects and methods : this is a retrospective study of patients with a preoperative tvs diagnosis of adenomyosis , leiomyoma , or combined . patients diagnosed with adenomyosis or combined adenomyosis and leiomyoma via tvs underwent hysterectomy . symptomatic patients diagnosed with adenomyosis and leiomyoma via tvs underwent myomectomy with excision of the surrounding myometrium which contained possible adenomyosis . following surgery , a histopathological examination was performed by the hospital pathologists . the microscopic diagnosis of the specimen was recorded.results:tvs diagnosis of adenomyosis was sensitive but not specific . tvs was sensitive , specific , and accurate in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma.conclusion:this study demonstrated that tvs is a valuable noninvasive method that should be utilized in the diagnosis of leiomyoma and combined adenomyosis and leiomyoma . tvs is sensitive , but is not specific in the diagnosis of adenomyosis only .

peptide amphiphiles are a fascinating class of bioactive molecules in which one or more lipid chains are attached to peptides , either bioderived , bioinspired , or designed . the lipid chains drive self - assembly , leading to nanostructures in which the functional peptide unit can be presented at high density , for example , on the surface of nanofibrils . the use of enzymes to modulate the self - assembly of peptide amphiphiles ( pas ) and other amphiphilic biomolecules such as amphiphilic peptides and peptide polymer conjugates has attracted considerable attention due to potential applications in biomedicine as well as catalysis and sensing . enzymes have been evolved by nature to perform many essential functions in biological systems . they have excellent specificity and activity under mild environmental conditions associated with living organisms . the topic of enzyme - controlled peptide self - assembly has been the subject of a recent comprehensive review . other reviews cover enzyme - driven ( biocatalyzed ) self - assembly of peptide derivatives , leading specifically to hydrogelation and the influence of enzymes on the aggregation of polymer - peptide conjugates . as well as enzyme - stimulated transitions in nanostructure , peptide systems can also undergo enzyme - mediated disassembly and enzyme - triggered self - assembly . by design of the peptide substrate , responsiveness to a wide variety of enzymes we have demonstrated a concept whereby pegylated peptide micelles prepared from an amyloid -peptide derived heptapeptide containing a diphenylalanine ( ff ) sequence attached to peg were enzymatically degraded by -chymotrypsin which selectively cleaves peptide bonds between aromatic residues . this released a hexapeptide ( containing a c - terminal f ) along with peg with an n - terminal f residue . this proof - of - principle work established a possible route to create an enzyme - responsive delivery system based on peg - coated particles which have relevance to biological applications since peg provides a sterically stabilized corona , enabling enhanced in vivo stability . here , we investigate the influence of the model serine protease -chymotrypsin on the self - assembly of the pa c16-kkffvlk . the peptide amphiphile ( pa ) has been designed based on the klvff core motif from the amyloid beta ( a ) peptide . this contains two f residues at the c terminus which drive self - assembly through hydrophobic and -stacking interactions . two additional lysine residues are incorporated between the hexadecyl lipid chain and the ffklvff motif to promote solubility and impart amphiphilicity . the serine protease -chymotrypsin is expected to cleave preferentially between the two f residues , although as discussed below an additional cleavage site is also identified . the pas corresponding to cleaved products c16-kkf and c16-kkff are shown to self - assemble into spherical micelles , quite distinct from the nanotubes / helical ribbons formed by c16-kkffvlk . we thus show that it is possible to develop enzyme - responsive pa systems with pronounced nanostructure transitions , leading in turn to macroscopic changes in the sample transparency . peptide amphiphile c16-kkffvlk was purchased from cs bio ( menlo park , ca ) . for the first batch , the molar mass by electrospray - ionization mass spectrometry was 1146.97 da ( expected 1147.57 da ) . the molar mass by es - msi was 1147.65 da and the purity was 99.73% by hplc . peptide amphiphile c16-kkff was synthesized by cs bio ( menlo park , ca ) and supplied as a tfa salt . the molecular weight was found to be 807.31 da ( expected 807.11 da ) as revealed by electrospray - ionization mass spectrometry . purity of the sample was 99.88% by analytical hplc in a 0.1% tfa water / acetonitirile gradient . peptide amphiphile c16-kkf was purchased from cs bio ( menlo park , ca ) and supplied as a tfa salt . electrospray - ionization mass spectrometry revealed a molecular weight of 659.73 da ( expected 659.94 da ) . purity was 99.35% by analytical hplc in a 0.1% tfa water / acetonitirile gradient and supplied as a tfa salt . peptide fvlk was custom synthesized by peptide synthetics ( peptide protein research ltd . the molecular weight was found to be 505.641 da by electrospray - ionization mass spectrometry . the sample was purified in acetonitrile and water containing 0.1% tfa prior to lyophilization and was supplied as a tfa salt . the sample was purified in acetonitrile and water containing 0.1% tfa prior to lyophilization and was supplied as a tfa salt . the enzyme -chymotrypsin , which has a molecular weight of 25 kda , was purchased from sigma - aldrich ( bovine pancreas extract ) . to characterize self - assembly in water , solutions were made by direct dissolution in ultrafiltered water , from a barnstead nanopure system . the ph of a 0.5 wt % solution of c16-kkffvlk was 4.18 , and the ph was 3.84 for a 1 wt % solution . direct - infusion electrospray mass spectra ( es - ms ) were obtained on a thermofisher scientific orbitrap xl instrument . the ms / ms experiments were performed with an isolated target mass of 648 da ( isolation width of 3 da ) , and fragmentation was achieved in the hcd trap with a fragmentation energy of 35 . a mixture of 1 wt % c16-kkffvlk and 2 wt % -chymotrypsin was prepared for mass spectrometry analysis . a vial containing the solution was then placed in a sonicator at 50 c for 1 h. this temperature was found to improve the activity of -chymotrypsin ( previous work showed increasing activity with temperature , but the highest temperature studied was 35 c ) . the pa / enzyme mixture was left to age for 4 months before mass spectrometry measurements were performed . spectra were recorded using a nexus - ftir spectrometer equipped with a dtgs detector and a multiple reflection attenuated total reflectance ( atr ) system . solutions of the pas and peptides in d2o ( 0.5 , 1 , and 2 wt % ) were sandwiched in ring spacers between two caf2 plate windows ( spacer 0.006 mm ) . spectra were recorded with a varian cary eclipse fluorescence spectrometer with samples in 4 mm inner width quartz cuvettes . the spectra were recorded from 460 to 600 nm using an excitation wavelength ex = 440 nm . measurements were performed on stalks prepared by drying filaments of the pas or peptides , prepared from 0.5 , 1 , or 2 wt % solutions . solutions of the peptide were suspended between the ends of wax - coated capillaries and dried . the stalks were mounted ( vertically ) onto the four axis goniometer of a raxis iv++ x - ray diffractometer ( rigaku ) equipped with a rotating anode generator and a saturn 992 ccd camera . the sample the x - ray wavelength was = 1.54 . the wavenumber scale ( q = 4 sin / , where 2 is the scattering angle ) was geometrically calculated using the size of each pixel in the detector screen ( 0.0898 mm ) and the sample detector distance . saxs data were collected on the biosaxs beamline bm29 at the esrf , grenoble , france . solutions containing 0.5 , 1 , or 2 wt % c16-kkffvlk were loaded in pcr tubes in an automated sample changer . further experiments ( including kinetics measurements ) on a 1 wt % sample were performed on beamline id02 at the esrf , grenoble , france . saxs data were collected with a frelon kodak ccd with a 1.2 m sample detector distance , and waxs data were measured simultaneously with an aviex ccd . solutions were injected using a syringe into enki ki - beam thin ( 0.05 mm ) wall 1.85 mm diameter polycarbonate capillaries which optimize background subtraction . heat / cool experiments were performed with the sample mounted in a quartz capillary instead of the enki polycarbonate capillary . cryo - tem was carried out using a field emission cryo - electron microscope ( jeol jem-3200fsc ) , operating at 200 kv voltage ( except for sample vlk for which the voltage was 300 kv ) . images were taken in bright field mode and using zero loss energy filtering ( omega type ) with slit width of 20 ev . vitrified specimens were prepared using an automated fei vitrobot device using quantifoil 3.5/1 holey carbon copper grids with the hole size of 3.5 m . just prior to use , grids were plasma cleaned using a gatan solarus 9500 plasma cleaner and then transferred into an environmental chamber of a fei vitrobot at room temperature and 100% humidity . thereafter , 3 l of sample solution ( 2 wt % concentration ) was applied to the grid and it was blotted twice for 5 s and then vitrified in a 1/1 mixture of liquid ethane and propane at temperature of 180 c . the most viscous gel ( 5 wt % concentration ) was blotted four times for 5 s. the grid with vitrified sample solution were maintained at liquid nitrogen temperature and then cryo - transferred into the microscope . our objective was to examine the effect of -chymotrypsin ( c ) in cleaving the pa c16-kkffvlk and to investigate the self - assembly of the product peptides and peptide amphiphiles . figure 1 shows an electrospray ionization mass spectrum for the pa / enzyme mixture , which reveals sharp peaks with m / z values of 660.50 and 506.33 da . these peaks are identified as c16-kkf and fvlk , respectively , since these values correspond to the expected molar masses , listed in the experimental section ( i.e. , ions with z = 1 are produced ) . these two fragments are expected to be released from c16-kkffvlk in the presence of c as the enzyme selectively catalyzes the hydrolysis of peptide bonds on the c - terminal side of phenylalanine , which in our peptide corresponds to cleavage between the two f residues . unexpectedly we observed an additional two peaks with high abundances with m / z values of 807.57 and 359.27 da . the former is ascribed to c16-kkff and the latter as the tripeptide vlk , since the m / z values correspond to the expected molar masses for these species ( again z = 1 ) . ms / ms fragmentation was employed to confirm the nature of the two unexpected fragments by sequentially knocking off amino acid residues from the c - terminus as shown in figure 2 . as the amino acids k then l and then v are removed from the c - terminus , a peak at 789.56 da ms / ms fragmentation indicates that c cleaves at an additional site between f and v residues and confirms the existence of the two fragments ( c16-kkff and vlk ) . es - ms for a mixture of 1 wt % c16-kkffvlk and 2 wt % -chymotrypsin . ms / ms fragmentation spectrum for c16-kkffvlk , sequentially removing lysine , leucine , and valine from the c - terminus . to further elucidate the enzymatic degradation products of the pa by -chymotrypsin , figure 3 shows spectra for the pa and the enzyme alone , as well as the mixture . it is clear that the cd spectrum from the mixture is completely different from either of the two components and can not be expressed as a superposition of the spectra from the species in the mixture . the spectrum for c alone is consistent with that previously reported by ourselves and others . the spectrum for c16-kkffvlk is dominated by electronic transitions associated with the stacking of the f residues , as discussed elsewhere . the expected cd spectrum for -sheet structures , with a negative minimum near 216 nm and a positive maximum in the 195202 nm range , we provide evidence in a recent paper that this pa does form -sheet fibrils and this is supported by further data provided herein , to be discussed shortly . cd spectra for 1 wt % c16-kkffvlk and 1 wt % -chymotryspin and their mixture ( 1 and 1 wt % ) . cd was also used to examine the self - assembly of peptides and pas corresponding to the cleavage products , c16-kkf and fvlk and c16-kkff and vlk , respectively . the spectra for the two pas ( figure 4a , b ) do not show features of canonical secondary structure such as -sheets . the spectra contain contributions arising from stacking of phenylalanine residues which as shown in our papers on phenylalanine - rich peptides and peptide conjugates can give rise to a positive maximum in the cd spectrum at 210220 nm . there is also a contribution from polyproline ii ( ppii ) structure in this region . the presence of a deep minimum in a cd spectrum at around 190 nm , along with a broad positive maximum at around 210 nm , is a signature of ppii ( collagen - like ) secondary structure . the spectra for vlk and fvlk in figure 4c and d show these features . the spectra for flvk also show features in the 240270 nm range associated with vibronic transitions of the phenylalanine chromophore . cd spectra for solutions of 0.5 , 1 , and 2 wt % for ( a ) c16-kkff , ( b ) c16-kkf , ( c ) vlk , and ( d ) fvlk . spectra covering the amide i and amide ii regions from 0.5 , 1 , and 2 wt % solutions of the pas and peptides are shown in figure 5 . the spectra for c16-kkffvlk show a peak at 1626 cm which is due to -sheet structure , along with a peak at 1672 cm which is due to bound tfa counterions . the spectra for c16-kkff and c16-kkf do not show peaks in the 16151635 cm range typical of -sheet structures but have broad peaks near 1641 cm . peak positions in this range are typically ascribed to random coil structure ; however , as discussed elsewhere , ppii structures can also give a peak in this region . all of the pas also exhibit broad peaks in the amide ii region centered on 1580 cm . the spectra for peptides fvlk and vlk in figure 5d , e show broad shoulder peaks in the 16401650 cm region due to random coil / ppii structure ( along with the peak due to bound tfa counterions ) . peaks in the amide i region are absent for the peptides indicating a lower extent of ordering of n ftir spectra for solutions of 0.5 , 1 , and 2 wt % for ( a ) c16-kkffvlk , ( b ) c16-kkff , ( c ) c16-kkf , ( d ) fvlk , and ( e ) vlk . fiber x - ray diffraction was also used to examine peptide secondary structure . diffraction patterns from dried stalks are shown in supporting information figure 1 along with meridional and equatorial intensity profiles . the peak positions were used to calculate d spacings and these are listed in supporting information table 1 . the pattern for c16-kkffvlk ( supporting information figure 1a ) shows features of a cross- structure with meridional reflections from a d = 4.83 spacing ( corresponding to the -strand spacing ) along with a series of equatorial reflections from the -sheet stacking distance and lateral packing of the pa molecules . a series of off - axis reflections close to the equator are due to the helical twisting of nanoribbons . the xrd pattern shows a high degree of alignment and multiple bragg peaks confirming the regular ordering of the pa within the nanotubes / ribbons . in contrast , the xrd patterns for c16-kkf and c16-kkff do not show orientation , and only present a small number of reflections . the pattern for c16-kkff indicates the lack of secondary structure for this pa since cross- features are not observed , just sharp peaks corresponding to the packing of the lipid chains along with a broad 5.7 ring due to the spacing of the molecules . unexpectedly , c16-kkf shows more order in its xrd pattern than c16-kkff with some cross- features ( i.e. , 4.79/4.65 spacings and a 13.4 peak ) although the 5.7 ring and the 4.11 peak from the packing of the lipid chains predominate . the self - assembled nanostructures of the pas ( c16-kkffvlk and pas corresponding to the fragments produced after enzymatic degradation ) and peptide fragments were examined by cryogenic transmission electron microscopy ( cryo - tem ) . c16-kkffvlk forms nanotubes coexisting with helical ribbons ( figure 6a and ref ( 22 ) ) . saxs data ( figure 7 ) confirm this assignment , as discussed in detail elsewhere . in complete contrast , c16-kkff and c16-kkf self - assemble into small ( approximately 5 nm diameter ) micelles . this was confirmed by saxs ( figure 8) since the intensity profiles were fitted by form factors corresponding to uniform spheres , yielding radii of 2.5 and 2.9 nm for c16-kkf and c16-kkff , respectively . cryo - tem did not reveal a significant extent of nanostructure formation for peptide fragments fvlk and vlk . there was some evidence for sparse fibril clusters for fvlk ( figure 6d ) , and for vlk occasional raftlike film structures were observed ( figure 6e ) . however , saxs on solutions of these two peptides ( data not shown ) revealed no scattering above background . the lack of significant structures for these two peptides is also consistent with ftir . the observed raftlike film structures for vlk may be due to the surface activity of this peptide . cryo - tem images for solutions containing 1 wt % ( a ) c16-kkffvlk , ( b ) c16-kkff , ( c ) c16-kkf , ( d ) fvlk , and ( e ) vlk . saxs profiles fitted to a spherical shell form factor model for ( a ) 0.5 wt % c16-kkf and ( b ) 0.5 wt % c16-kkff . the difference in morphology of the aggregates of c16-kkffvlk and the fragments c16-kkf and c16-kkff leads to changes in sample transparency as shown in figure 9 . the large nanotube / ribbon structures formed by c16-kkffvlk cause light scattering and a partly cloudy appearance , whereas the micellar structures of c16-kkf and c16-kkff show higher transparency . confirmation that addition of enzyme to a c16-kkffvlk solution leads to a clear solution due to the cleavage process producing very small micelles is provided by the image shown for the mixture in figure 9 . image of samples ( 1 wt % ) in vials from left to right : c16 - kkffvlk , c16-kkff , c16-kkf , and c16-kkffvlk with added c ( 2 wt % ) . as discussed in more detail elsewhere , c16-kkffvlk undergoes a thermoreversible transition from nanotubes coexisting with helical ribbons ( unwound nanotubes ) at 20 c to twisted tapes at higher temperature ( 55 c ) . the oscillations in the data at 20 c are characteristic of a nanotube / ribbon bilayer structure as analyzed by form factor fitting . these disappear on heating , showing the break - up of the nanotubes / ribbons . however , on recooling , the oscillations develop slowly over a period of hours . the enzymatic cleavage of a pa using the model protease -chymotrypsin has been demonstrated . es - ms reveals that c attacks two cleavage sites within the pa and preferentially cleaves between the two f residues as expected , leading to a higher abundance of both c16-kkf and fvlk . the second cleavage process occurs at the c terminus of the second f residue in c16-kkffvlk to produce c16-kkff and vlk . we have shown that the pas corresponding to the cleavage products c16-kkf and c16-kkff assemble into very different structures than the parent pa which forms nanotubes and helical ribbons at room temperature . in contrast to c16-kkffvlk , the two pas with shorter peptide headgroups lack substantial defined secondary structure ( there were some signs of -sheet content in the xrd pattern for c16-kkf , possibly due to reduced hydrophobicity compared to c16-kkff ) . indeed , a phase diagram assembled for pas on the basis of molecular dynamics computer simulations anticipates the formation of spherical micelles when hydrogen bonding interactions are weak . the near absence of intermolecular hydrogen bonded -sheets for c16-kkf or c16-kkff presumably reflects the insufficient length of the peptide headgroup , i.e. there are insufficient residues to participate in extensive hydrogen bonding networks . the change in nanostructure from c16-kkffvlk to c16-kkf and c16-kkff leads to macroscopic changes in sample appearance which might be useful for a simple enzyme sensing system , although more sensitive methods may be available . we have recently investigated the self - assembly of the pa c16-kttks which forms extended -sheet nanotapes . considering in addition the findings reported here , our results on lysine - rich pas suggest that a minimal sequence capable of forming -sheets may require around five residues , provided that the peptide has both highly soluble hydrophilic residues along with hydrophobic residues . however , this is not a general rule since , for example , we have recently shown that a pa c16-ah with only a dipeptide -alanine - histidine headgroup can self - assemble into -sheet fibrils in water . neither of the peptide fragments vlk nor fvlk form extensive self - assembled nanostructures , although tripeptides are able to do so provided they have sufficient amphiphilicity ( e.g. , if they contain two hydrophobic phenylalanine residues ) . our own work also shows that a hexapeptide with one terminal phenylalanine does not self - assemble whereas a homologous heptapeptide with two c - terminal phenylalanines is capable to form -sheet fibrils . one interesting application of our lysine - rich pas may be antimicrobial agents . the antimicrobial activity of pas c16-kxk ( where x is a , g , l or k or k , i.e. d - lysine ) and c16-kk and c16-k has been investigated , and this was correlated to their self - assembly properties . the most active pa ( against microbes ) c16-kkk formed very small oligomers in contrast to c16-kgk and c16-klk which formed fibrils and c16-kak which formed 10 nm diameter micelles . our results highlight the ability of enzymes to modulate the self - assembly of pa systems and the ability to tune nanostructure through use of enzymatic triggers . our proof - of - concept work may be extended to create other enzyme - responsive assemblies or in applications where release of peptide or pa fragments is beneficial . specifically , release of micelles in response to enzymatic degradation of extended nanostructures ( which have been shown to offer enhanced circulation times in vivo ) may be useful in delivery of actives .

the enzymatic cleavage of a peptide amphiphile ( pa ) is investigated . the self - assembly of the cleaved products is distinct from that of the pa substrate . the pa c16-kkffvlk is cleaved by -chymotrypsin at two sites leading to products c16-kkf with fvlk and c16-kkff with vlk . the pa c16-kkffvlk forms nanotubes and helical ribbons at room temperature . both pas c16-kkf and c16-kkff corresponding to cleavage products instead self - assemble into 56 nm diameter spherical micelles , while peptides fvlk and vlk do not adopt well - defined aggregate structures . the secondary structures of the pas and peptides are examined by ftir and circular dichroism spectroscopy and x - ray diffraction . only c16-kkffvlk shows substantial -sheet secondary structure , consistent with its self - assembly into extended aggregates , based on pa layers containing hydrogen - bonded peptide headgroups . this pa also exhibits a thermoreversible transition to twisted tapes on heating .

participants were autoantibody - positive relatives of t1d patients from the dpt-1 ( n = 670 ) and tnnhs ( n = 991 ) cohorts . dpt-1 participants were all islet cell autoantibody ( ica ) positive , whereas tnnhs participants were all positive for gada , ia-2a , miaa , and/or ica . both studies were approved by institutional review boards at all participating sites , and written informed consent or assent as appropriate were obtained in both studies . two - hour oral glucose tolerance tests ( ogtts ) were performed at baseline and at 6-month intervals in both cohorts . oral glucose ( 1.75 g / kg ; maximum , 75 g of carbohydrate ) was administered after fasting samples were obtained ; glucose and c - peptide samples were then obtained every 30 min . plasma glucose was measured by the glucose oxidase method . in dpt-1 , c - peptide was measured by radioimmunoassay , whereas in the tnnhs , the tosoh assay was used . values from the two assays were similar in split samples ( r = 0.961 ; tosoh = 0.96 * rai + 0.1 ; n = 564 ) . undetectable fasting c - peptide values ( < 0.2 ng / ml ) were assigned a value of 0.1 ng / ml . the methodology for performing autoantibody measurements in the tnnhs has been reported previously ( 12 ) . the autoantibodies obtained in the tnnhs include ica , gada , miaa , ia-2a , and znt8a . the dptrs ( 7 ) , developed from the dpt-1 cohort , is based on a proportional hazards model that includes the glucose sum of 30- , 60- , 90- , and 120-min values divided by 100 , the c - peptide sum of 30- , 60- , 90- , and 120-min values divided by 10 , log fasting c - peptide , log bmi , and age . there is a curvilinear relation between risk and the dptrs . as dptrs values increase above 6.50 , the risk estimates increase more steeply ( 8) . dysglycemia was defined as any of the following on the baseline ogtt : a fasting glucose value between 110 and 125 mg / dl ( impaired fasting glucose ) ; a 30- , 60- , and/or 90-min value 200 mg / dl with a 2-h value < 140 mg / dl ( indeterminate ) ; or a 2-h value between 140 and 199 mg / dl ( impaired glucose tolerance [ igt ] ) . participants were informed if they had a dysglycemic ogtt ; however , no treatment was recommended . analyses included the following : a comparison of the cumulative incidence of t1d between normoglycemic individuals with dptrs values > 7.00 and those with dysglycemia ; a comparison of the cumulative incidence of t1d between dptrs values < 7.00 or > 7.00 among those with dysglycemia and among those with two or more autoantibodies ; a comparison of the cumulative incidence of t1d between the tnnhs and dpt-1 among those with dptrs values > 7.00 and among those with dysglycemia ; and a comparison of the reliability between dptrs values > 7.00 and dysglycemia . the 7.00 threshold had previously been shown to identify high - risk individuals in the overall dpt-1 and tnnhs cohorts ( 8) . the p values are two - sided . a p value < 0.05 was considered to be statistically significant . participants were autoantibody - positive relatives of t1d patients from the dpt-1 ( n = 670 ) and tnnhs ( n = 991 ) cohorts . dpt-1 participants were all islet cell autoantibody ( ica ) positive , whereas tnnhs participants were all positive for gada , ia-2a , miaa , and/or ica . both studies were approved by institutional review boards at all participating sites , and written informed consent or assent as appropriate were obtained in both studies . two - hour oral glucose tolerance tests ( ogtts ) were performed at baseline and at 6-month intervals in both cohorts . oral glucose ( 1.75 g / kg ; maximum , 75 g of carbohydrate ) was administered after fasting samples were obtained ; glucose and c - peptide samples were then obtained every 30 min . plasma glucose was measured by the glucose oxidase method . in dpt-1 , c - peptide was measured by radioimmunoassay , whereas in the tnnhs , the tosoh assay was used . values from the two assays were similar in split samples ( r = 0.961 ; tosoh = 0.96 * rai + 0.1 ; n = 564 ) . undetectable fasting c - peptide values ( < 0.2 ng / ml ) were assigned a value of 0.1 ng / ml . the methodology for performing autoantibody measurements in the tnnhs has been reported previously ( 12 ) . the autoantibodies obtained in the tnnhs include ica , gada , miaa , ia-2a , and znt8a . the dptrs ( 7 ) , developed from the dpt-1 cohort , is based on a proportional hazards model that includes the glucose sum of 30- , 60- , 90- , and 120-min values divided by 100 , the c - peptide sum of 30- , 60- , 90- , and 120-min values divided by 10 , log fasting c - peptide , log bmi , and age . there is a curvilinear relation between risk and the dptrs . as dptrs values increase above 6.50 , the risk estimates increase more steeply ( 8) . dysglycemia was defined as any of the following on the baseline ogtt : a fasting glucose value between 110 and 125 mg / dl ( impaired fasting glucose ) ; a 30- , 60- , and/or 90-min value 200 mg / dl with a 2-h value < 140 mg / dl ( indeterminate ) ; or a 2-h value between 140 and 199 mg / dl ( impaired glucose tolerance [ igt ] ) . participants were informed if they had a dysglycemic ogtt ; however , no treatment was recommended . analyses included the following : a comparison of the cumulative incidence of t1d between normoglycemic individuals with dptrs values > 7.00 and those with dysglycemia ; a comparison of the cumulative incidence of t1d between dptrs values < 7.00 or > 7.00 among those with dysglycemia and among those with two or more autoantibodies ; a comparison of the cumulative incidence of t1d between the tnnhs and dpt-1 among those with dptrs values > 7.00 and among those with dysglycemia ; and a comparison of the reliability between dptrs values > 7.00 and dysglycemia . the 7.00 threshold had previously been shown to identify high - risk individuals in the overall dpt-1 and tnnhs cohorts ( 8) . the p values are two - sided . a p value < 0.05 was considered to be statistically significant . the 991 tnnhs participants were significantly older than the 670 dpt-1 participants ( mean age sd : 18.5 13.3 years [ median 13.0 years ] vs. 13.8 9.6 years [ median 11.1 years ] ; p < 0.001 ) . in the tnnhs , the median follow - up of those diagnosed and those not diagnosed was 1.4 and 2.0 years , respectively . whereas 125 had igt alone , 29 had an indeterminate ogtt alone , and 7 had impaired fasting glucose alone . figure 1 shows that a dptrs threshold > 7.00 identified tnnhs participants with normoglycemia at baseline who were at substantial risk for t1d . the cumulative incidence of tnnhs participants with normoglycemia and dptrs values > 7.00 was comparable to the cumulative incidence of those with dysglycemia . the 3-year risk estimates were 0.38 for those with dptrs values > 7.00 and 0.33 for those with dysglycemia . tnnhs participants with normoglycemia and dptrs values > 7.00 were much younger than those with dysglycemia ( 8.1 4.9 years for dptrs > 7.00 vs. 19.6 14.3 years for dysglycemia ; p < 0.001 ) . shown are cumulative incidence curves for t1d of tnnhs participants with normoglycemia ( ngt ) and the cumulative incidence curves are comparable . n.s . , not significant . when those in the tnnhs with dysglycemia were dichotomized according to dptrs values > 7.00 or < 7.00 ( fig . 2 ) , there was a marked difference between the cumulative incidence curves ( p < 0.001 ) . the 3-year risk estimate was 0.46 for those > 7.00 , whereas the 3-year risk estimate was only 0.16 for those < 7.00 . the 3-year risk for those with normoglycemia and dptrs values < 7.00 was 0.08 . shown are cumulative incidence curves of tnnhs participants with dysglycemia after they are dichotomized according to dptrs values < 7.00 or > 7.00 . there is a large difference in the cumulative incidence ( 3-year estimates : 0.16 for < 7.00 and 0.46 for > 7.00 ) . we also assessed the differences in risk according to the 7.00 dptrs threshold among individuals with two or more autoantibodies in the tnnhs . there was again a marked difference in risk ( p < 0.001 for difference in cumulative incidence curves ) . individuals with dptrs values > 7.00 ( n = 87 ) had a 3-year risk estimate of 0.55 , whereas those with dptrs values < 7.00 ( n = 209 ) had a 3-year risk estimate of 0.16 . the degree of consistency in estimating risk between dpt-1 and the tnnhs is shown for those with dptrs values > 7.00 in fig . whereas there was no significant difference in the cumulative incidence of t1d between dpt-1 and tnnhs participants for those with dptrs values > 7.00 , the cumulative incidence for those with dysglycemia was much higher in dpt-1 than in the tnnhs ( p < 0.001 ) . shown are cumulative incidence curves of tnnhs and dpt-1 participants who either had dptrs values > 7.00 ( a ) or dysglycemia ( b ) . among those with dptrs values > 7.00 , the cumulative incidence was similar between the tnnhs and dpt-1 , whereas among those with dysglycemia , the cumulative incidence was markedly lower in the tnnhs than in dpt-1 . reliability was also compared between dysglycemia and the 7.00 dptrs threshold . of those who had dysglycemia at baseline in the tnnhs of the 77 , 41 ( 53% ) had dptrs values < 7.00 at baseline . of those with dptrs values > 7.00 at baseline , a smaller proportion , 42 of 177 ( 24% ) , reverted to having values < 7.00 at the next visit . of the 42 , 22 ( 52% ) had normoglycemia at baseline . of those with dptrs values > 7.00 who reverted to dptrs values < 7.00 , the glucose sum declined significantly ( p < 0.001 ) . there was a decline in the c - peptide sum that was of borderline significance ( p = 0.05 ) . we have performed proportional hazards regressions to examine associations of t1d with dysglycemia and with the dptrs as single variables . each was highly predictive of t1d when included alone ( p < 0.001 for both ) . however , when both were included in a model , while t1d and the dptrs were still strongly associated ( p < 0.001 ) , there was no longer a significant association between t1d and dysglycemia ( p = 0.783 ) . risk estimates are indicated at 2 , 3 , and 4 years of follow - up for those above certain dptrs thresholds at baseline in the tnnhs . if the dptrs threshold of 7.00 was used instead of dysglycemia for the selection of prevention trial participants , more would have been diagnosed ( 71 vs. 62 ) with a smaller number entered ( 191 vs. 221 ) . other thresholds could be selected according to the desired number of participants and their level of risk . for example , if the 6.75 threshold was chosen in place of dysglycemia for the selection of participants , appreciably greater numbers would have been entered ( 253 vs. 221 ) and diagnosed ( 85 vs. 62 ) even though the risk was still comparable . risk estimates of t1d for dysglycemia and dptrs thresholds at baseline in the tnnhs ( n = 991 ) we examined the occurrence of t1d for those above dptrs thresholds when dysglycemia was absent and for those below dptrs thresholds when dysglycemia was present ( supplementary table 1 ) . whereas 22 of 64 ( 39% ) were diagnosed of those normoglycemic with a dptrs value > 7.00 , only 13 of 94 ( 13% ) were diagnosed of those dysglycemic with a dptrs value < 7.00 . the findings indicate that a reliance upon dysglycemia as a demarcation of risk in autoantibody - positive populations could result in a less - than - optimal classification of risk for prevention trials . the risk of certain individuals with normoglycemia could actually be higher than some with dysglycemia . the findings also show that the risk implications of dysglycemia can vary markedly according to the particular population that is studied . in addition , the presence of dysglycemia can be inconsistent when ogtts are repeated in individuals . it is evident that the dptrs can improve the accuracy of risk classification when it is used in conjunction with dysglycemia for prediction . however , the data in table 1 suggest that it might be advantageous to use dptrs thresholds in place of dysglycemia for prevention trials . in addition to improving accuracy , dptrs thresholds provide a choice of target populations with different risks . if greater risk homogeneity is desired for a trial , bounded categories could be used . risk estimates of specific dptrs categories have previously been reported ( 8) ; these can be used for reference . the dptrs provides selection from a risk continuum , whereas dysglycemia only offers a dichotomous selection . the presence of multiple autoantibodies has been used as an indicator of higher risk for t1d . however , those with dptrs values < 7.00 were at much lower risk than those with dptrs values > 7.00 . a recent article ( 12 ) examined prediction by an autoantibody risk score in tnnhs participants that takes both positivity and level into account . in that article , the area under the receiver operating characteristic curve was higher for the dptrs than the autoantibody risk score . also , when the autoantibody risk score and the dptrs were both included in a regression model , the dptrs was still highly predictive . risk can vary substantially for a given number of autoantibodies , depending upon which specific autoantibodies are positive ( 13 ) . it is likely that risk prediction will be improved further with the refinement of autoantibody prediction ( 14,15 ) and the integration of autoantibody and metabolic information . however , it is relatively weak in comparison with other predictors , both overall ( 7 ) and among those with normoglycemia ( 6 ) . moreover , the performance of intravenous glucose tolerance testing is cumbersome for participants and labor intensive for staff . individuals with normoglycemia identified as being at substantial risk for t1d were much younger than those with dysglycemia . this finding suggests that the t1d risk of children can be particularly underestimated when dysglycemia is used as an indicator of risk and highlights the importance of the consideration of age in assessing the risk of t1d . the dptrs is useful for identifying normoglycemic individuals at high risk in large part because it includes age , which is inversely related to the risk for t1d ( 7,8 ) . the lack of consistency in the cumulative incidence curves between the tnnhs and dpt-1 among those with dysglycemia shows that a dependence upon glucose indices alone for risk estimation can be misleading . the greater consistency of dptrs values > 7.00 is possibly attributable to the consideration of other factors associated with risk . the frequent reversion from the dysglycemic state to the normoglycemic state in the tnnhs is consistent with previous findings in dpt-1 in which there were frequent fluctuations between states of glycemia during the progression to t1d ( 20 ) . the lower reversion rate from dptrs values > 7.00 to < 7.00 suggests that dptrs thresholds are more reliable indicators of risk than dysglycemia . thus , the findings are not necessarily generalizable to other populations , such as those at genetic risk for t1d . however , since dpt-1 participants were selected on the basis of autoantibodies that differ from those in the tnnhs , the findings show that the dptrs has general utility for improving the risk classification of autoantibody - positive populations . although no treatment was recommended , it is possible that some could have undertaken treatment on their own . dysglycemia has been shown to be a frequent precursor to t1d ( 4,21 ) , and an understanding of its development in the pathogenesis of diabetes is of importance . however , data now suggest that the prediction accuracy of t1d can be improved well beyond the predictive information provided by glycemia status . the findings indicate that the dptrs can refine prediction and ultimately improve the accuracy of t1d risk classification .

objectivewe studied the utility of the diabetes prevention trial - type 1 risk score ( dptrs ) for improving the accuracy of type 1 diabetes ( t1d ) risk classification in trialnet natural history study ( tnnhs ) participants.research design and methodsthe cumulative incidence of t1d was compared between normoglycemic individuals with dptrs values > 7.00 and dysglycemic individuals in the tnnhs ( n = 991 ) . it was also compared between individuals with dptrs values < 7.00 or > 7.00 among those with dysglycemia and those with multiple autoantibodies in the tnnhs . dptrs values > 7.00 were compared with dysglycemia for characterizing risk in diabetes prevention trial - type 1 ( dpt-1 ) ( n = 670 ) and tnnhs participants . the reliability of dptrs values > 7.00 was compared with dysglycemia in the tnnhs.resultsthe cumulative incidence of t1d for normoglycemic tnnhs participants with dptrs values > 7.00 was comparable to those with dysglycemia . among those with dysglycemia , the cumulative incidence was much higher ( p < 0.001 ) for those with dptrs values > 7.00 than for those with values < 7.00 ( 3-year risks : 0.16 for < 7.00 and 0.46 for > 7.00 ) . dysglycemic individuals in dpt-1 were at much higher risk for t1d than those with dysglycemia in the tnnhs ( p < 0.001 ) ; there was no significant difference in risk between the studies among those with dptrs values > 7.00 . the proportion in the tnnhs reverting from dysglycemia to normoglycemia at the next visit was higher than the proportion reverting from dptrs values > 7.00 to values < 7.00 ( 36 vs. 23%).conclusionsdptrs thresholds can improve t1d risk classification accuracy by identifying high - risk normoglycemic and low - risk dysglycemic individuals . the 7.00 dptrs threshold characterizes risk more consistently between populations and has greater reliability than dysglycemia .

tumors derive from a single cell clone but consist of heterogeneous cell subpopulations with diverse features and functions . sequencing analysis has revealed that tumors accumulate mutations , the majority of which do not provide selective benefits , while a small subset of mutations drive cancer development by conferring a selective advantage . a limited number of subclones with a selective advantage survive treatment with anticancer drugs or radiation . such subclones can initiate tumors when inoculated into immunocompromised mice and are known as cancer stem or cancer - initiating cells ( cics ) . cics can lead to tumor recurrence because they escape apoptosis by effluxing antitumor drugs and degrading reactive oxygen species ( ros ) , which are related to radiation - induced apoptosis . cics are generally defined on the basis of their ability to initiate a tumor in a transplantation assay in immunocompromised mice . in leukemia , breast , lung , and colon cancers , however , recent reports reveal that in some tumor types , such as melanomas , the majority of tumor cells can initiate tumors . asymmetrical division of stem cells yields a stem cell and a more highly differentiated cell . as in the case of physiological stem cells , cics are derived from cics but not from non - cics under normal conditions . however , under some conditions , such as severe hypoxia , non - cics can yield cics ; this phenomenon is known as plasticity . the dynamic exchange between cics and non - cics may enable tumors to survive under unfavorable conditions . therefore , cics may not be a distinct cell type but may be a mode or phenotype of tumor cells . this review briefly describes isolation of cics from tumor cell populations and the microenvironmental factors that regulate cic phenotypes in uterine cancer and lymphoma . cics express high levels of cell membrane - associated abc transporters . when stained with hoechst 33342 dye and incubated for approximately 1 hour in contrast , a small population with high efflux ability eliminates hoechst 33342 dye ( hoechst 33342-low ) . the upper part shows an image of hoechst 33342 staining , in which hoechst 33342 dye is designated by stars . the lower part shows the dot - blot pattern of flow cytometers , in which hoechst 33342-high cells are contained in the mp , whereas hoechst 33342-low cells in the sp . upper part shows the image of cm - h2dcfda staining , in which ros is designated as flowers . when treated with hydrogen peroxide , most tumor cells are stained with cm - h2dcfda , but a small population remains unstained . the upper part shows an aldefluor assay , in which aldh - high population is figured out in the box ( red part ) . these boxed cells are diminished when treated with n , n - diethylaminobenzaldehyde , the inhibitor of aldh . the lower part shows the results of immunohistochemical analysis using an anti - aldh antibody , in which a portion of uterine endometrioid carcinoma cells is aldh - positive . ) . cics express high levels of cell membrane - associated atp - binding cassette ( abc ) transporters , which play important roles in efflux of antitumor drugs . when stained with hoechst 33342 dye , most tumor cells retain the dye and are termed hoechst 33342-high . however , a small population with high efflux ability eliminates hoechst 33342 dye and are termed hoechst 33342-low . most tumor cells are hoechst 33342-high , and are termed the main population ( mp ) , whereas hoechst 33342-low cells are known as the side population ( sp ) . flow cytometers equipped with ultraviolet lasers can be used to distinguish the mp and sp , which enable isolation of sp cells . because sp cells can efflux compounds efficiently , cics are considered to be involved in the sp . patrawala et al . reported that the sp cells in several tumor cell lines are more tumorigenic than mp cells . this finding is consistent with the concept that cics are enriched in the sp . cics express high levels of cell membrane - associated abc transporters . when stained with hoechst 33342 dye and incubated for approximately 1 hour in contrast , a small population with high efflux ability eliminates hoechst 33342 dye ( hoechst 33342-low ) . the upper part shows an image of hoechst 33342 staining , in which hoechst 33342 dye is designated by stars . the lower part shows the dot - blot pattern of flow cytometers , in which hoechst 33342-high cells are contained in the mp , whereas hoechst 33342-low cells in the sp . upper part shows the image of cm - h2dcfda staining , in which ros is designated as flowers . when treated with hydrogen peroxide , most tumor cells are stained with cm - h2dcfda , but a small population remains unstained . the lower part shows the staining of endometrioid carcinoma cells . the upper part shows an aldefluor assay , in which aldh - high population is figured out in the box ( red part ) . these boxed cells are diminished when treated with n , n - diethylaminobenzaldehyde , the inhibitor of aldh . the lower part shows the results of immunohistochemical analysis using an anti - aldh antibody , in which a portion of uterine endometrioid carcinoma cells is aldh - positive . the second method of isolating cics is based on their ability to degrade ros ( fig . upon x - ray irradiation , tumor cells produce a large quantity of ros and undergo apoptosis . cics express high levels of ros - degrading enzymes , such as superoxide dismutase , which facilitate their escape from apoptosis by degrading intracellular ros . intracellular ros can be stained with 5-(and 6)-chloromethyl-2,7-dichlorohydrofluorescein diacetate ( cm - h2dcfda ) . when treated with hydrogen peroxide , most tumor cells are stained with cm - h2dcfda , but a small population remains unstained . these cm - h2dcfda - low cells are more tumorigenic than cm - h2dcfda - high cells , and cics are enriched in the cm - h2dcfda - low population . s lymphoma cell lines , most foxo3a - expressing cells are contained in the cm - h2dcfda - low population . a small subpopulation of hodgkin s lymphoma cells expressing foxo3a can degrade ros efficiently and is resistant to apoptosis . sternberg ( rs ) cells ; however , foxo3a - expressing cells are small mononucleated cells . the l1236 and l428 hodgkin s lymphoma cell lines include both small mononucleated and giant multinucleated cells . in these cell lines , multinucleated cells are derived from mononucleated cells , but mononucleated cells are not derived from multinucleated cells . these findings suggest that some mononucleated cells , but not rs cells , are the cics of hodgkin s lymphoma . several such markers , such as cd133 and cd44 ( cd44v isoform ) , have been reported ; aldehyde dehydrogenase ( aldh ) is one of the most commonly used . high aldh activity is present in not only normal stem cells , such as hematopoietic and neural stem cells , but also various types of cics . indeed , immunohistochemical analysis using an anti - aldh antibody revealed that a portion of uterine endometrioid carcinoma cells are aldh - positive , and cases with high aldh activity have an unfavorable prognosis ( fig . aldh - high endometrioid carcinoma cells are resistant to apoptosis , mobile , and highly invasive . sp and ros - degrading cells can only be detected while alive . by contrast , aldh - high cells can be detected immunohistochemically in formalin - fixed paraffin - embedded tissues . uterine endometrioid carcinoma cells usually express steroid hormone receptors , such as estrogen and progesterone receptors . however , double staining of clinical samples with anti - aldh and anti - hormone receptor antibodies revealed that aldh - high cells are negative for these hormone receptors . aldh - high endometrioid carcinoma cells appear to be immature and negative for differentiation markers , such as estrogen and progesterone receptors . because aldh - high cells are readily isolated by flow cytometry , their proteome can be investigated using mass spectroscopy ( table 1table 1 . list of proteins preferentially expressed in the aldh - high population of uterine endometrioid adenocarcinoma cells ) . these cells also exhibit impaired akt phosphorylation and matrix metalloproteinase-2 activation , which account for their impaired proliferation and invasion , respectively . elevated expression of s100a4 is related to myometrial and lymphatic invasion in well- to moderately - differentiated endometrioid carcinoma . notably , strong and diffuse expression of s100a4 is observed in tumor tissues with a microcystic , elongated , and fragmented ( melf ) pattern , which is associated with a highly invasive phenotype . however , aldh is not an almighty marker of cics , because , for example , aldh is highly expressed in stromal cells , such as dendritic cells and macrophages , in lymphoma . this suggests the presence of microenvironmental factors that induce or inhibit aldh expression and suggests that intercellular signaling between tumor cells might regulate aldh expression . indeed , phosphorylated smad-2 , an indicator of tgf- signaling activation , is present at higher levels in aldh - low than -high endometrioid carcinoma cells . tgf- family members regulate cell fates in developing embryos ; for example , lefty determines the left similarly , the development or differentiation of tumor cells might be controlled by tgf- family members . among the tgf- receptor ligands immunohistochemical analysis of clinical samples has revealed nodal - high tumor cells to be aldh - low cells , and vice versa . these findings suggest that nodal inhibits aldh expression via stimulation of tgf- signaling in uterine endometrioid carcinoma cells . lymphomas can be categorized using the surface markers of differentiation status expressed by lymphocytes . for example , peripheral t - cell lymphoma cells express the t - cell marker cd3 , and diffuse large b - cell lymphoma cells express the b - cell markers cd79a and cd20 . lymphoplasmacytic lymphoma ( lpl ) is a rare indolent disease that affects the bone marrow , and is associated with expression of the b- and plasma - cell markers cd20 and cd138 . mwcl-1 cells are derived from lpl , and express cd20 and cd138 as surface markers . mwcl-1 cells include three subpopulations : cd20 cd138 , cd20 cd138 , and cd20 cd138 cells . the first subpopulation expresses neither b- nor plasma - cell markers , the second subpopulation expresses only b - cell markers , and the third subpopulation expresses both b- and plasma - cell markers . when cultured , cd20 cd138 cells yield all three subpopulations , but cd20 cells do not yield cd20 cd138 cells ( fig . mwcl-1 cells derived from lpl include three subpopulations : cd20 cd138 , cd20 cd138 , and cd20 cd138 cells . when cultured , cd20 cd138 cells yield all three subpopulations , but cd20 cells do not yield cd20 cd138 cells . the cd20 cd138 subpopulation expresses the chemokine receptor cxcr7 , the ligand of which is cxcl12 . the number of cd20 cd138 cells increases in a time- and dose - dependent manner when mwcl-1 cells are cultured in the presence of cxcl12 . ) . cd20 cd138 cells have higher ros degradation and in vitro colony formation activities than cd20 cd138 and cd20 cd138 cells . when cultured in the absence of serum or the presence of an anticancer drug , cd20 cd138 cells immunohistochemical analysis of clinical samples has indicated that lpl tumor cells undergoing apoptosis are cd138 . the production of all three subpopulations , efficient ros degradation and in vitro colony formation activities , and resistance to apoptosis suggest that cd20 cd138 cells are candidate cics in lpl . mwcl-1 cells derived from lpl include three subpopulations : cd20 cd138 , cd20 cd138 , and cd20 cd138 cells . when cultured , cd20 cd138 cells yield all three subpopulations , but cd20 cells do not yield cd20 cd138 cells . the cd20 cd138 subpopulation expresses the chemokine receptor cxcr7 , the ligand of which is cxcl12 . the number of cd20 cd138 cells increases in a time- and dose - dependent manner when mwcl-1 cells are cultured in the presence of cxcl12 . as described above , cics are plastic under stress conditions , in which non - cics are transformed to cics . indeed , hypoxia induces conversion of cd20 cd138 cells to cd20 cd138 cells , whereas normoxic conditions do not . among the proteins preferentially expressed by the cd20 cd138 subpopulation , expression of the chemokine receptor cxcr7 it is possible that the conversion from cd20 cd138 to cd20 cd138 cells is mediated by signaling via cxcr7 ( fig . this is plausible because the number of cd20 cd138 cells increases in a time- and dose - dependent manner when mwcl-1 cells are cultured in the presence of cxcl12 , a ligand of cxcr7 . recently , a constitutively active mutation of cxcr4 , a receptor related to cxcr7 , has been reported in lpl . the fact that cics are resistant to apoptosis and related to aggressive behavior is consistent with the notion that the cics of lpl utilize the cxcl12-cxcr4/cxcr7 axis . cics , which are resistant to anticancer drugs and escape from apoptosis , may be a good therapeutic target for cancers . however , they are , under some circumstances , derived from non - cics due to their plasticity . therefore , the regulatory mechanism of cics should be determined . reagents that block the signals controlling cics may interfere with their plasticity , leading to their complete elimination . the tgf- family member nodal regulates aldh activity in uterine endometrioid carcinoma , whereas signaling via the chemokine receptor cxcr7 regulates cics in non - hodgkin s lymphoma . therefore , investigation of the cics of each tumor type is necessary to facilitate development of novel therapeutics . aldh - high tumor cells express a large number of factors , some of which play roles in invasion and some of which play roles in mobility . although aldh - high cells are invasive and mobile , these characteristics are likely mediated by different factors . genome - editing techniques will enable determination of the function of such factors in cics , which will facilitate development of novel therapeutics .

tumors derive from a single cell clone but consist of heterogeneous cell subpopulations with diverse features and functions . a limited number of subclones with a selective advantage can initiate tumors when inoculated into immunocompromised mice , and are called cancer - initiating cells ( cics ) . cics can be isolated from the bulk of tumors on the basis of their characteristics , such as high reagent efflux , degradation of reactive oxygen species , and aldehyde dehydrogenase ( aldh ) activity . under normal conditions , new cics are produced by existing cics rather than non - cics . however , under stress conditions , non - cics can occasionally produce cics , a phenomenon known as plasticity . the dynamic exchange between cics and non - cics may enable tumors to survive under unfavorable conditions . cics are located in a small portion of tumors . this suggests that microenvironmental factors induce or inhibit the cic phenotype , which might be regulated by intercellular signaling between tumor cells . this review describes isolation of cics from tumor cell populations and the microenvironmental factors that regulate cic phenotypes in uterine cancer and lymphoma .

some forms , such as declarative memory , are explicit memories of objects , places , or events . others forms , such as non - declarative memory , are implicit , like habits , skills , or priming , and do not require conscious awareness ( gilbert et al . , 2001 ; squire , 2004 ) . while this has been an established taxonomy of learning , the boundaries are often vague and difficult to demarcate . for instance , in associative learning there is a fine - grained distinction between declarative associative learning , which is dependent on the conscious association of events , and non - declarative unconscious associative learning , which occurs without awareness of the link between the related meaningful events ( shanks , 1995 ) . in an associative learning task , when two stimuli are systematically presented in a temporal sequence , a new relationship between these two items is learned . in classical trace learning , a neutral conditioning stimulus ( cs ) precedes and , therefore , causes the subsequent prediction of an emotionally charged or noxious stimulus ( unconditioned stimulus , us ; figure 1 ) . during a differential eye - blink trace conditioning task , a specific tone ( cs+ ) warns of a puff of air to the eye , whereas another tone ( cs ) does not . the presence of an anticipatory eye - blink response , which is a conditioned response ( cr ) to the cs+ tone , is highly correlated with participants verbal report of the relationship between the stimuli presented ; this is known as the contingency ( dawson and reardon , 1973 ; clark and squire , 1998 ) . in contrast , during a delay conditioning task , for which there is no gap between the neutral stimulus ( cs ) and the puff of air to the eye , blink responses ( cr ) are elicited despite the lack of awareness of the contingency ( clark and squire , 1998 , 1999 ; figure 1 ) . furthermore , trace , but not delay , conditioning is strongly influenced by an expectancy of the us ( clark et al . , 2001 ) . differential trace ( upper graph ) and delay conditioning ( lower graph ) for the eye - blink task . in trace conditioning there is a silent period between a tone and a puff of air to the eye . the cs+ tone is presented for 250 ms , followed by a 500-ms silent gap , before a 100-ms puff of air to the eye ( aversive stimulus ) . a second neutral stimulus tone , cs , is presented alone . in delay conditioning , the tone lasts for 850 ms , covering the silent gap and co - terminating with the puff of air . consequently , delay conditioning has been considered a hallmark of non - declarative learning and is systematically used as an associative learning task in vertebrates and invertebrates ( lavond et al . , 1993 ) . in contrast , human trace learning is dependent on conscious awareness of the contingency between stimuli ( christian and thompson , 2003 ) . moreover it is this fact that has made human trace learning a potential turing - test of consciousness ( koch , 2004 ) . though this turing - test potential has been influential in behavioral neuroscience , three arguments could pose a serious challenge to this notion : ( 1 ) trace conditioning can be learnt by almost every animal , even invertebrate sea slugs ( glanzman , 1995 ) ; ( 2 ) trace conditioning can be elicited using subliminal stimuli ( esteves et al . , 1994 ) ; and ( 3 ) clinically defined unconscious patients might learn trace conditioning ( bekinschtein et al . it is in addressing these three arguments that we will characterize the theoretical boundaries of consciousness of learning . the evolution of wings can be used as an analogy for the evolution of conditioning . during the evolution of animals on this planet , wings appeared at least three times from three different ancestors . primitive wingless insects known as bristletails used long antennae - like filaments at the ends of their bodies to glide down to tree trunks from forest canopies ( yanoviak et al . , 2009 ) . the second appearance , around 150 million years ago , involved the development of wings on dinosaurs . some dinosaurs started to evolve lighter skeletons ; their wrists changed and feathers grew to form wings ( lewin , 1983 ; sullivan et al . , 2010 ) . though dinosaurs evolved wings , which gave birth to modern birds their capacity to fly has a different origin and ancestry than flying insects . the third appearance of wings developed in bats ( simmons et al . , 2008 ) . though the three different wings serve a similar function , that is to fly , they were not derived from a common ancestor . moreover , they are not controlled by the same machinery , and they do not obey the same rules . is the same true of conditioning ? has a conditioning mechanism appeared only once in evolution ? while the well preserved molecular machinery may suggest this , findings at the systems level are disparate and may suggest a different conclusion ( barco et al . , molecular mechanisms underlying acquisition and consolidation of memory are in fact preserved in most species . however , learning relies on different network mechanisms in humans and sea slugs ( takehara et al . , 2003 ) . the plasticity of arrays of neurons and the ubiquitous evolutionary pressure of associative learning makes it extremely difficult to disentangle whether conditioning emerged only once during the course of evolution . it could be that conditioning emerged several times from several ancestral sources just as wings did . we are only left with the certainty that associative learning can be instantiated in simply a few neurons , as in sea slugs , or in millions of neurons , as in mammals . humans show the highest degree of behavioral flexibility among animals , probably through a flexible network that relies on a frontoparietal hub . this flexibility , however , comes at a cost that results in slow , limited computational capacity . trace conditioning does not seem to escape from this rule ( zylberberg et al . , 2010 ) . arbitrary associations in humans are instantiated in this routing system that appears to be intrinsically related to consciousness ( zylberberg et al . , 2011 ) . on the contrary , in the sea slug these temporary associations may rely on direct connections ( grol et al . , motor relations which bypass the central routing system ( grol et al . , 2006 ) . trace conditioning in sea slugs , where consciousness is not required , does not necessarily imply that consciousness does not play a role in human trace conditioning . alone , it does not imply more than , for example , a bacteria in need of oxygen breathing under water makes implications about human respiration . the sea slug aplysia californica only needs a few neurons to perform trace conditioning , while rabbits require hippocampal , frontal cortex , and cerebellar networks ( christian and thompson , 2003 ) . in contrast , rabbits seem to be more flexible than gastropods in their learning abilities , but not more than insects , i.e. , fruit flies , ( heisenberg et al . , 2001 ) . in humans , as in rodents , trace conditioning is dependent of the hippocampus ( james et al . , 1987 ; moyer et al . , 1990 ; clark and squire , 1998 ) while delay conditioning can be elicited without hippocampi ( ivkovich and stanton , 2001 ; woodruff - pak and disterhoft , 2008 ) . delay conditioning seems to rely primarily on a functional cerebellum ( mccormick and thompson , 1984 ; mauk and thompson , 1987 ; gerwig et al . , 2007 ) . functional imaging studies have shown that the hippocampus is activated by both trace and delay conditioning , but it is significantly more activated by trace ( cheng et al . , 2008 ) . these studies of neuroanatomy and the function of associative conditioning may indicate declarative memory s high dependence on the hippocampi and neocortex , as opposed to non - declarative memory s need for these structures , which is minimal ( eichenbaum and cohen , 2000 ) . further evidence of a causal link between awareness of the contingency and trace conditioning comes from human and non - primate mammalian data . in humans the variability in trace conditioning responses and learning seems to be linked to attention ( lovibond and shanks , 2002 ; carter et al . , 2003 ) , suggesting a strong modulation by central cognitive processes . in healthy volunteers , increasing attentional load parametrically this elegant experiment used the classic n - back task to engage attentional resources and working memory . the engagement of these dramatically decreases the anticipatory responses to the us in trace conditioning . interestingly , the interference paradigm used in humans to decrease conditioning has been replicated using mice ( han et al . , 2003 ) , demonstrating a similar functional frontotemporal network supporting successful trace conditioning in a non - primate mammalian species . in this study mice heard a tone and received a foot shock immediately after ( delay conditioning ) or , alternatively , the mice received a silent gap between tone and shock ( trace conditioning ) . two additional groups of mice experienced two lights flashing as interference for the tone shock conditions . learning was impaired by light interference in trace but not in delay conditioning . in mice the light flashing acted as cognitive interference , just as working memory load might cause interference in trace conditioning with humans . in both populations , i.e. , in humans and mice , the fact that attention is a key component of conscious learning and also that trace conditioning is affected by awareness may indicate that trace conditioning is a type of conscious learning . there is general agreement that exposure to a contingency between conditioned stimulus ( cs ) and us will create an associative process called conditioning ( dickinson , 1980 ; rescorla , 1988 ) . the difference between human and non - human animal models is that the former can easily produce a verbal or motor report of the relationship between the cs and us . if humans can form an internal representation of the contingency and verbalize it or make a voluntary response , then this behavior is taken as evidence of conscious awareness ( lovibond and shanks , 2002 ) . what if the cs is masked , or more generally , not accessible to verbal reports ? this question is central to current theoretical discussions of the role of conscious processing in trace conditioning . the single - process model , asserts that a sole propositional learning process mediates expression cr and the expectancy of us ( full network mapping ; lovibond and shanks , 2002 ) . the dual - process model , however , claims that these behavioral responses , both cr expression and us expectancy , are expressions of two independent learning processes ( partial network mapping ; perruchet , 1985 ; morris et al . , 1999 ; some studies find evidence in support of the single - process model ( daum et al . , 1991 , 1992 ; manns et al . , 2000a , b ; weike et al . , 2007 ) . while the series of experiments performed by perruchet , destrebecqz , cleeremans , and colleagues ( destrebecqz and cleeremans , 2001 ; destrebecqz et al . , 2005 , 2010 ; perruchet et al . , 2006 ) and experiments performed by others ( ohman and soares , 1993 , 1994 , 1998 ; ohman et al . , 1995 ; weidemann et al . , the discussion is centered on the verbal reports of awareness of the contingencies ( in questionnaires , motor evaluation or subjective ratings ) and the measures of cr . firstly , post - training questionnaires may elicit metacognitive process . therefore the participant may verbally report the contingencies , not because they noticed them during the learning phase , but because they were forced to think about the contingencies after conditioning had finished . in this case it is difficult to know when conscious learning of the relationship between the stimuli occurred . it could have been during learning or it could have been when that participant was prompted about the relationship between stimuli . secondly , if awareness is measured online during the experiment , as to avoid a post hoc metacognitive process , then participants may then take note of the contingencies of which they were previously unaware . the measurement of both verbal reports of contingencies and the cr is critical . in a series of studies , ohman et al . ( 1995 ) and ohman and soares ( 1993 , 1994 , 1998 ) argue that conditioning of electro - dermal responses to electric shocks can occur with masked , unconsciously perceived , stimuli . it has been found that unconsciously perceived stimuli only elicited a cr when fear - relevant stimuli , such as spiders and snakes ( ohman and soares , 1993 ) or angry faces ( esteves et al . , 1994 ) , were presented as cs+ . in support of ohman s claims , one recent study showed that sensitivity to masking conditions was related to the cr of a masked cs but not an unmasked cs ( cornwell et al . , 2007 ) . in this study , sensitivity to the masked condition was a marker of unconscious processing , i.e. , if participants were not aware of the masked item then this was taken to indicate that items were processed at an unconscious level . the depth of unconscious processing of the cs was linked to the intensity of the cr . weak perception of the cs through masking may not elicit conscious recognition of it , but the cs may still be above an identification threshold . however , there are criticisms of these findings . one criticism is that the measure used to assess perceptual awareness of the cs may not be sufficiently sensitive to identify participants with residual awareness of stimulus features ( pessoa , 2005 ; graziano and sigman , 2009 ) . additionally , some fear relevance effects in backward masking conditioning , as observed by ohman , could be due to selective sensitization rather than unconscious associative processes . some methodological concerns have also been raised concerning the extent to which participants were truly unaware of the stimuli ( lovibond and shanks , 2002 ) . firstly , not all verbal discriminative responses indicate awareness ; discrimination of stimuli above chance levels does not necessarily imply conscious awareness ( merikle and daneman , 2000 ; wiens and ohman , 2002 ) . another highly debated experiment was performed by nez and de vicente ( 2004 ) , they have also showed that cr can be elicited when masked words are paired with a mild shock , and that this response is , as it was in the studies by ohman , related to the participants detection threshold . however , the results of this study are somewhat difficult to interpret as a higher proportion of participants in the unconscious masked condition produced a cr than in the conscious group . this study evaluated masked words paired with electric shocks ; they used either a detection threshold , i.e. , was the stimulus a word or a blank ? , or an identification threshold , i.e. , was it word1 , word2 or not a word ? when participants failed to detect a stimulus in the tachitoscope , half exhibited a cr above learning criterion ( four out of eight ) . yet , when participants were above detection threshold ( conscious ) only 11% ( 2 of 18 ) showed learning . there was a higher instance of conditioning , using a detection threshold , when stimuli were are presented unconsciously and when cr was measured using autonomic nervous system signals like skin conductance . on the contrary , when an identification threshold was applied , i.e. , subjects had to differentiate between two words or two non - words , only 10% ( 1 of 10 participants ) of participants in the unconscious condition exhibited cr , but 58% ( 7 out of 12 ) of participants in the conscious group exhibited cr . these contradictory results point to two different learning systems , the unconscious system , which bypasses central processes and consciousness - related workspaces . it also possibly directly links the early visual system with the autonomic nervous system and the conscious associative system , where the activation of the frontoparietal cortices may influence the autonomous nervous system giving rise to a different signal and a different type of learning . it is indeed the case that the variability , speed , and regularity of the cr was higher in the conscious identification group as compared to the unconscious detection learners ( nez and de vicente , 2004 ) . there are , however , caveats to this study s design that must be considered when discussing the conclusions . firstly , in the identification threshold condition , words were repeated and could have therefore been deprived of their meaning . when a word is repeated it becomes easy to rely on low level features , such as the letter array , to determine the word s identity without the need to access its meaning . the participants decisions in this condition may not have been based on anything more than a surface features . another methodological problem of this study is that the variability of the perceptual threshold was high and several subjects were therefore excluded leading to a small sample size . the final subgroups of learners were 2/18 and 4/8 for conscious and unconscious detection of a word / blank , respectively , and 7/12 and 1/10 subjects for conscious and unconscious identification of the word , respectively . the low number of participants complicates the statistical analysis and make conclusions difficult to extrapolate to other cases . when an electric shock is used as the aversive stimulus , as opposed to a puff of air to the eye or a loud tone , it may induce a general increase in arousal . this may , change detection thresholds and act as a confounding factor , leading to difficulty in interpreting the results . in short , there seems to be consistent evidence showing that trace conditioning can also be elicited by unconscious stimuli with a strong emotional content , which is not accessible for verbal report . there may be some specific residual forms of trace learning that can be mediated by the most likely candidates : unconscious processing and emotional stimuli . however , evidence of subliminal abstract , non - emotional stimuli eliciting trace conditioning is not conclusive . careful and well - designed experiments are needed to robustly deliminate the boundaries of consciousness thresholds of stimuli in a given task . their ability to elicit trace learning above and below this threshold should certainly be included in the agenda of highly relevant experiments in the next years . if trace conditioning is taken as an indirect test of awareness , then a clinically defined unconscious patient , i.e. , those in a vegetative state ( vs ) , that shows crs ( learning ) should be considered conscious . conversely , if a clinically defined unconscious patient shows anticipatory responses in trace conditioning , then this learning may not necessarily be indicative of conscious awareness ( bekinschtein et al . , 2009 ) . they lack any behavior consistent with conscious awareness , such as , they do not follow someone with their eyes or head across the room , and they do not gesticulate or react to signs , words or commands . however , we used a classic differential trace conditioning eye - blink paradigm in 13 vs patients to investigate whether these unconscious patients might show learning ( bekinschtein et al . , 2009 ) . moreover , we found a subset of these vs patients did show learning ( see figure 2 ) . two vs could appropriately produce cr to cs+ and not to cs , and another four patients showed non - discriminatory anticipatory responses , i.e. , producing the cr to both stimuli types . patients that demonstrated learning eventually recovered by regaining awareness , as opposed to those patients that did not show learning . it is our belief that the patients that showed learning were partially conscious at the time of testing , but were unable to produce overt voluntary responses . in a recent trace conditioning study , using aversive noises and pleasant fanfares measuring skin conductance response , scott et al . ( 2011 ) , showed that only participants attending to the stimuli and able to catch the rule of the experiment showed crs . if this paradigm is applied in the same disorders of consciousness patients group as the trace eye - blink conditioning , it may show convergence in conscious - dependent learning , and helping to better define the learning consciousness relationship . anticipatory learning in normal and anesthetized participants , and in vegetative state patients during trace differential conditioning . bars show mean muscle activity for baseline of the cs+(bs cs+ ) , cs+ and baseline of the cs(bs cs ) , cs. learning is in arbitrary units . sedated subjects show no learning ; vegetative state patients , as a group , show less muscle activity between cs+ and its baseline , and to cs. it is crucial to consider alternate hypotheses related to trace conditioning and the limited capacity of consciousness . limited capacity implies that to learn trace conditioning , progressive gain of awareness of the contingency is required by maintaining a free global workspace clear of other contents ( dehaene and naccache , 2001 ) . the use of this limited space for other events or processes may weaken the link that establishes the association between the cs and the us . this in turn would prevent the associative relationship from being established . in our original study these sedated healthy participants showed no learning of the contingency , or cr , for eye - blink trace conditioning . the sedative ( propofol ) that anesthetized these participants ensured that they were indeed unconscious , and the levels of drug caused a significant decrease in absolute cerebral blood flow . in particular the propofol - related variations in the thalamic blood flow appeared to be linked to the midbrain reticular formation , thus suggests a close functional relationship between the two brain structures while unconscious ( fiset et al . , 1999 ) . these deeply sedated participants showed true unconsciousness through both diminished awareness and arousal ( wakefulness ) . moreover , the participants under low doses of propofol had a completely disorganized system for global integrated processing ( davis et al . , 2007 ; stamatakis et al . , 2010 ) . on the contrary , unconscious vs patients show dissociated wakefulness and awareness ; they are regarded as being awake but not aware ( jennett and plum , 1972 ) . it could be that some vs patients retain partial functionality of the networks that support the acquisition of trace conditioning , but this is not enough to produce volitional movements . sea slugs can learn trace conditioning , but they do not show a form of learning that reveals the flexibility typically displayed in conscious forms of learning ( van den bussche et al . , 2008 ; heinemann et al . , 2009 ; see dehaene and changeux , 2011 for a review ) . instead , these mollusks use the minimal numbers of systems necessary for successful associative learning . as far as we know sea slugs are not conscious , that is they are not conscious in the same way as humans are . hence the mere observation of trace conditioning learning in this species does not provide evidence that trace conditioning is not an adequate signature of consciousness . quite the opposite is true ; humans are extremely sensitive to context and are continuously interpreting all incoming stimuli in multiple ways . this capacity for over - interpretation may help in social interaction , goal - oriented behavior and possibly changes the way that trace conditioning is encoded and processed . the expression of trace learning in humans through a display of non - stereotyped representations of the contingency relies on an entirely distinct neural architecture several studies have used masked or subliminally presented stimuli in order to make cs impossible to report ( unconsciously perceived ) . this type of design has been used to explicitly determine whether conscious perception of the stimuli is necessary to achieve trace learning . unfortunately , the results are inconclusive about whether trace learning , generally , is achievable through subliminal stimuli or under some specific circumstances . when it is not neutral , but of negative valence , the masked stimuli seems to be processed up to the point of forming an association with the us , despite not being reported , detected , or discriminated . the fact that clinically defined unconscious patients show trace learning suggest that they may have partial capacity for conscious awareness , as trace learning is dependant on some form of conscious awareness of the contingency . this result is further strengthened by data from sedated participants that show drug induced unconsciousness does not produce anticipatory responses above the baseline ( bekinschtein et al . , 2009 ) . trace conditioning remains very much linked to awareness of the contingency between cs and us . we believe it is a combination of the timing between stimuli and the variations on the cs that will allow us to better frame , over the next few years , the boundaries between this basic form of learning and conscious awareness . the aforementioned analyses leads us to propose here three conditions which should be met in order for trace conditioning to be used as a test for conscious awareness : ( 1 ) a relatively stable , sustained attention to the stimuli , ( 2 ) a low central processing load to avoid interference and ( 3 ) a well defined stimuli , perceptually discernible and close in presentation time . if ( 1 ) is not met , then attention will deviate from the stimuli , and the creation of the association between cs and us will be disrupted . this would lead to sparse and inconsistent demonstration of the cr ( armony and dolan , 2002 ) . if ( 2 ) is not met then working memory capacity will be saturated by another task . learning will then decrease , and as a result there will be low awareness of the contingencies . in this respect , trace conditioning seems to require the central resources of the attentional system in order for the association to be established , and this seems to be paired with awareness ( carter et al . , 2003 ) . if ( 3 ) is not met , then the stimuli will be ambiguous and the trace that is established will not be long - lasting or robust . this will lead to partial learning only and high variance in cr ( lovibond and shanks , 2002 ; sehlmeyer et al . , we would like to raise one final important point that has not yet been emphasized . it is of paramount importance if trace conditioning is to be used in practical terms . clark and squire ( 1998 ) established that in order to observe an anticipatory eye - blink response participants must be aware of the contingency at a level that is sufficient for a verbal report . the contrary , however , is not true ( both in the original clark and squire data and in our data making more than 200 participants combined ) : some participants show verbal reports of the contingency , but do not show the cr of an anticipatory associative response . this simply may be because participants react in different ways to the anticipation of the air puff when they are aware that it is imminent . the most frequent reaction is a contraction of the muscle prior to the air puff , however , some participants may control this spontaneous response by relaxing and , inhibiting their blinks or by simply doing nothing . hence , an improved statement is needed . while the absence of learning does not provide information concerning the participants degree of awareness the presence of trace learning may be taken as evidence in favor of conscious awareness . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

classical ( trace ) conditioning is a specific variant of associative learning in which a neutral stimulus leads to the subsequent prediction of an emotionally charged or noxious stimulus after a temporal gap . when conditioning is concurrent with a distraction task , only participants who can report the relationship ( the contingency ) between stimuli explicitly show associative learning . this suggests that consciousness is a prerequisite for trace conditioning . we review and question three main controversies concerning this view . firstly , virtually all animals , even invertebrate sea slugs , show this type of learning ; secondly , unconsciously perceived stimuli may elicit trace conditioning ; and thirdly , some vegetative state patients show trace learning . we discuss and analyze these seemingly contradictory arguments to find the theoretical boundaries of consciousness in classical conditioning . we conclude that trace conditioning remains one of the best measures to test conscious processing in the absence of explicit reports .

world health organization estimates that one million serious unintentional poisonings occur every year and an additional two million people are hospitalized for suicide attempts with pesticides . india is a predominantly agrarian country where pesticides are routinely used for farming . according to data available from national poison information centre india , suicidal poisoning with house - hold agents ( ops , carbamates , pyrethrinoids , etc . ) recent data from national crime bureau of india shows suicide by consumption of pesticides account for 19.4% and 19.7% of all cases of suicidal poisoning in the year 2006 and 2007 respectively . op compounds inhibit acetylcholinesterase resulting in accumulation of acetylcholine ( ach ) and overstimulation of cholinergic synapses . patients die mostly from respiratory failure and lung injury , although there is variability in the clinical symptoms and signs depending on nature of compounds , amount consumed , severity , time gap between exposure , and presentation in the hospital . common clinical features of op poisoning include the following : respiratory : increased bronchial secretions , bronchospasm , chest tightness , dyspnoea , cough eyes : blurred vision , conjunctival injection , dimness of vision , miosis gastrointestinal : cramping , diarrhea , nausea , vomiting urinary : incontinence cardiovascular : bradycardia , hypotension exocrine glands : hyperamylesia , increased salivation muscle fasciculation , cramping , weakness , diaphragmatic paralysis , respiratory failure , tachycardia , hypertension . thus we can appreciate that a patient with op poisoning presents with a variety of signs and symptoms . in india , unconscious patients are often brought by their relatives and neighbors who are unable to provide correct information regarding the nature of the particular poison to which the patient was exposed . in such cases the diagnosis of op poisoning is based on clinical features as observed by the treating physicians . the clinical features also help to determine the severity of poisoning which is of prognostic importance . a literature search was conducted which consisted of a medical literature analysis and retrieval system online ( medline ) database search ( accessed on 16.4.2009 ) and a world wide web search ( search engine : google , accessed on 16.4.2009 ) using the following keywords : organophosphorus poisoning , clinico - epimiological features , study . the search revealed that only a few studies have considered the clinical and epidemiological features of op poisoning.[611 ] our study was conducted in this backdrop to explore the clinical features and epidemiological characteristics of patients presenting with op poisoning . respiratory : increased bronchial secretions , bronchospasm , chest tightness , dyspnoea , cough eyes : blurred vision , conjunctival injection , dimness of vision , miosis gastrointestinal : cramping , diarrhea , nausea , vomiting urinary : incontinence cardiovascular : bradycardia , hypotension exocrine glands : hyperamylesia , increased salivation muscle fasciculation , cramping , weakness , diaphragmatic paralysis , respiratory failure , tachycardia , hypertension . thus we can appreciate that a patient with op poisoning presents with a variety of signs and symptoms . in india , unconscious patients are often brought by their relatives and neighbors who are unable to provide correct information regarding the nature of the particular poison to which the patient was exposed . in such cases the diagnosis of op poisoning is based on clinical features as observed by the treating physicians . the clinical features also help to determine the severity of poisoning which is of prognostic importance . a literature search was conducted which consisted of a medical literature analysis and retrieval system online ( medline ) database search ( accessed on 16.4.2009 ) and a world wide web search ( search engine : google , accessed on 16.4.2009 ) using the following keywords : organophosphorus poisoning , clinico - epimiological features , study . the search revealed that only a few studies have considered the clinical and epidemiological features of op poisoning.[611 ] our study was conducted in this backdrop to explore the clinical features and epidemiological characteristics of patients presenting with op poisoning . the study was conducted in a tertiary care medical college and hospital in west bengal , india . all consecutive patients presenting in the emergency department of the hospital with history and clinical evidence of op poisoning during the study period were considered eligible for participation in the study . patients attending the emergency department with history of exposure to op compounds were initially evaluated and resuscitated for maintaining airway , breathing , and circulation . informed consent was obtained from eligible patients / legally authorized representatives ( if the patient was unconscious ) . after completing the medicolegal formalities careful history was taken from the patients / legally authorized representatives ( in case the patient was unconscious ) following which clinical examination was carried out to determine the common clinical features of op poisoning . data collected were analyzed in computer by using the statistical package for social sciences ( spss ) program version 10 . data analysis was done by using descriptive and inferential statistical methods : frequency , percentage , means , standard deviation ( s.d . ) . a two - tailed p - value less than 0.05 was considered to be statistically significant . the study was conducted in a tertiary care medical college and hospital in west bengal , india . all consecutive patients presenting in the emergency department of the hospital with history and clinical evidence of op poisoning during the study period were considered eligible for participation in the study . patients attending the emergency department with history of exposure to op compounds were initially evaluated and resuscitated for maintaining airway , breathing , and circulation . informed consent was obtained from eligible patients / legally authorized representatives ( if the patient was unconscious ) . after completing the medicolegal formalities careful history was taken from the patients / legally authorized representatives ( in case the patient was unconscious ) following which clinical examination was carried out to determine the common clinical features of op poisoning . data collected were analyzed in computer by using the statistical package for social sciences ( spss ) program version 10 . data analysis was done by using descriptive and inferential statistical methods : frequency , percentage , means , standard deviation ( s.d . ) . a two - tailed p - value less than 0.05 was considered to be statistically significant . a total of 968 patients presented in the emergency department of the institute during the study period with the clinical features of op poisoning . mean age of the persons presenting in the emergency with clinical features of op poisoning is 34.47 years ( means.d . : poisoning with suicidal intent was more common ( 82.02% ) than the accidental one ( 17.98% ) ( p - value < 0.0001 ) . majority of the patients were housewives ( 42% ) followed by farmers ( 33.99% ) , shopkeepers ( 9.93% ) , laborers ( 8.14% ) , students ( 6.2% ) . the mean interval between poison consumption and admission to hospital was 4.4 hours ( means.d . : 4.42.29 ) . regarding the type of poison consumed by the patient , methyl parathion was the most common poison consumed by the patients ( 35.74% ) followed by diazinon ( 28.62% ) , chlorpyriphos ( 19.52% ) , dimicron ( 16.12% ) . the signs and symptoms with which the patients presented are enumerated in table 2 . in the present study , nausea and vomiting is the most common symptom reported by 85.02% of patients followed by abdominal cramps ( 47.93% ) , while miosis is the most common sign observed in 91.94% patients . following admission , a total of 56 ( 5.78% ) patients died during their hospital stay . respiratory failure was the primary cause of death in 21 patients ( 37.5% ) followed by cns depression ( 33.92% ) , cardiac arrest ( 21.44% ) , and septicemia ( 7.14% ) . baseline demographic parameter of the patients of op poisoning ( n=968 ) clinical features of patients suffering from op poisoning at presentation in the present study , majority of the patients belonged to the young age group ( means.d . : females show a clear preponderance over male with a male ( m ) : female ( f ) ratio of 1:1.38 . this finding is in agreement with studies conducted in turkey ( m : f=1:1.47 ) , nepal ( m : f=1:2 ) . however , in the study conducted in chennai by shivaprasad et al . , ( 2001 ) , male patients ( 74% ) outnumbered female ( 26% ) . majority of the affected patients in our study are housewives ( 42% ) which is at par with the study conducted in turkey ( housewives 47.3% of total sample ) . in the present study , poisoning with suicidal intent was more common ( 82.02% ) than the accidental one ( p - value < 0.0001 ) . this is in congruence with studies conducted in nepal , turkey , gulbarga where poisoning with suicidal intent accounts for 95.24% , 75.9% , and 97.25% of total cases of op poisoning respectively . the mean interval between poison consumption and admission to the hospital was 4.4 hours ( means.d . : 4.4 2.29 ) . in studies conducted at chennai , maximum patients ( 89.69% ) presented within 6 hours . regarding the type of poison consumed by the patient , methyl parathion was the most common poison ( 35.74% ) followed by diazinon , chlorpyriphos , dimicron . , carbamates however , in a study conducted in turkey , dichlorvos was the most common one . this variation in the type of poison consumed can be attributed to the regional availability of pesticides in different countries . clinical presentation depends on the specific op involved , the quantity absorbed , and the type of exposure . in our study nausea and vomiting was the most common symptom ( 85.02% ) while miosis ( 91.94% ) was the most common sign . a comparison between the clinical features as observed in our study with earlier studies has been listed in table 3 . the mortality rate in our study was 5.78% which is comparable to the study done in turkey ( 9.1% ) . the present study showed that majority of the patients were of young age with females outnumbering males . poisoning with suicidal intent was more common than accidental . nausea and vomiting was the most common symptom reported by the patients while miosis was the most common sign observed by the treating physicians of the research team .

background : organophosphorus ( op ) poisoning is a major health problem all over the world , particularly in the developing countries.aim:the present study aims to explore the clinical and epidemiological features found in patients presenting with op poisoning.materials and methods : a 1-year cross - sectional study was conducted on patients presenting with clinical features of op poisoning in a tertiary care medical college.results:a total of 968 patients presented during the study period . poisoning with suicidal intent ( 82.02% ) was more common than the accidental one ( 17.98% ) . majority of the patients were housewives ( 42% ) followed by farmers , shopkeepers , laborers , students . methyl parathion was the most common poison consumed by the patients ( 35.74% ) followed by diazinon , chlorpyriphos , dimicron . nausea and vomiting ( 85.02% ) was the most common symptom while miosis was the most common sign observed in 91.94% patients . a total of 56 patients of op poisoning died ( 5.78% ) with respiratory failure being the primary cause of death followed by cns depression , cardiac arrest , and septicaemia.conclusion:the present study showed that majority of the patients were of young age with females outnumbering males . poisoning with suicidal intent was more common than accidental . nausea and vomiting was the most common symptom reported by the patients while miosis was the most common sign observed by the treating physicians of the research team .

village indigenous birds are constantly exposed to immunosuppressive conditions such as aflatoxicosis and infectious bursal disease virus . in addition , management and ecological factors such as confinement , climatic and seasonal fluctuations , poor feeding , and worm infestations have been associated with stress and reduced immune response . stressful factors have been reported to cause functional and morphological changes in chickens . in tanzania , it was observed that newcastle disease ( nd ) was a greater problem in villages with ducks . earlier reports indicated that newcastle disease virus ( ndv ) persisted for a long time in a flock of ducks in a village situation in indonesia . however , the factors leading to shedding of the virus by the carrier ducks are not well documented . it was hypothesized that immunosuppression of immunised carrier ducks does not influence persistence of ndv in these birds . in this experiment , thus , the aim of the present study was to determine the effect of immunosuppression on the viral persistence and immune status of ducks . it was designed to simulate field situation where ducks that have varying levels of ndv antibodies undergo immunosuppression in the presence of high ndv challenge . one - day - old indigenous ducklings were hatched from the duck flock maintained at the university of nairobi premises . all the birds were reared in isolation and transferred to experimental units at one year of age . they were wing tagged , tested , and confirmed to be free of ndv and respective antibodies . water and food were provided ad libitum . a kenyan virulent newcastle disease virus isolate ( vndv ) was obtained from the repository maintained at the university of nairobi and characterized by standard methods . the inactivated vaccine was prepared by mixing 40% formalin and allantoic fluid with a titer of 2 of vndv in a ratio of 1 : 40 , that is , formalin to virus . the reparation was kept at room temperature ( 24c to 26c ) for 24 hours before use . all the ducks were vaccinated via an initial dose of 1 ml of the vaccine intramuscularly on the thighs and a booster of 0.5 ml of the same vaccine 16 days later . the live virulent kenyan newcastle diseases virus isolate , previously characterized by standard methods , was later used to challenge vaccinated and naive ducks . dexamethasone ( dexamethasone sodium phosphate and sodium methyl hydroxybenzoate , coophavet , france ) was used to stress ducks in this study . the respective groups of ducks were injected intramuscularly with the dexamethasone , following the protocol of corrier et al . modified as follows : the dosage was given at the rate of 2 mg per kilogram of body weight per day for 4 days continuously , then the ducks were rested for 2 days and the injections resumed at the same dosage for 2 more days . sixty - four ducks were vaccinated with 1 ml of inactivated nd vaccine intramuscularly and 14 days later , they were bled from the brachial vein and sera prepared . they were later boosted with a single dose of 0.5 ml of the nd inactivated vaccine and bled 7 days later . seven days after the booster dose , the ducks were divided into two groups , each with 32 birds , namely , low antibody level group ( 1 : 32 ) and medium antibody level group ( 1 : 64 ) . each group of 32 ducks was further subdivided into 4 minigroups , as follows : ( i ) immunosuppressed and challenged ( 1a , 2a ) , ( ii ) immunosuppressed only ( 1b , 2b ) , ( iii ) challenged only ( 1c , 2c ) , and ( iv ) not challenged nor immunosuppressed ( 1d , 2d ) . another group ( group 3 ) of 30 nonimmunized ducks were subdivided into 4 groups . groups 3a and 3c had 12 ducks each while 3b and 3d had 3 birds each . immunosuppression was done before respective groups were inoculated intranasally with 0.2 ml of undiluted amnioallantoic fluids of vndv having a titer of 1 : 1024 . five birds from each of the challenge groups and all the 3 ducks from each control group were sampled throughout the experimental period ( 28 days ) . the samples were taken on days 0 , 1 , 4 , 8 , 14 , and 28-after inoculation ( p.i . ) . blood for serum was sampled each time from the five ducks in each challenge group , and the three ducks from each of the controls . further , two ducks from each of the ndv challenged groups were killed serially and brain , kidney , lung , cecal tonsils , liver , and spleen collected separately from each bird . the tissues were processed for nd viral recovery using chicken embryo fibroblasts , while serum samples were tested for ndv - specific antibodies by hemagglutination inhibition ( hi ) test . virus recovery from the tissues was carried out in primary chicken embryo fibroblasts as described by oie . analysis of variance was performed using sas software ( sas institute inc . , cary , nc , usa , 2002 - 2003 ) to determine the treatments ' main effects and the interaction between time ( days ) and treatment , on various responses . immunosuppressed virus - challenged ducks ( group 1a ) had low mean antibody levels ( 5.0 ) up to day 4 after - inoculation ( p.i . ) compared with day 0 ( 4.5 ) . thereafter , there was marked increase ( from 4.5 to 7.0 ) in antibody titers up to 14 days p.i . after day 14 p.i . , there was a slight decrease ( 6.9 ) in antibody levels up to 28 days p.i . although the levels were still higher than any period between day 0 and 8 p.i . the nonimmunosuppressed virus - challenged group ( 1c ) had a moderate increase ( 5.0 to 6.0 ) in antibody levels from day 1 up to day 14 p.i . , after which there was a decrease to day 0 level titers by day 28 p.i . the immunosuppressed group ( 1b ) had marked decrease in antibody titers from day 1 to 4 and gradual decrease ( 5.0 to 3.8 ) up to day 28 p.i . the immunosuppressed virus - challenged group for the medium antibody level ducks ( 2a ) had a gradual decline ( 6.0 to 5.7 ) in antibody titers up to day 4 followed by an increase in antibody titers ( 6.9 ) up to day 14 p.i . this was followed by a marked decrease ( 6.1 ) and by day 28 p.i . the nonimmunosuppressed virus - challenged group ( 2c ) showed a slight decrease ( from 6.0 to 5.9 ) in the antibody titer followed by a gradual decrease and then an increase up to the end of the experiment . from day 1 up to day 4 after - inoculation , the immunosuppressed , immunised noninfected ( 2b ) group showed a more rapid decrease ( 6.0 , 5.2 , 4.8 , 3.7 , 2.3 , and finally 2.0 ) in antibody levels as compared ( 6.0 , 4.7 , 4.5 , 3.8 , 2.7 , and finally 2.2 ) to the nonimmunosuppressed controls ( 2d ) . in general , all the nonchallenged , but immunized control ducks showed decrease in antibody titers with time ( figure 1(b ) ) . the immunosuppressed virus - challenged group ( 3a ) had a gradual antibody response ( from 0.0 to 6.5 ) up to the end of the experimental period . the nonimmunosuppressed virus - challenged group ( 3c ) showed a massive increase ( 0.0 to 6.6 ) in antibody levels similar to immunosuppressed virus - challenged group 3a . the group 3c also had a marked decrease ( from 6.6 to 4.6 ) in antibody titres after day 14 p.i . and by 28 days p.i . , the titers were quite low . negative control ducks ( 3b and d ) , sampled at the same time , were negative for ndv antibodies ( figure 1(c ) ) . for days 4 , 8 , 14 , and 28 p.i . antibody titres of the following groups were found to be significantly different ( p < 0.05 ) . when considering the antibody levels elicited in the different duck - groups , with respect to their initial antibody levels , both sets ( low - antibody group and medium - antibody group ) , and both the immunosuppressed and non - immunosuppressed ducks showed higher responses in ducks that were challenged with virulent virus than in those that were not challenged . all the control naive ducks ( groups 3b and 3d ) did not sero - convert . immunosuppressed medium - antibody - level , challenged with ndv ducks ( group 2a ) versus immunosuppressed medium - antibody - level , nonchallenged ( group 2b ) , was lowest in the latter group . nonimmunosuppressed low antibody level , challenged with ndv ducks ( group 2c ) versus 2d , the latter group had lower levels of antibodies . in addition , antibody titres of group 1a versus 1d were significantly different ( p < 0.05 ) on day 14 , being lower in the latter . all the control nave ( groups 3b and 3d ) birds did not seroconvert . on day 1 after inoculation , ndv titers were recorded in liver tissues of group 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) ducks only . on day 4 , high titres of the ndv were recorded in the kidneys ( figure 2(a ) ) . on day 8 p.i . , ndv was isolated in the liver , kidneys , caecal tonsils , and lungs of all treatment groups ( figure 2(b ) ) . the highest ndv titers were recorded in the liver and kidney tissues of immunosuppressed medium ( 2a ) and nonimmune ( 3a ) challenged ducks and nonimmunosuppressed , low - antibody - level challenged ducks ( 1c ) . no ndv was isolated by day 14 p.i . in the brain and spleen from any of the groups . high ndv titres were recorded from the liver tissues of ducks in all treatment groups . however , titres were recorded in the cecal tonsils only in group 2a on day 14 p.i . and in groups 1a and 1c at day 28 p.i . in addition , the immunosuppressed ducks of groups 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) and 2a ( medium - antibody - level group , immunosuppressed and challenged with vndv ) yielded the highest ndv titres as compared to other treatment groups . newcastle disease virus was recovered from the brain and lung on day 28 p.i from immunosuppressed ducks only ( figure 2(d ) ) . there have been comparatively few sequential virological studies on the pathogenesis of nd in ducks and the reported studies involved only fully susceptible chickens [ 8 , 9 ] . reports in other studies have documented frequent isolation of virulent ndv from captive caged birds , healthy wild birds , and village chickens [ 1012 ] . in some cases , the ducks expressed clinical nd as a result of confinement that was understood to have induced stress . our findings indicate that ndv carrier status in nonnatural hosts such as ducks would possibly , under stress , recrudescence virulent virus from sequestered sites in the kidney , liver , and caecal tonsils , leading to virus release in faecal and respiratory exudates . the excreted virus would set up an infectious index case in chickens , thus maintaining ndv endemicity . immunosuppression induced by injection of dexamethasone in the three treatments influenced the pattern of antibody response and the ndv recovery rate . the immunosuppressed ducks that had low and medium antibody level showed a decrease in antibody titers up to day 4 after challenge with ndv . the nonimmunosuppressed virus - challenged ducks of low - to - medium antibody level had an increase in antibody titres up to day 14 p.i . the nonimmunized ducks manifested increased antibody titres after day 4 p.i . and had a massive increase in antibody levels as compared to immunosuppressed challenged group . in the present study , the number of immunosuppressed ducks that yielded the nd virus was higher compared to the nonimmunosuppressed . the prechallenge antibody titers may therefore play a significant role in shedding off the virus as well as clinical manifestation of the disease . . noted that a few hours of treatment with low concentrations of synthetic glucocorticoid ( analogue dexamethasone ) are sufficient to inhibit the synthesis of interferon , a virus inhibitor . this may , in part , explain the observation that treatment with glucocorticoids increased virus yield and lethality in mice infected with coxsackie virus . our present study using ducks concurs with those of asdell and hanson who showed that prior treatment of chickens with dexamethasone lead to massive nd virus multiplication . there was significant difference in geometric mean antibody titers between the immunosuppressed ducks of group 1a and nonimmunosuppressed counterparts ( group 1d ) and also between immunosuppressed ducks of group 3a and nonimmunosuppressed group 3d . this means that whereas dexamethasone seems to have an effect on immune system of ndv - infected ducks , the prechallenge titres also play a major role in the immune response of immunosuppressed birds in that immunosuppression of ducks with high viral titers allows virus multiplication making ducks better carriers . the effect of immunosuppression with dexamethasone in ducks appears to be the same as that induced by aflatoxin in chicks with ndv infections . chickens fed on aflatoxin produced lower antibody levels when compared to the non - aflatoxin - treated ones . the nonchallenged preimmunized ducks had a progressive decrease in antibody levels suggesting that if they were to be exposed to the virus , they could come down with the nd or if the antibody titers were within the protective levels ( 2 to 2 ) , they might not develop clinical disease but instead may remain as virus carriers . the fact that the ducks in these experiments had high levels of antibodies may not necessarily prevent subclinical infection and excretion of virulent virus as supported by other studies elsewhere . based on these results , it is possible to assume that immunosuppressed ducks carrying ndv are more likely to shed virus under stress than nonimmunosuppressed ducks . furthermore , the ducks showed progressively declining antibodies titres to very low levels making them more susceptible to infection by challenge nd virus , thus leading to clinical disease and virus excretion . the excreted virus would contaminate the birds ' environment and be transferred to susceptible chickens and other birds . thus , immunosuppression seems to have epidemiologically significant effects on ndv carrier ducks having varying antibody titer levels .

this study was carried out to verify the possibility that ducks are sources of newcastle disease ( nd ) virus infection for chickens in mixed flocks . immunosuppressed ( is ) and non immunosuppressed ( nis ) birds , at three different antibody levels ( medium , low and absent ) were used ; the titres having been induced through vaccination , and immunosuppression done using dexamethazone . each of the 3 respective groups was further divided into 2 groups of about 12 ducks each : one challenged with velogenic nd virus ; the other not challenged . selected ducks from all groups had their antibody titres monitored serially using hemagglutination inhibition test , while two birds from each of the challenged groups were killed and respective tissues processed for nd viral recovery , using chicken embryo fibroblasts . in general , antibody titres of is and nis challenged ducks were significantly higher than their unchallenged counterparts ( p < 0.05 ) . non - challenged pre - immunised ducks had a progressive decrease in antibody levels ; non - immunised ducks did not seroconvert . newcastle disease virus was isolated from livers and kidneys of the challenged ducks throughout the experimental period ; indicating a possibility of viral excretion , especially when the birds are stressed . it , therefore , provides another possible model of viral circulation within mixed flocks .

rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease affecting predominantly joints , synovial membranes , articular cartilages , and subchondral bones . disease progression is attributed to increases in reactive oxygen species ( ros ) and oxidative stress ( os ) in the lesion sites . proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , regulate the inflammatory and immune responses and play a pivotal role in the disease . overproduction of nitric oxide ( no ) , as a result of induction of inducible nitric oxide synthase ( inos ) due to enhanced production of these cytokines , is associated with persistent inflammation and tissue destruction in experimental arthritis models , including rheumatoid arthritis [ 4 , 5 ] . a number of inflammation stimuli , including tnf- , il-1 , il-6 , or ros , can activate proinflammatory pathways involved in ra pathogenesis , concerning predominantly nuclear factor-b ( nf-b ) , mitogen activated protein kinases ( mapks ) , or janus kinases / signal transducers and activators of transcription ( jak / stat1/3 ) [ 68 ] . this results in translocation of relevant downstream transcription factors from the cytoplasm to the nucleus , where they activate messenger rna ( mrna ) expression of target genes , including il-1 , tnf- , inos , and 12/15-lipoxygenase ( lox ) , leading to overproduction of corresponding proteins . cytokines released into the synovium reach also the systemic circulation and act in other tissues and organs such as lungs , vascular tissue , liver , and heart . several recent investigations reported damage of vital organs with various degrees of impairment , considered to be secondary complications of ra and a major predictor of mortality in ra patients . increasing evidence is pointing to the critical role of the liver in modulating the immune response in autoimmune and chronic inflammatory diseases including ra [ 5 , 11 , 12 ] . the hepatic biochemical and immunological alterations are associated with and influenced by changes in the oxidative state of liver cells . adjuvant - induced arthritis ( aa ) in rats not only is an experimental model of polyarthritis but also induces pathological changes in a variety of other tissues , including the liver and spleen . it is a useful tool to study immunopathologic processes , autoimmune chronic inflammation , and inflammatory cachexia in rodents . in addition , at the molecular level , mrna profiling suggests that this model is also similar to human ra , particularly in tissue gene expression and in the activation of regulatory pathways [ 11 , 14 ] . numerous studies reported natural polyphenols as potential therapeutic agents of diseases caused by os and inflammation [ 1517 ] . n - feruloylserotonin ( n - f-5ht , n - feruloyl-5-hydroxy - tryptamine ) is a conjugated serotonin , a member of the indole hydroxycinnamic acid amides , with serotonin ( 5-ht ) and ferulic acid ( fa ) as representative components of its structure . hydroxycinnamic acid amides of serotonin , synthesized by serotonin n - hydroxycinnamoyltransferase , are present in several vegetables and wild - growing plants whose seeds are used in herbal medicine in eastern countries [ 1820 ] . in cell - based studies , under short - term high - glucose conditions , n - f-5ht exerted an inhibitory effect on overproduction of mitochondrial superoxide by acting as scavenger of superoxide . n - f-5ht attenuated the upregulation of mrna and proteins of ros - dependent adhesion ( vascular cell adhesion protein-1 ( vcam-1 ) ) and migration factors ( monocyte chemoattractant protein-1 ( mcp-1 ) ) , crucial in early atherosclerosis lesions in human aortic endothelial cells , and inhibited the activation of transcription factor nf-b . furthermore , n - f-5ht showed a protective effect on ros - related neuronal damage by decreasing the activity of proapoptotic caspase-3 . n - f-5ht isomers isolated from seeds of leuzea carthamoides were shown to inhibit protein kinase c / ii activation and decrease the oxidative burst of human whole blood and isolated neutrophils in vitro . n - f-5ht was also found to have a protective effect against ldl oxidation and atherogenesis in experimental animals and in human studies [ 2426 ] . methotrexate ( mtx ) , used as a standard drug in our study , represents the most frequently used pharmacotherapy of ra in clinical practice . its administration is , however , limited due to its toxic side effects [ 27 , 28 ] . yet application of a combination therapy of mtx with other potential immunomodulators , synthetic drugs or natural substances [ 3032 ] , might elevate the therapeutic efficacy : decrease the dose of mtx and thus its side effects . in our previous study , we showed that administration of n - f-5ht to mtx - treated arthritic rats lowered the dose of mtx for the required sustained antirheumatic impact . in this study , we focused on the therapeutic impact of n - f-5ht and mtx administered in monotherapy and on details of the inflammatory state in the arthritic rat liver with the aim to elucidate the molecular mechanisms of their effect . one of the possible clarifying approaches is to study the mrna expression of key proinflammatory markers ( il-1 , tnf- , and inos ) in the liver of treated and untreated arthritic rats . further , it is of particular interest to expand our knowledge on the effect of n - f-5ht and mtx in the aa model , which in turn should allow extrapolations of these results to ra patients . to this aim we evaluated also conventional arthritic parameters ( hpv , arthritic score , body weight change , and weight of the liver ) along with changes in plasmatic levels of il-1 and crp and the activity of 12/15-lox in the liver . adult male lewis rats weighing 160180 g were obtained from charles river wiga , germany . the experimental protocol was approved by the ethics committee of the institute of experimental pharmacology and toxicology and by the slovak state veterinary and food administration in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and was in line with slovak legislation . to induce a rat model of adjuvant arthritis ( aa ) , rats were intradermally injected with a suspension of heat - inactivated mycobacterium butyricum in incomplete freund 's adjuvant ( difco laboratories , detroit , mi , usa ) . the third group comprised adjuvant arthritis rats treated with methotrexate ( methotrexat ebewe sol inj 20 mg/2.0 ml ) in oral dose of 0.4 mg / kg twice a week ( aa - mtx ) . the fourth group comprised adjuvant arthritis rats treated with n - feruloylserotonin dissolved in suspension of methylcellulose tween 80 at a dose of 3 mg / kg / day orally ( aa - n - f-5ht ) . drugs were administered orally by gastric gavage from day 0 ( the day of treatment ) to day 28 of the study . blood for plasma preparation was taken by retroorbital puncture on day 14 and by cardiac puncture on day 28 under deep ketamine / xylazine anesthesia . after the animals had been sacrificed under deep ketamine / xylazine anesthesia , tissues for liver and spleen homogenate preparation were taken at the end of the experiment ( day 28 ) . blood in heparinized tubes for plasma preparation was centrifuged at 3000 rpm for 15 minutes at 4c . fraction of four isomers of n - f-5ht ( table 1 ) was isolated from the seeds of leuzea carthamoides ( wild ) dc by solvent extraction . this was then followed by column chromatography on silica gel and hplc separations under conditions previously reported [ 35 , 36 ] . the hind paw volume ( hpv ) was recorded on days 14 , 21 , and 28 with the use of an electronic water plethysmometer ( ugo basile , comerio , varese , italy ) . calculation of the increase in hind paw volume in ml assessed the intensity of the edema . the arthritic score was measured as the total score of hpv ( ml , max . points 5 ) + diameter of scab in the site of mb application , measured in parallel to the spinal column ( mm , max . body weight change ( bwc ; g ) was measured on days 1 , 14 , 21 , and 28 . bwc was calculated as the difference of the body mass measured on days 14 , 21 , and 28 to the body weight measured at the beginning of the experiment ( day 1 ) . for the determination of rat crp concentration in plasma ( g / ml ) , the elisa kit from immunology consultant laboratories , inc . the reaction of secondary biotin - conjugated anti - rat crp antibody was evaluated by streptavidin - hrp . the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 450 nm ( microplate reader , labsystems multiskan rc ) . for the determination of il-1 concentration in plasma , the elisa kit from r&d systems quantikine was used . rat cytokine present in the samples binds to anti - rat cytokine antibodies absorbed in the microwells . the reaction of secondary biotin - conjugated anti - rat cytokine antibody is evaluated by hrp . the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 490 nm in comparison with the reference wavelength of 620 nm ( microplate reader mrx ii ) . concentration of proteins in liver homogenates was determined by using the bradford method and expressed in mg / ml of enzyme preparation ( cytosolic fraction from rat lung and liver tissues ) . linoleic acid ( 99% , sigma - aldrich , usa ) was used as a substrate prepared in solubilized state as described in the concentration of 0.2143 100.7143 10 m. the assay of lox was monitored for 60 seconds as an increase in the absorbance at 234 nm , reflecting the formation of hydroperoxylinoleic acid . for the lox activity assay , an uv / vis spectrometer perkin - elmer lambda 35 ( usa ) was used . the reaction medium contained a 50 mm tris - hcl buffer ( ph 7.0 ) , 2.5 l of the enzyme , and solubilized linoleic acid . total rna was isolated from the rat liver and spleen using rnazol rt ( sigma - aldrich ) and converted into complementary dna ( cdna ) using the primescript rt reagent kit ( takara ) following the protocols of the manufacturers . amplification and detection of cdna of reference and target genes were performed on a 7300 real - time pcr system ( applied biosystems ) using hot firepol evagreen qpcr mix plus ( rox ) ( solis biodyne ) . relative mrna expressions of il-1 , tnf- , and inos were analyzed using the ct value method . the sequences of the primers were designed and checked using primer3 and oligo analyzer 1.0.3 ( table 2 ) . mean and sem values were calculated for each parameter in each group ( 810 animals in each experimental group ) . statistically significant differences among treated , untreated , and control groups were tested using parametric analysis of variance ( anova ) . post hoc tests ( tukey - kramer ( anova ) ) were applied in situations where differences among groups were significant at the level of significance = 0.05 . after post hoc testing , the following significance levels were specified : extremely significant ( p < 0.001 ) , highly significant ( p < 0.01 ) , significant ( p < 0.05 ) , and not significant ( p > 0.05 ) . antioxidant properties of polyphenols including n - f-5ht have been reported [ 21 , 40 , 41 ] . nevertheless , the n - f-5ht impact on chronic inflammatory and os - inducing arthritis , which could widen the possibilities of the ra therapy , remains to be elucidated . in our previous study in the model of aa , n - f-5ht in the dosage of 15 mg / kg markedly potentiated the therapeutic effect of low - dose ( nontherapeutic dose ) mtx ( 0.3 mg / kg ) on arthritic ( hind paw volume and arthritic score ) and inflammatory parameters ( il-17 , mcp-1 , and crp ) , yet it resulted in insignificant effect in monotherapy . as data about the optimal n - f-5ht dose in the rat model are scarce , we decided to study two doses of n - f-5ht : ( i ) when 15 mg / kg exceeded the physiologically acceptable concentration , we used 3 mg / kg , and ( ii ) when 15 mg / kg was too low to reach the maximal effect , we used 30 mg / kg . unexpectedly , contrary to the lower dose of n - f-5ht , the higher dose exhibited minor effect on the parameters examined and/or these varied strongly among the animals . for this reason , this report shows only the data evaluating the lower dose of n - f-5ht . in this study , we used the therapeutic dose of mtx ( 0.4 mg / kg ) with the intention to compare each mechanism of action of mtx and n - f-5ht , both evaluated in monotherapy . the significant rise in arthritic parameters , arthritic score , and hpv confirmed the arthritis in our model in rats . the arthritic score showed an increase in the untreated arthritic group compared to the control group on all days monitored ( aa versus co , day 14 , p < 0.01 ; day 21 and day 28 , p < 0.001 ; table 3 ) . at the end of the experiment , the arthritic score was almost doubled in the aa group compared to controls . a trend toward reduction was observed after administration of n - f-5ht to aa animals on day 28 , but the effect was not statistically significant . the treatment with mtx significantly reduced the arthritic score on observation days 21 and 28 , compared to the untreated arthritic group , proving the therapeutic potential of the applied dose of mtx ( aa - mtx versus aa , day 21 , p < 0.05 ; day 28 , p < 0.01 ; table 3 ) . similarly , the change in hpv showed an increase in the untreated arthritic group compared to the control group on days 21 and 28 ( aa versus co , day 21 , p < 0.01 ; day 28 , p < 0.05 ; table 3 ) . the administration of n - f-5ht induced no modification of hpv of the arthritic animals on any day monitored . mtx therapy significantly reduced the observed swelling on days 21 and 28 compared to the untreated arthritic group ( aa - mtx versus aa , day 21 and day 28 , p < 0.001 ; table 3 ) . the muscle wasting condition due to high catabolic activity , known as rheumatoid cachexia , occurring in approximately two - thirds of all patients with ra , is mediated by tnf- and il-1 in ra . papers published over the past years confirmed that oxidative metabolism was considerably enhanced in the liver of adjuvant - induced arthritis in rats [ 4346 ] . rats used in this study revealed signs of cachexia ( table 3 ) . a significant decrease in body weight change ( bwc ) the bwc of the arthritic rats was 56% on day 14 , 19% on day 21 , and 27% on day 28 ( aa versus co , days 14 , 21 , and 28 , p < 0.001 ; table 3 ) of the bwc of healthy controls . n - f-5ht treatment led to a significant increase of bwc on day 28 ( aa - n - f-5ht versus aa , p < 0.05 ; table 3 ) . the administration of n - f-5ht in arthritic animals did not change these parameters on any of the days observed . the liver weights were significantly lower ( aa - mtx versus aa , p < 0.05 ; table 3 ) only in the group of rats treated with mtx . the reduced weight of the liver in mtx - treated rats was assumed to be the result of inhibition of the pathway of de novo dna synthesis by mtx [ 47 , 48 ] . in summary , the statistical significance of 3 mg / kg of n - f-5ht treatment was determined only for bwc . the arthritic score revealed a trend toward the positive effect increasing with time , indicating a late onset of n - f-5ht action ( table 3 ) . as expected , significant differences were found in the arthritic score and hpv in the arthritic animals treated with the therapeutic dose of 0.4 mg / kg mtx compared to those treated with the low dose of 0.3 mg / kg mtx . il-1 , a prototypic proinflammatory cytokine , is a major mediator of the inflammatory cascade in ra , which is involved in the mechanisms leading to progressive joint destruction . in the model of aa , the early phases of the disease seem to be characterized by a systemic increase of il-1 . the plasmatic level of il-1 , a protein of multiorgan origin , was significantly increased in arthritic animals compared to the control group in the early phase of aa , on day 14 ( aa versus co , p < 0.001 ; figure 1(a ) ) , ascertaining the presence of inflammation . administration of mtx did not lead to a significant change of plasmatic il-1 concentration ; only a trend toward reduction was observed on day 14 . it is noteworthy that n - f-5ht treatment resulted in a significant decrease of il-1 level in plasma ( aa - n - f-5ht versus aa , p < 0.05 ; figure 1(a ) ) . this result is interesting , as this molecule was reported to be relevant in driving the transition from the acute phase to the chronic irreversible phase of the disease and it has been suggested that it could be the target of early intervention to stop the course toward the chronic form of the disease . the blocking il-1 protects bone and cartilage from progressive destruction in ra and its inhibition could be effective in the treatment of this disease . activation of t and b cells , macrophages , and inflammatory mediators tnf- , il-1 , and il-6 aggravates the oxidative damage of the vital organs in rheumatoid arthritis , such as the liver . the liver , in turn , influences the systemic inflammation via producing inflammatory cytokines and mediators such as tnf- , il-1 , il-6 , no , crp , and lox . il-6 , il-1 , and tnf- promote the synthesis of crp in hepatocytes via stat3 [ 51 , 52 ] and nf-b pathways . the level of the systemic inflammatory parameter crp in plasma , resulting from liver synthesis , was increased significantly in the group of arthritic animals compared with control animals in the chronic phase of the disease on experimental day 28 ( aa versus co , p < 0.001 ; figure 1(b ) ) . administration of n - f-5ht and mtx significantly reduced the plasmatic levels of crp on day 28 compared to the untreated group of arthritic animals ( aa - n - f-5ht versus aa , p < 0.05 ; aa - mtx versus aa , p < 0.05 ; figure 1(b ) ) . interaction of crp with fc - gamma receptors ( fcr ) fcri and fcriia is known to promote the production of proinflammatory cytokines , resulting in the amplification loop of inflammatory reaction . these processes are initiated through the induction of the receptor activator of nuclear factor-b ligand ( rankl ) protein and direct stimulation of osteoclastogenesis , causing a loop between inflammation and bone destruction in ra . crp enhances both the proinflammatory response and bone destruction . in the treatment of ra , a lowered crp level thus not only is a significant parameter in terms of disease progression elimination but also has a direct impact on decreasing the degree of bone destruction . alterations in the oxidative state lead to the activation of nf-b and nf-b - dependent genes , such as lox . the enzyme 5-lox catalyzes the conversion of arachidonic acid to leukotrienes , whose production has been associated with inflammation in arthritis . suppression of 5-lox expression ameliorates clinical parameters in ra and aa [ 56 , 57 ] . increased levels of nf-b in the lung and liver as well as increased activity of lox in the lung highlight the importance of extra - articular manifestations of aa . in our experiment , liver 12/15 lox activity increased in arthritic animals in comparison to healthy animals ( aa versus co , p < 0.001 ; figure 1(c ) ) . the effect of n - f-5ht on the activity of 12/15-lox in liver homogenate was comparable with that of mtx . after administration of mtx or n - f-5ht , a significant decrease to control levels was assessed in the liver of the aa group ( aa - n - f-5ht versus aa , p < 0.001 ; aa - mtx versus aa , p < 0.001 ; figure 1(c ) ) . thus the anti - inflammatory effect of n - f-5ht in aa was supported by the ability of the molecule to inhibit 12/15-lox activity . similar to this result , recent observations also reported that several other flavonoids may act as lox inhibitors . in aa , the gene expression levels of tnf- and inos produced in the liver were reported to increase [ 60 , 61 ] . also , in our study , the levels of tnf- and inos mrna expressions were significantly increased in arthritic animals ( both p < 0.001 , aa versus co ; figures 2(a ) and 2(b ) ) . it was proposed that these modifications in the liver of arthritic rats not only were a consequence of the metabolic alterations caused by the disease , especially the increased oxidative metabolism , but also depended on increased inflammatory parameters in the liver . the same agents that increase oxidative metabolism , tnf- , il-1 , il-6 , and others , are responsible for increasing the activity of inos in several tissues . an increase of inos activity as a consequence of elevated inos mrna expression was considered to play a dominant role in the pathogenesis of ra . no generation by inos induced in chondrocytes in the initial stage of aa may play a key role in triggering the subsequent events in arthritis . in general , the use of nos inhibitors has been shown to exert beneficial effects in experimentally induced arthritis . however , which types of cells expressing inos are associated with the induction or progression of adjuvant - induced arthritis via no generation remains uncertain . mrna expression of inos in rat liver was reduced following mtx ( aa - mtx versus aa , p < 0.001 ; figure 2(a ) ) and n - f-5ht treatment ( aa - n - f-5ht versus aa , p < 0.01 ; figure 2(a ) ) . the effect of mtx treatment on tnf- protein and mrna expression differs among studies , depending on the conditions of the given study , concerning gender of patients , type of cell line , duration of treatment , mtx dose , and so forth . in our study in the rat aa model , administration of mtx attenuated significantly the mrna expression of tnf- ( aa - mtx versus aa , p < 0.01 ; figure 2(b ) ) . in many patients , however , mtx treatment does not result in lower tnf- plasma concentration . when mtx fails to produce an adequate response , newer therapies are used in combination with mtx . blocking tnf- with anti - tnf- monoclonal antibodies significantly decreased the signs and symptoms of ra compared to placebo in ra patients with active disease receiving mtx [ 65 , 66 ] . thus , the n - f-5ht - driven significant reduction of tnf- mrna expression ( a - n - f-5ht versus aa , p < 0.01 ; figure 2(b ) ) suggests an intriguing effect on ra treatment , calling for deeper investigation . increase of mrna expression was observed for il-1 in the liver of arthritic animals ( aa versus co , p < 0.001 ; figure 3(a ) ) as expected . administration of mtx did not lead to significant attenuation of il-1 transcription in the liver . this is in concert with previous studies of mtx function in different types of cells ( e.g. , human peripheral blood mononuclear cells and murine peritoneal and splenic cells ) [ 67 , 68 ] . on the other hand , mtx exhibits another mechanism of il-1 function inhibition , which involves blocking the binding of il-1 to il-1 receptor in the membrane of peripheral blood cells ( monocytes , lymphocytes , and granulocytes ) . contrary to mtx , treatment with n - f-5ht led to a substantial inhibition of il-1 gene expression ( aa - n - f-5ht versus aa , p < 0.01 ; figure 3(a ) ) . further , we examined il-1 mrna expression in the main immunocompetent organ , in the rat arthritic spleen , which has not been studied previously in terms of the aa model , related to il-1 expression . we observed il-1 mrna expression activation comparable to that in the liver ( aa versus co , p < 0.001 ; figure 3(b ) ) . interestingly , both mtx and n - f-5ht exhibited a significant and remarkably stronger inhibition of il-1 mrna expression in comparison to that in the liver ( aa - mtx versus aa , p < 0.01 ; aa - n - f-5ht versus aa , p < 0.001 ; figure 3(b ) ) . in the spleen of n - f-5ht treated rats , the relative mrna expression decreased even to control level . besides other events , mtx treatment leads to suppression of nf-b , a heterodimer consisting of two subunits p65 and p50 , one of the most prominent inflammatory transcription factors activated in ra . this was confirmed in our previous work , along with the finding that also n - f-5ht ( 15 mg / kg ) suppressed the activation of nf-b ( p65 ) in the arthritic rat liver [ 21 , 33 ] . interestingly , combination therapy ( mtx + n - f-5ht ) potentiated the effect of a single drug , suggesting different mechanisms leading to nf-b inhibition . mtx driven reduction of cytokine transcription was attributed to abrogation of ib kinase activation and thereby suppression of ib ( nf-b inhibitor ) phosphorylation and degradation , resulting in retaining the inactive nf-b form in cytoplasm . however , the contribution of n - f-5ht to nf-b pathway suppression needs to be further investigated . studies of the proposed pathways involved in the transcription of tnf- , il-1 , and inos in ra could help evaluate the mechanism of action of these drugs [ 68 , 71 , 72 ] . the gene expression of inos is mostly under the control of synergistically activating nf-b ( il-1 and tnf- stimulated ) and stat1 ( ifn- stimulated ) key proinflammatory signals in the liver . in contrast to inos , tnf- does not contain the stat binding element in its promoter region . the inhibition of tnf- and inos transcription observed in our study might be mostly attributed to the suppressed nf-b pathway for both mtx and n - f-5ht [ 33 , 64 , 70 ] . however , the contribution of ap-1 to tnf- and stat1 for inos can not be excluded . mtx - dependent suppression of nf-b was reported [ 33 , 70 , 73 , 74 ] , but in other cases mtx was not found to be effective in the attenuation of arthritic - increased mrna expression of il-1 [ 68 , 75 ] . taking into account our results , where mtx treatment did not lead to inhibition of il-1 mrna expression in the arthritic liver in contrast to the significant n - f-5ht impact , yet treatment of both mtx and n - f-5ht decreased the presumably nf-b - dependent lox activity and inos and tnf- transcription to a similar extent , the involvement of n - f-5ht in another pathway for transcription regulation of this cytokine in the arthritic liver should be considered . after analysis of the reported pathways involved in the regulation of il-1 mrna expression , we hypothesized that tnf--driven ap-1 transcription factor activation or jak / stat3 pathway activated via il-6 or ifn- might play a role ( [ 7 , 8 , 71 , 72 , 76 , 77 ] , figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7509653 ) . papers reporting involvement of other polyphenols in anti - inflammatory regulation , for example , resveratrol , claim that these compounds exhibit their anti - inflammatory effect through suppression of nf-b and jak / stat signaling pathways [ 78 , 79 ] . the enhanced influence of mtx and n - f-5ht on il-1 transcription in the spleen in comparison to the liver may be the consequence of different predominance of inflammatory pathways in this organ , presumably with a stronger nf-b contribution . details about the relevance of these pathways and the role of n - f-5ht in the transcription regulation of il-1 , inos , and tnf- in the liver and other organs in ra are to be further elucidated . the present study contributed additional evidence about the beneficial effect and mechanism of action of n - f-5ht and of mtx on a systemic inflammatory process in the liver and its association with the pathogenesis of adjuvant arthritis . n - f-5ht treatment led to amelioration of inflammatory parameters tested ( plasmatic crp and il-1 protein levels , liver lox activity , and liver and spleen cytokine expression ) . however , this did not result in a significant change of hpv , although a trend of improvement of the arthritic score was observed after 28 days . a synergistic effect of tnf- and il-1 was shown to influence the balance between protein degradation and protein synthesis causing among others an increase in resting energy expenditure and net efflux of amino acids from muscle to liver . the significant increase of bwc in n - f-5ht treated rats , probably sign of the partial improvement of rheumatoid cachexia , might be the result of lowered mrna expression of tnf- and il-1 determined in the arthritic liver . moreover , taking into account the reported association of weight loss with the il-1 production by splenic cells , the n - f-5ht mediated attenuation of increased il-1 mrna expression in the arthritic spleen might contribute to this complex process . the contribution of the affected expression of tnf- and il-1 originating from other organs can not be excluded and is to be further elucidated . unexpectedly , chronic daily treatment with a high concentration of n - f-5ht ( 30 mg / kg ) exhibited either a minor effect on the parameters examined and/or a strong variation among the animals ( not shown ) and that in contrast to a much lower concentration ( 3 mg / kg ) . since n - f-5ht possesses a serotonin ( 5-hydroxytryptamine , 5-ht ) moiety , the question if there might be some interplay between effects of these two molecules on ra pathogenesis is to be raised . since n - f-5ht inhibited the increase of cytosolic free ca concentration in rat vascular smooth muscle cells induced by serotonin mediated by 5-ht2 receptors , it was hypothesized that at a sufficient concentration n - f-5ht may act as a competitive antagonist , which displaces serotonin from its binding site . intake of a high concentration of a 5-ht2 receptor antagonist may lead to a variety of effects : it may influence the receptor density , even enhance the effect of serotonin , or lead to desensitization and with time to receptor resistance ( through inhibitory feedback due to binding - induced enhanced production of serotonin ) . interestingly , serotonin is known not only as a neurotransmitter . increasing but contradictory reports associate serotonin with immunoinflammatory pathways in the periphery . serotonin , via its 5-ht2a , 5-ht2b , and 5-ht3 receptors , has been implicated to have both proinflammatory and anti - inflammatory roles in a number of studies of rheumatoid arthritis [ 8488 ] . the reported effects of 5-ht receptor antagonist on macrophage - like synovial cells encourage the interest to study the effect of n - f-5ht from this point of view . to confirm this hypothesis , a precise characterization of interaction between n - f-5ht and 5-ht receptors is to be done . on comparing the effects of the two drugs , administration of mtx ( 0.4 mg / kg ) or n - f-5ht ( 3 mg / kg ) was found to lead to a decrease of the main plasma marker of systemic inflammation crp , the liver origin protein , and to inhibition of proinflammatory lox in the liver . the impact of mtx and n - f-5ht on mrna expression of tnf- , il-1 , and inos in the liver and on the level of crp in plasma was mentioned at the conference . mtx and n - f-5ht reduced the arthritis - increased transcription of tnf- and inos in the liver to a comparable extent . we suppose that the inhibition of tnf- and inos transcription might be mostly attributed to the suppressed nf-b pathway for the two drugs [ 21 , 33 , 70 ] . as previously reported [ 67 , 68 ] and also proven by our study , mtx was not able to diminish the arthritic - induced il-1 mrna transcription in the liver . this handicap might be compensated by coadministration of n - f-5ht , since this drug was shown to lower the level of proinflammatory cytokine il-1 in plasma in the acute phase of aa and to attenuate significantly the elevation of il-1 mrna expression in the arthritic rat liver and spleen in the chronic phase . detailed studies are required to confirm the hypothesis that n - f-5ht might function through potentially different mechanisms of inhibition of the inflammatory pathway nf-b and not through mtx , as well as the possibility of an additional pathway influencing il-1 transcription under control of n - f-5ht but not mtx . the confirmation would support n - f-5ht as a promising agent for the treatment of ra in combination therapy with mtx . the positive effect was shown in our previous study , where n - f-5ht markedly potentiated the therapeutic effect of low - dose mtx . as the therapeutic dose of mtx was used in this study and the purpose of combination study is to lower the mtx dose to decrease the side effects of this drug , the effect of combination therapy was not included . oral daily intake of n - f-5ht could overcome the inconvenient administration and high costs of biological therapy using il-1 monoclonal antibody , which was shown in clinical trials to be superior to placebo in combination with mtx in reducing signs , symptoms , and radiographic progression in patients with advanced ra [ 91 , 92 ] . future studies of n - f-5ht mechanisms of action should shed more light on the immunomodulatory function of this natural polyphenol . it is to be expected that n - f-5ht is able to positively affect the activity of other markers of inflammation and oxidative stress not only in the liver and spleen but also in other organs ( lung , brain , etc . ) , a hypothesis to be tested by future work . however , to establish the optimal dosing in light of the effects achieved is of primary importance .

rheumatoid arthritis ( ra ) is a chronic inflammatory disease , leading to progressive destruction of joints and extra - articular tissues , including organs such as liver and spleen . the purpose of this study was to compare the effects of a potential immunomodulator , natural polyphenol n - feruloylserotonin ( n - f-5ht ) , with methotrexate ( mtx ) , the standard in ra therapy , in the chronic phase of adjuvant - induced arthritis ( aa ) in male lewis rats . the experiment included healthy controls ( co ) , arthritic animals ( aa ) , aa given n - f-5ht ( aa - n - f-5ht ) , and aa given mtx ( aa - mtx ) . n - f-5ht did not affect the body weight change and clinical parameters until the 14th experimental day . its positive effect was rising during the 28-day experiment , indicating a delayed onset of n - f-5ht action . administration of either n - f-5ht or mtx caused reduction of inflammation measured as the level of crp in plasma and the activity of lox in the liver . mrna transcription of tnf- and inos in the liver was significantly attenuated in both mtx and n - f-5ht treated groups of arthritic rats . interestingly , in contrast to mtx , n - f-5ht significantly lowered the level of il-1 in plasma and il-1 mrna expression in the liver and spleen of arthritic rats . this speaks for future investigations of n - f-5ht as an agent in the treatment of ra in combination therapy with mtx .

a recent institute of medicine ( iom ) report on this subject acknowledges that the rates and impact of medication errors are huge but are poorly understood . the president of the institute of safe medication practices ( ismp ) , michael cohen , in his testimony to a committee of the us congress estimated that the dollar cost of adverse drug events was about $ 200 billion across all settings . in ambulatory settings , medication errors and adverse drug events ( ades ) are one of the most important safety issues . a study based on the national ambulatory medical care survey ( namcs ) found that office - based physicians prescribed at least 1 inappropriate medication to nearly 8% of the elderly who received prescriptions . another study of ambulatory elderly patients with polymorbidity and associated polypharmacy documented that 35% reported experiencing at least one ade within the previous year . gurwitz and colleagues have estimated ( by extrapolation ) that medicare enrollees alone suffer approximately 500,000 preventable ades per year . a 2003 report of a multidisciplinary group ( composed of the ahrq - supported medical group management association center for research , the centers for medicare and medicaid services , and the partnership for patient safety ) draws attention to the fact that while safety risks are widespread in ambulatory settings , there has been insufficient attention paid to them . some estimates suggest that ambulatory settings are at least equally important as inpatient settings , with up to 200,000 avoidable deaths annually in the united states of america ( usa ) alone [ 7 , 8 ] . lack of awareness of the type , the incidence , and consequences of errors in any setting is one of the most important barriers to reducing these errors and improving quality of care . the most commonly used method for estimating vulnerabilities in healthcare is to retrospectively collect and count errors through voluntary reporting systems ( often referred to as these are fraught with difficulty due to underreporting ( according to iom 's 1999 report , only 5% of known errors are typically reported and then there are unknown errors ) and abuse ( e.g. , reports filed and counterfiled as a means of retaliation against colleagues ) . error reporting often does not promote understanding of the organizational structure and processes of care . instead it tends to be associated with blame and shame , and frequently results in antagonism between team members undermining mutual respect , trust , and cooperation . the patient safety and quality improvement act ( 2005 ) was introduced in large part to stimulate increased error reporting through the creation of patient safety organizations ( psos ) . bates and colleagues have described difficulties involved in defining and quantifying errors ; they report that even direct observational studies , which are highly labor intensive , often miss errors . an alternative approach that is prospective , rather than retrospective , and encourages involvement of all teammembers for identifying and prioritizing safety and quality problems invokes failure modes and effects analysis ( fmea ) . this has been widely used in other high - risk industries and has been advocated by the iom as a means of analyzing a system to identify its weaknesses ( failure modes ) , possible consequences of failure ( effects ) , and to prioritize areas for improvement . we have adapted and tailored this methodology to allow for the levels of resources and expertise available in ambulatory settings , and developed an instrument that has been shown to be effective in a variety of these settings . the details of the rationale and processes behind this instrument termed safety enhancement and monitoring instrument that is patient centered have done , that a vital step toward creating the medical home is to close the physician staff divide so as to maintain communication and coordination . they draw attention to the fact that currently practice meetings are universally unpopular despite their indispensability . our trm methodology , invoking the paradigm of complex adaptive systems , is designed to aid formation of central attractors in the form of self - empowered effective learning teams with a common vision to help their complex microsystems to adapt and thrive [ 1721 ] ; thriving systems are endowed with simple rules , shared vision , and opportunities for team members to innovate . an outcome - oriented team has to be enabled to ( a ) own and identify vulnerabilities in their settings , ( b ) design and implement interventions tailored for its settings , ( c ) monitor the efficacy of these interventions , and ( d ) continue the never ending journey in pursuit of safe care , that is , continuing quality improvement . interestingly , a recent study by quinn et al . found that physicians from practices that were involved in evaluation of quality improvement activities had significantly less isolation , stress , and dissatisfaction . objectivethe primary objective of this study was to evaluate the impact of the prospective team resource management methodology ( trm ) , based on the semi - p instrument , ( with and without the use of a practice enhancement associate ( pea ) ) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades . the primary objective of this study was to evaluate the impact of the prospective team resource management methodology ( trm ) , based on the semi - p instrument , ( with and without the use of a practice enhancement associate ( pea ) ) onthe number of preventable ades in a vulnerable population aged 65 and above;the severity of these preventable ades . the number of preventable ades in a vulnerable population aged 65 and above ; the severity of these preventable ades . this was a cluster randomized trial in which 12 practices in the upstate new york practice - based research network were each randomized to one of 3 states ( 4 practices each ) : ( 1 ) team resource management intervention ; ( 2 ) team resource management intervention with pea ; ( 3 ) no intervention ( comparison group ) . these can be repeated to make a cycle , as shown in figure 1 , for continuous quality and safety improvement . this anonymous survey is an opportunity for all staff to freely express opinions about the care processes in their setting . this survey asks about each of the steps in the medication use process . to help orient staff to the overall process , the survey uses a diagram ( figure 2 ) that shows who and what is involved in the processes and how they work ( or are supposed to work ) together . each page of the survey is about a different area and consists of a list of errors or causes of error that can occur in that area . the lists are based on review of the literature and consultation with practicing physicians and nursing leaders . the survey asks staff to think about each of the errors in turn and , for each , to indicate their opinion about how often it occurs and , when it does happen , how severe the consequences usually are . the diagram of the testing process is included on each page , with red highlighting to show which part of the process is being asked about . if willing , they can mark their job category ( provider , nursing , or administrative ) . scores ( called hazard scores obtained by multiplying frequency of each error with its respective severity of consequence ) are calculated for each error in the survey , based on respondents ' answers to the frequency and severity questions . the items are then listed in rank order ( highest to lowest ) and presented to staff in a graphical format in a group meeting for their discussion . this helps the team to form a common vision and consensus regarding which problems need to be addressed first . in further group meetings , staff discuss the prioritized problems and work together to design solutions , keeping in mind the resources available and the capabilities of their unique setting . teams are formed to implement the solutions , with clear allocation of responsibilities and an agreed time schedule . she had completed a master 's degree in nutrition but with no prior experience working with practices . she participated in all study - related team meetings at each practice and made herself available to each practice for up to half a day a week throughout the study period to support safety and quality improvement activities ( not limited to study - related activities ) . the main contribution of the pea was to follow through on plans developed by the practice team , that is to support stages 3 and 4 in the trm cycle outlined above . examples of pea activities include developing patient education materials , preparing draft protocols , and collecting resources for patient prescription assistance . the rate and severity of ades and preventable ades were measured by chart review for the two 12-month periods before and after the start of the intervention . eligible patients were those aged 65 years and above , who had at least one visit for cardiovascular disease ( icd-9-cm codes 390459 ) during the measurement period and at least one visit for any diagnosis in the prior year . if there were 200 or fewer eligible charts at a site , all were screened , otherwise a random sample of 200 was taken . the chart review was performed using a trigger tool method that involves 2 steps , namely a screening step and a review step [ 5 , 2332 ] . the screening step involves identifying the presence of certain chart findings , known as triggers , that are known to be possible evidence of an adverse event . in this study , the triggers of interest are those that might represent an adverse drug event ( ade ) . examples include an elevated inr ( often associated with adverse effects of warfarin ) and abrupt discontinuation of a medication ( that sometimes occurs because of an ade ) . in our study research assistants performed the screening step , using a previously published ade trigger tool that we adapted from the work of others [ 5 , 25 , 26 ] . in the second step , known as the review step , a physician and pharmacist reviewed the identified triggers to determine for each trigger : ( 1 ) whether an ade took place and if so , ( 2 ) whether the event was preventable , ( 3 ) the stage of the medication use process where the ade originated ( prescribing , dispensing , administration , and monitoring ) , and ( 4 ) the effect on the patient ( none / minimal , mild , moderate , or severe ) . examples of preventable errors include missed allergy , wrong dosage , errors of dispensing , administration errors , and failure to order or complete laboratory monitoring . the physician and pharmacist worked together , reviewing , discussing , and recording their consensus opinion of each trigger . when they were unable to reach consensus a final determination was made by a third reviewer . if they identified any potential or actual harm that had not been previously recognized and addressed , they notified the primary care physician or site medical director . the study protocol was approved by the social and behavioral sciences institutional review board of the state university of new york at buffalo . part way into the study , one of the comparison group practices withdrew from the study , citing concerns over the administrative burden of the chart reviews ( the practice was undergoing administrative changes ) . table 1 shows the characteristics of the 11 sites that completed the study . at baseline , of these , 1066 ( 54.1% ) had triggers , yielding a total of 2898 triggers . this far exceeded our expectation and therefore posed a practical problem due to the effort ( and therefore cost ) associated with reviewing this number of triggers . therefore , we elected to reduce the sample by randomly eliminating a proportion of patients at each site , sufficient to reduce the number to approximately 100 patients per site . this yielded a total of 1125 patients , of whom 598 patients had one or more triggers , all of which underwent review . a similar process was used to make the endpoint data manageable , yielding 1050 patients , of whom 564 had triggers that were reviewed . table 2 summarizes the rates of preventable ades ( normalized per 100 patient - years ) for each arm of the study . for each arm , we compared the pade rate ( per 100 patient - years ) at the two time points ( after versus before ) by means of a paired t test with sites as the unit of analysis . as shown in table 2 , in the intervention with pea group there was a statistically significant decrease in the overall rate of pades after the intervention compared to before ( 7.4 per 100 patient - years versus 12.6 , p = 0.018 ) and in the rate of moderate or severe ( combined ) pades(1.6 versus 6.4 , p = 0.035 ) . in the comparison arm and the unaided intervention arms , analysis of variance ( anova ) with study arm and time as the factors and total pades as the outcome variable showed no significant interaction between arm and time . for the outcome of moderate or severe pades , the interaction term was significant ( p = 0.023 ) supporting the notion that the intervention with pea practices had a greater reduction in moderate / severe pades over time than the comparison group . the trm approach , when aided by a pea , demonstrated a significant reduction in pades , especially those with the highest severity . the reduction in pades represents a significant improvement in patient safety through the collective efforts of practice staff guided by a structured trm process . it appears that the additional resource offered in the form of a pea was important , as the non - pea practices did not achieve the same statistically significant improvement that the pea practices did . while the mechanism of action of the pea is not clearly understood , it is reasonable to surmise that the extra human resource represented by the pea enabled practices to more effectively implement their planned interventions . the addition of a pea may have helped to minimize the incremental burden on teams that are already overburdened . while we did not formally evaluate this , it is the authors ' observation that the humanistic self - empowerment approach used in this study helped to energize front - line workers to maintain and continually improve quality . staff commented that seeing other people 's perspectives ( as reflected in semi - p results ) helped to improve mutual understanding , leading to consensus . further , we believe that by closing the physician staff divide , and encouraging closer communication and coordination between team members in addressing practice issues , the trm approach can achieve progress toward the creation of the medical home . firstly , the outcome measure is based on a trigger tool methodology that has limited sensitivity . in fact , the sensitivity of the trigger tool is unknown because there is no gold standard against which to compare it . this means that the rates of pades reported should not be seen as estimates of true rates . instead , they represent only a subset of pades , that is , those that are identifiable by the trigger tool . however there is no reason to expect that the sensitivity of the trigger tool would vary between study groups or over time so the validity of the study findings is not jeopardized . another significant weakness is the small size of the study . having only 3 - 4 practices in each arm limited the power . in addition , further exploration of the role of the pea is required to establish the most important active ingredients so that these can be deployed in an efficient way .

background . most safety issues in primary care arise from adverse drug events . team resource management intervention was developed to identify systemic safety issues to design and implement interventions to address prioritized issues . objectives . evaluate impact of intervention on rates of events and preventable events in a vulnerable population . design . cluster randomized trial . 12 practices randomly assigned to either : ( 1 ) intervention ; ( 2 ) intervention with practice enhancement assistants ; ( 3 ) no intervention . the intervention took 12 months . main outcome measure . rate and severity of events and preventable events measured using a trigger tool chart review method for the 12-month periods before and after the start of the intervention . results . in the intervention with assistants group there was a statistically significant decrease in the overall rate of events and in the rate of moderate / severe events . analysis of variance with study arm and time as the factors and moderate / severe events as the outcome showed a significant interaction between arm and time supporting the notion that the intervention with assistants practices had a greater reduction in moderate / severe preventable events . conclusions . the intervention had a significant effect on medication safety as estimated using a trigger tool . further exploration of role of assistants and trigger tool is warranted .

in 2007 , the world health organization ( who ) launched its initiative for the global elimination of congenital syphilis , with the goal that by 2015 at least 90% of pregnant women are tested for syphilis and at least 90% of seropositive pregnant women receive adequate treatment . subsequently in september 2010 , paho member states approved a strategy and plan of action for the elimination of mother - to - child transmission ( mtct ) of hiv and congenital syphilis by resolution 50/12 at the 50th directing council meeting [ 2 , 3 ] . one of the three targets set was reduce the incidence of congenital syphilis to 0.5 cases or less per 1,000 live births by 2015 . the caribbean community ( caricom ) health ministers endorsed this initiative . in adults syphilis is a complex sexually transmitted disease that has a highly variable clinical course . clinically the primary lesion ( chancre ) presents mainly as an anogenital ulcer that appears 990 days after exposure , although extra - anogenital sites such as the lip , tongue , and tonsils from oral sex and kissing , nipple from kissing or wet nursing of infected babies , and finger with minor abrasion from touching infectious lesions can occur . secondary syphilis presents with generalised rash affecting the palms and soles , generalised lymphadenopathy , and orogenital mucosal lesions , including snail tract ulcers and condylomata lata . gummatous syphilis ( sometimes known as benign tertiary syphilis ) can involve organs or supporting structures and can result in infiltrative or destructive lesions leading to granulomatous lesions or ulcers ( e.g. , skin ) or perforation / collapse of structure ( e.g. , palate and nasal septum ) or organomegaly . late neurosyphilis can cause meningovascular syphilis leading to stroke syndromes and parenchymal involvement leading to general paresis and tabes dorsalis . cardiovascular syphilis involves the aortic arch , which can lead to angina from coronary ostitis , aortic incompetence , and aortic aneurysm . there is sparse data reflecting the true occurrence of congenital syphilis ( cs ) in trinidad . in 1990 ali reported that during the period from january 1985 to december 1988 , 28 cases of cs were diagnosed shortly after birth at one major hospital in trinidad with an average annual incidence of 1.5 per 1000 live births , seen at that particular institution . subsequently paho reported 45 cases of congenital syphilis in 2009 for the entire country , a rate of 2.3 per 1000 live births ; both pieces of evidence suggest that cs remains a public health challenge . diagnosis and prevention of mother - to - child transmission of syphilis ( mtct ) are feasible , inexpensive , and cost - effective in nearly every situation evaluated . in fact congenital syphilis is preventable if a single dose of 2.4 million units of benzathine penicillin g is given to a pregnant woman with the infection during the first two trimesters of pregnancy [ 911 ] . the challenge is to find such women . in order to monitor progress towards the goals of the elimination initiative in the region , paho requested that member countries gather information on several key programmatic elements . in trinidad there are two tier systems of health care both operating independently of each other , a public health care system ( phcs ) , and a private health care system ( prhcs ) . the phcs is funded by the ministry of health ( moh ) and therefore all services are free as opposed to the prhcs which is based on a fee for service model . the largest provider for the diagnosis and treatment of sexually transmitted infections is the moh through a dedicated vertical program delivered at the queen 's park counselling centre ( qpcc ) . the laboratory services delivered by this program extend to prhcs and therefore offer a reliable source of data from which syphilis occurrence in trinidad and can be estimated . the aim of this study therefore is to describe the current status of acquired syphilis in trinidad , trends over the first decade of the 21st century , and a spatial analysis of syphilis cases , to inform strategies for achieving the elimination of syphilis . the qpcc is a repository for all clinical and laboratory data for syphilis for the entire population of 1.3 million . thus all clinical cases and laboratory investigations associated with a diagnosis of syphilis or cs were eligible for entry into the study . we defined syphilis and cs using the cdc surveillance case definitions , which include clinical features as well as a laboratory diagnosis . in particular for syphilis clinical features included one or more of the following : chancres consistent with primary syphilis , localized or diffuse mucocutaneous lesions often associated with generalized lymphadenopathy and , a reactive serologic test ( nontreponemal : venereal disease research laboratory ( vdrl ) or treponemal ( fluorescent treponemal antibody absorbed ) ) . similarly for cs the clinical features occur in an infant or child such as hepatosplenomegaly , rash , condyloma lata , snuffles , jaundice ( nonviral hepatitis ) , pseudoparalysis , anaemia , or oedema ( nephrotic syndrome and/or malnutrition ) and stigmata ( e.g. , interstitial keratitis , nerve deafness , anterior bowing of shins , frontal bossing , mulberry molars , hutchinson teeth , saddle nose , rhagades , or clutton joints ) in older children . this was followed by laboratory confirmation using the treponema pallidum particle agglutination assay ( tppa ) . the results of all serum samples tested by the laboratory for vdrl between 2009 and 2012 were reviewed . vdrl positive samples using standard methods and quantitative vdrl testing were eligible for entry into the study . a positive vdrl supported by a positive confirmatory test was used to tract the clinical records to ensure that a case of syphilis met the case definitions . from the clinical records , data on age , gender , ethnicity , and address ( without street number ) we also used secondary data published by the moh for the period 19942009 to demonstrate trends . this data reports only the recorded number of cases for each calendar year and published by the moh in the annual reports of the moh . the street addresses of established syphilis cases diagnosed , between 01/01/2010 and 31/12/2012 only , were collected and mapped using arcmap 10.0 gis software ( esri , redlands , ca , usa ) . the geocoded addresses were transformed into a density map , using the spatial analyst kernel density tool areas with the highest density of syphilis ( defined as areas with greater than ten cases per square mile over the three - year period ) and were designated as hot spots . in addition we calculated a standardized incidence ratio ( sir ) with 95% confidence intervals ( ci ) using the approach by vandenbroucke . all data was stored , retrieved , and analysed under strict confidential cover using spss versus 18 . data published by the ministry of health ( moh ) for the period 20042009 revealed a decline in aggregate cases of syphilis during this period ( figure 1 ) . this decline was observed in both males and females . since there were no published data by the moh for 20102012 we reviewed the laboratory records at the qpcc to determine the number of laboratory confirmed cases for the period ( 20102012 ) . in addition in order to validate the most recent data published by the moh , that is , 2009 , we also reviewed the clinical and laboratory records at the qpcc for 2009 . we found the number of cases of syphilis for 2009 was 507 and not 130 as reported by the moh . overall the number of cases of syphilis for the period 200912 averaged 428 , with cumulative incidence rates varying between 39 and 29 per 100 000 population for this period . there were 64 confirmed cases , 37 ( 57.8% ) males and 27 ( 42.2% ) females . more than half ( 14 , 52% ) of the females who tested positive came from samples received from prenatal clinics . for the first time data were collected on the sexual orientation of the positive cases . there were more cases of syphilis among heterosexuals ( 47 , 73.4% ) than homosexuals ( 11 , 17.2% ) or bisexuals ( 6 , 9.4% ) . these numbers should be interpreted cautiously since it is reasonable to assume ( although unknown ) that the heterosexual population is much larger than bisexuals or homosexuals . in addition risk taking behaviour , attitudes , and practices among these groups are unknown . using only data reported by moh in fact it appears that in 2009 trinidad had almost reached the target of 0.5 cases per 1000 live births . three hot spots were identified ( beetham gardens , san juan / laventille , and arima ) using the kernel method ( figure 3 ) . arima has the second highest population density of 2890 km on the island ; both beetham gardens and san juan / laventille also have high population densities of > 1500 km . hence all the areas identified as hot spots were urban and densely populated . the major finding of the study was the high levels of occurrence of syphilis still occurring in parts of trinidad . in fact between 2009 and 2012 the average number of cases per annum was 428 with an incidence rate as high as 39 per 100 000 population . the highest rates of syphilis were predominantly found in urban men and women in their sexually most active years , that is , between the ages of 15 and 35 . on average , women become infected at a younger age than men . consequently , the prevalence of active syphilis is higher among young women than their male peers , a finding that has also been observed for hiv , herpes simplex virus type 2 ( hsv-2 ) , and other stds [ 1517 ] . however in developed countries such as the usa , for instance , there were 46 042 cases of syphilis in 2011 and the rate of primary and secondary syphilis increased by 3.8% among men ( from 7.9 to 8.2 cases per 100,000 men ) during 2010 - 2011 [ 18 , 19 ] . on the other hand , the rate decreased to 9.1% among women ( from 1.1 to 1.0 cases per 100,000 women ) , while in western europe rates have fell below 5 per 100 000 in the majority of countries [ 2123 ] . the implication of this finding is clearly that young women are at high risk , and this is of special concern as a high proportion of women become pregnant or commence childbearing before reaching 20 years of age , thus sustaining prenatal transmission and congenital syphilis . these findings support the implementation of educational programs among adolescents to encourage safer sexual behavior and improved treatment seeking behaviour . based on this evidence we advocate for adolescent sexual health education programs to be implemented in the school curriculum . another important finding of the study is the large discrepancy in data reported by the moh for 2009 , that is , 130 reported cases versus 507 cases actually collected from a review of laboratory data . national health data are required for planning and evaluation of service delivery [ 2426 ] . this planning and evaluation is critical in developing countries where the majority of health services are provided through national programs and the limited funds must be used efficiently and effectively [ 24 , 25 , 27 ] . in these settings , high data quality is important to ensure that decisions reflect program needs and direct health professional education priorities [ 2528 ] . poor data quality not only contributes to poor decisions and loss of confidence in the systems but also threatens the validity of impact evaluation studies . while the study did not focus on the reasons for this discrepancy , several studies have reported inconsistencies in data reporting as well as poor support mechanisms to ensure data quality as it traverses several levels of governmental bureaucracy [ 3032 ] . for example a study done in nepal found that data obtained from the facility registers were lower than the data reported at the district level , showing a tendency of overreporting to the higher levels . other studies showed that errors in reporting were due to lack of supervision and feedback from the superior levels as well as inadequate incentives to health workers [ 3032 ] . the quality of data , and consequently of the information system , must be seen in a broader perspective focusing not only on technicalities ( data collection tools and the reporting system ) but also on support mechanisms . another important finding was the widening gap in the occurrence of syphilis among men and women . historically more men than women contracted the disease but by 1997 - 98 women began exceeding men and a decade latter this gap was the largest . this finding has two implications ; first it is more likely for the disease to be sustained and propagated if women continue to be harbouring the infectious agent and are agents for transmission and secondly the risk of cs remains . while significant gains have been made with congenital syphilis , as of 2009 we are still above the targeted incidence of congenital syphilis , that is , 0.5 cases/1,000 live births . three hot spots were identified using spatial analysis techniques , as well as a sir of 1.6 . the sir is generally calculated to provide an estimate of the likelihood that an excess of cases exists in the population of concern ( hot spots ) compared to the general or reference population . the measure is typically interpreted as an excess number of cases in this instance as much as 60% . there were several limitations to the study mainly the unavailability and difficulty to retrieve all data , which may hinder the exact burden of the disease . in addition because case definitions for congenital syphilis vary widely by country , measuring the attainment of the specific targets of the global initiative may be difficult . in a young child , the possibility of sexual abuse should be considered as a cause of acquired rather than congenital syphilis , depending on the clinical picture ; this data could not be determined . although congenital syphilis includes cases of syphilitic stillbirths we were unaware of the existence of such cases and hence it is not included in the study . in conclusion the occurrence of syphilis in trinidad continues to be high ; this is against a background in which syphilis screening and treatment can cost less than us $ 1 per syphilis test and us $ 0.50 per penicillin dose , and health economists estimate that this is among the most cost - effective public health interventions in existence . in addition elimination of congenital syphilis as a public health problem by 2015 is an important and attainable component of global efforts to achieve the millennium development goals ( mdgs ) 4 ( reduce child mortality ) , 5 ( improve maternal health ) , and 6 ( combat hiv / aids , malaria , and other diseases ) .

objective . to describe the current epidemiological features of syphilis and congenital syphilis in trinidad , 20092012 . methods . all laboratory confirmed syphilis cases diagnosed through a vertical program in the ministry of health , between 1/1/2009 and 31/12/2012 , were identified . all relevant data were collected including address which was geocoded and mapped using arcgis 10.0 ( esri ) . both spatial techniques and standardized incidence ratios were used to determine hot spots . results . the annual cumulative incidence rate for syphilis remains high varying from 39 per 100 000 population in 2009 to 29 per 100 000 in 2012 . we identified 3 hot spots , in urban areas of trinidad . young men and particularly young women in childbearing age 1535 living in urban high density populations were commonly infected groups . conclusion . the incidence of syphilis continues to be very high in trinidad . new initiatives will have to be formulated in order to attain the global initiative to eradicate syphilis by 2015 .

they say passing a kidney stone is the worst pain you could ever have . i still feel it now , intense as ever , 10 years later . dan vento is talking about the time his daughter suffered a relapse of an uncommon form of cancer called osteosarcoma . but a year later , jennifer s left knee began to swell and hurt again , then her thigh , then her back . the cancer not only had returned but was now all over jennifer s body , and it had happened so quickly that even the doctors were caught off guard . metastatic tumors invaded jennifer s bones , obstructed her airways and destroyed her vision . overnight , their little girl had become unrecognizable , from the cancer and from the aggressive treatments a swollen , lumpy mass with tubes and iv lines hooked up to beeping machines . they stood by , feeling as if the cancer were ripping through their bones too . it was almost a relief at first ; the ventos could nt bear watching their daughter suffer another day . but when they saw jennifer in the little coffin , when they saw the coffin lowered into the ground , when they saw the earth covering the coffin from that day on , their pain would never end . i tried to be strong for my wife and kids , dan vento explained . if they saw me crumble , how would they be able to keep going ? but despite the strong facade , vento was crumbling . i felt weak and lightheaded all the time , like i might pass out , and needed to grab onto something though not physically imposing he was short and stocky he was a self - made man from the bronx who owned a successful chain of grocery stores and always got whatever he wanted , at work and at home . when jennifer died , however , he did nt seem to want much anymore and found it increasingly difficult to concentrate . is what dan vento experienced as he watched his daughter suffer , when he buried her , and now , 10 years later , still entangled in grief pain ? he certainly thinks so and uses the word just as he did when he spent an agonizing night in the er with kidney stones . so do many other people who undergo similar trauma , as well as those who suffer pain in psychiatric illnesses like depression and schizophrenia . scientists who study pain and doctors who treat pain consider the experience a strictly physical phenomenon , in the sense that it can only be caused by injury to the body . pain occurs when receptors on nerve cells in the skin and internal organs detect potentially damaging stimuli , a pinrick , for example , or high temperatures ( melzack and wall 1983 , pp . the nociceptors ( from the latin nocere , to injure ) then signal the brain , which assesses the threat and coordinates a series of protective responses . this highly effective biological warning system that prevents further damage and aids in healing is something we ca nt live very well without . just think of patients who are unable to feel pain , those with genetic defects and those with diseases that affect nerve transmission like diabetes and leprosy ; the benefits of life without pain are easily outweighed by the negatives of progressive injury to the body and premature death ( see brand and yancey 1997 ) . dan vento has suffered no physical injury . nor have patients who experience the psychic pain that accompanies acute depression . nor have cancer patients ( and their parents ) who experience the overwhelming fear and anxiety and isolation that accompany the physical symptoms of their illnesses . their nociceptors , at least with respect to these particular feelings , remain silent , sending no distress signal to the brain . therefore , their feelings are not really pain but something categorically different , what the professionals prefer to call suffering or anguish ( cassell 1991 , pp . 3046 ) . and therefore , one will find no mention of grief or depression in medical classification schemes of pain . even psychiatrists are wary of speaking about pain in their patients , reserving it only for those rare and strange cases of psychogenic pain or somatoform pain disorder that is , physical - like pain localized to a part of the body that has not been injured , the modern - day equivalent to what freud termed hysteria or conversion reaction ( dsm 3 , rev . ; american psychiatric association 1987 ) . the bottom line is that the psychological pain experienced by dan vento and millions of patients with acute depression is an oxymoron or , at best , a metaphor . how can there be such a gulf between the layperson and the expert , especially with regard to such a common part of life ? and if the experts are right , how could ordinary people like dan vento as well as our language professionals celebrated writers like william styron and joan didion , for example , who wrote so eloquently about pain in depression ( darkness visible ) and grief ( the year of magical thinking)have gotten things so wrong ? unless of course they havent . unless it s not the layperson but the expert who is confused . perhaps one s instinctive tendency to see pain more broadly , as a category that incorporates both physical and psychological varieties , may be more enlightened than the expert s narrower conception . perhaps there are good reasons for speaking of pain in the setting of grief or depression or schizophrenia or divorce or the nonphysical suffering that accompanies illness . in the first place , there is a wealth of subjective evidence what people feel and think and then convey to others through language . when we ask people about certain aversive emotional experiences and listen to their words , we find that they not only use the generic word pain to label these experiences , but also describe them in the same ways they describe physical pain . now pain of any kind is notoriously difficult to express . there are problems conceptualizing the experience because it is perceptually inaccessible ( we ca nt see or touch pain ) and because , unlike other inner states , it is not always linked to external objects that we can see or touch ( like the person who makes us angry or the dog that makes us scared ) ( scarry 1985 , pp . one is forced to think about pain indirectly , through metaphor : we imagine a more knowable object linked to the pain and then speak of the experience in terms of that object . by far the most common metaphor used to describe physical pain is the weapon ( scarry 1985 , pp . lengthy lists of similar adjectives can be found on the mcgill pain questionnaire , created in the 1970s to help patients communicate their feelings to doctors . pain can be described as piercing , drilling , burning , grinding , throbbing , stinging , squeezing , and so on . each of the descriptors implies the presence of a weapon or weapon - like object that can injure the body the drill that drills , the fire that burns . and since most patients have never been stabbed or shot or are not being stabbed or shot at the moment of pain , they are using these terms figuratively to objectify what would otherwise be difficult to pin down and represent ; now they could see pain and describe how it feels by talking about knives and guns and the damage they can do the body . dan vento , silenced for so long by the incapacitating pain of loss , will eventually open up to a psychiatrist . it felt like a bomb , he explained , that exploded inside of him , obliterating everything in his body . at other times , he felt the damage was occurring more slowly and methodically , as if there were a swarm of parasites eating away at his organs . but either way , the result was the same for vento : he was being emptied out from the insidegutted was the word he used until all that was left was a big , raw gaping wound . when her husband died and she was flooded with grief , joan didion saw giant waves . in her memoir , she writes that she felt as if she were being battered by destructive waves , paroxysms , sudden apprehensions that weaken the knees and blind the eyes and obliterate the dailiness of life ( didion 2005 , pp . 2728 ) . for kay redfield jamison , a psychiatrist who suffers from manic depression , it spins around her mind faster and faster , out of control , until it explodes , splattering blood everywhere ( jamison 1996 , p. 80 ) . listening to the language of pain of all kinds , we discover a shared felt structure that the weapon metaphor effectively captures ( biro 2010 , pp . whether triggered by grief and depression or kidney stones and spinal injury , pain reads like a story in three parts:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{weapon } } \to { \text { injury } } \to { \text { withdrawal}}. $ $ \end{document } in pain we feel as if there must be some weapon - like object ( bomb , swarm of parasites , giant wave , centrifuge ) that is moving toward and threatening us ; that when it strikes , it will injure , possibly even destroy us ; and that we must get away from it or shield ourselves at all costs . even when there is nothing coming at us , when there is no injury , when we remain motionless , we feel the movement , the injury and the desire to run . whatever happens that makes us feel these things the loss of a loved one or the physical destruction of cancer we experience pain . the subjective evidence for the existence of emotional pain is compelling , especially since there is no objective way to verify and characterize someone else s pain . although we can attach a person to a functional magnetic resonance imaging ( fmri ) device , observe the blood flow to pain centers in the brain and then infer its presence , the only definitive test is a person s word : i feel pain or i do nt . actually , most experts grudgingly acknowledge the inescapably subjective nature of pain . in an addendum to their universally accepted definition of painan unpleasant sensory and emotional experience associated with actual or potential tissue damagethe international association for the study of pain ( iasp 2007 ) concedes that people do report pain for strictly psychological reasons and that , since such reports ca nt be distinguished from instances where there is a physical cause , they should be taken at face value : if people regard their experience as pain and if they report it in the same ways as pain caused by tissue damage , it should be accepted as pain . but despite the concession , the iasp does not make room for the pain experienced by dan vento , joan didion , or kay redfield jamison on their extensive classification schemes of pain disorders . while complex regional pain which affects somewhere of the order of 626 people in 100,000 ( de mos et al . 2007)appears on the list , the vastly more common pain occurring in grief or depression does not . for physicians and scientists that will only pay lip service to the subjective argument , however , there is now mounting objective evidence for broadening our notion of pain . since the introduction of gate control theory in the 1960s , the link between tissue damage and pain has progressively weakened . we now have a better understanding why there can be severe injury and no pain ( wounded soldiers in battle ) and , conversely , no injury and severe pain ( migraine , fibromyalgia ) . this happens , as prominent pain scientists ronald melzack and patrick wall have explained , because there are psychological factors one s culture and past experiences , our emotional and cognitive states , the context of pain that can intensify or dampen the nociceptor signal before it registers in higher brain centers ( melzack and wall 1983 , pp . moreover , many cases of chronic pain seem to occur without any direct nociceptor stimulation at all . neuropathic pain results when a dysfunctional nervous system fires spontaneously or misinterprets ordinary sensory stimuli as noxious ( woolf and mannion 1998 ) . in tic dolouroux , for example , the movement of a feather across the face can trigger spasms of intense pain . a second strand of evidence comes from our growing understanding of how the brain processes pain . most important for this discussion , there are distinct areas in the brain that process the sensation of pain ( its quality , location , intensity ) and our feelings about the sensation ( the narrative of its aversiveness ) ( price 2000 ) . further , the sensory center ( in the somatosensory cortex ) and the affective center ( in the anterior cingulate and insula cortices ) are not only spatially apart but dissociable : that is , a person can have the sensation of pain but not feel pain ( grahek 2007 , pp . we can observe this in patients undergoing minor surgery with medication that makes them indifferent to being cut with a scalpel . even more dramatic is a rare group of patients whose affective pain centers ( or the connections to those centers ) have been destroyed . in the case of pain asymbolia , patients can still sense a needle prick ( because the nociceptor signal registers in the somatosensory cortex ) but will laugh at its insignificance ( because the signal is not processed by the anterior cingulate cortex ) . these instances of disconnect between the sensation and the feeling of pain tell us that despite the complexity of pain which involves sensations and behavior , feeling , cognition and memory the critical component is feeling . if we do nt have the feelings that dan vento had when the kidney stone was passing through his ureter that something bad was happening to him , that that something was damaging his body , and that he must do whatever he could to avoid further damage then pain loses its biological value . because they laugh at pain rather than run from it , pain asymbolia patients will likely fare no better than patients with congenital or acquired pain insensitivity . in fact , i would argue that if we do nt feel pain , there s no point using the term at all . leprosy patients , soldiers on the battlefield , sedated patients undergoing surgery , pain asymbolia patients they may experience unpleasant sensations but they do nt feel pain and do nt take protective measures . everything is contingent on the feeling of pain . if tissue damage is not necessary to feeling pain and if there is a special affective center in the brain devoted to such feeling , why ca nt that center be activated by means other than the nociceptor pathway ? why is nt it possible that noxious psychological stimuli stimuli that threaten the emotional well - being of a person , like the loss of a child or the pain of depression or the suffering of cancer patients find their way to the anterior cingulate gyrus , making us feel the same way we do when we experience physical pain ? naomi eisenberger and her colleagues at ucla have recently developed a clever model of psychological pain that can be studied objectively ( eisenberger et al . normal subjects played a video ball - tossing game while their brains were monitored by fmri . when the subjects were excluded from the virtual game , they experienced distress that correlated with increased blood flow to the anterior cingular and insular cortices , exactly the same pattern that would have occurred had they been stuck by a needle . the greater the social distress generated , the more active these affective pain centers became . studies done on saddened and grieving subjects produced similar results ( gundel et al . 2003 ) . it appears that the layperson s intuition about pain is being borne out by science ; psychological pain seems to run on the same neural tracks as physical pain . and why should nt it ? just as physical stimuli that can damage our bodies prompt certain feelings and responses , so too should psychological stimuli that can damage our psyche like the loss of a child or the intrinsic symptoms of depression . just as we need to rest the body to protect ourselves from further harm , so too should we protect the mind . is this just a semantic issue , a disagreement between two sets of language users that , in the end , does nt have any adverse consequences ? it matters because the disagreement reflects a much larger issue : the rigid mindset of the scientific community , which sees the world in a certain way and wo nt allow for deviation , even from dissenters within its own ranks . science focuses its spotlight exclusively on the objective world , what can be studied , quantified , and explained . because it seems resistant to such inquiry , the subjective realm has been traditionally off limits , something that can only be appreciated on much looser terms by the humanities and the because of their position in the intellectual hierarchy , there is a trickle - down effect , which carries over to the practical science of medicine and to the culture at large . in the case of pain , there is only one kind , the real or physical kind that can be objectively verified by observing nociceptor activity or finding lesions on a cat scan . other experiences that may feel like pain but can not be linked to tissue damage are not pain . much more subjective and less transparently material , they are therefore derivative , less important , and better labeled something else ( suffering or anguish ) . while psychological pain may be unpleasant , the fact remains that it is in our heads , not our bodies . as we continue to unfold the logic of the objectivist ( and dualistic ) paradigm which has now thoroughly permeated our cultural consciousness those who suffer without any physical corroboration to show for it inevitably begin to appear suspect . they are either crazy ( mentally ill ) , deceitful ( because there is no real pain ) or weak ( everything is painful to such people ) . they do nt need pain doctors or pain medication , but psychiatrists and priests . disregarding for a moment that that all pain is in the headeven dan vento s kidney stone pain , which he localized to the right side of his pelvis the truth is that psychological pain is often more intense and dangerous than the real thing . for dan vento , his bout with kidney stones , among the most painful of all medical conditions , was nothing compared to the pain of grief . similarly , lucy grealy tells us in her memoir , autobiography of a face ( 1995 ) , that she would much rather face the pain of cancer and its treatments than the far worse pain of feeling deformed and lonely ( pp . 7 , 170 , 186 ) . in fact , many such sufferers welcome , even court , physical pain , feeling that it actually alleviates their emotional pain to a degree . and when , unalleviated , the pain becomes too much to bear , some will choose to end it by ending life . suicide rates are significantly higher in the setting of grief and depression than they are in the setting of physical pain ( schneidman 1998 ) . in addition to relegating psychological sufferers to second - class status , sufferers of chronic pain conditions like migraine , lower back pain , and fibromyalgia find themselves somewhere in limbo between real pain and the derivative , mental kind . on the one hand , their pain seems physical ( because it is localized to a part of the body ) , but on the other , it has more in more in common with psychological distress ( because there is no detectable injury ) . for a long time , medicine had no idea what to do with these patients , and so they drifted from doctor to doctor without finding relief . although their lives have improved with the introduction of pain specialists and pain clinics , chronic pain patients are still often tormented by the insidious logic of the objectivist perspective ( see heshusius 2009 , pp . some , in fact , resort to self - mutilation to legitimize their pain in the face of ongoing skepticism from family members and doctors : you see now , they will say , pointing to their slashed arms , the pain is not in my head , it s real ( see padfield 2003 , pp . i bring up the seriousness of psychological pain and the limbo - like situation of chronic pain conditions because , like the work of a growing number of scientists , it goes against the grain of the prevailing mindset . perhaps , then , we should change this mindset and broaden our outlook . instead of privileging one type of pain over another , let s approach them in a more inclusive , democratic spirit , in which all pains are created equal . or better yet , let s view pain as occurring on a continuum or spectrum that runs from one ideal ( pain linked solely to physical injury ) to another ( pain linked solely to psychological injury ) . it accommodates our belief that the feeling of pain can arise from injury to the body as well as injury to the mind . it also accommodates our experience of the considerable overlap between the two varieties , that there is never pure physical or pure psychological pain but always combinations . those suffering from grief and mental illness often have somatic complaints : dan vento felt the loss of his daughter in his gut ; william styron s descent into depression was accompanied by sleeping and breathing problems ( styron 1992 , pp . at the same time , patients in physical pain inevitably suffer emotionally ; cancer patients routinely feel terrified , helpless and lonely ( cherney et al . moreover , the benefits of adopting a broader perspective go beyond validating and valuing our lived experiences . there are practical implications . for science , it would mean more support for the transformative work of researchers like joseph ledoux and antonio damasio into the subjective realm of feelings and emotions ( ledoux 1996 ; damasio 1999 ) after all , these experiences are as material as the beating of the heart and the dna molecule , even though at the moment we do nt precisely know how to translate neural activity ( brain language ) into mental states ( mind language ) . now that we know it shares neurological substrates with physical pain , scientists will no doubt look to extend the work of eisenberger by finding the nociceptor pathways of psychological injury : how are feelings of grief or depression detected and transmitted to the anterior cingular cortex ? and how could the signals be modifed ? dan vento s prolonged grief what psychiatrists classify as complicated grief has much in common with certain chronic pain states . in both instances , the injury has long past and yet the reverberating pain circuit , no longer serving any biological purpose , continues . are there similar mechanisms at work here , and might they be manipulated to help vento escape from his self - destructive rut ? there would also be changes in the clinical realm , improving the way doctors treat pain . some patients may require more attention to the body ; others , to the mind ; the majority , to both . here take , for example , the placebo effect in clinical trials , in which a fake pill has been shown to relieve pain on the order of 1530% of cases . most investigators view the phenomenon as a contaminant that must be eliminated to assess the efficacy of the real drug . but why not switch frames , as benedetti ( 2009 ) has urged , and focus just as diligently on the reality of the psychological factors that are equally effective , in some cases even more so ( pp . 6 , 30 ) ? why not try to harness and enhance these factors to help patients ? this same novel way of thinking led dewall et al . ( 2010 ) to administer physical pain medication ( acetaminophen ) to people suffering from psychological hurt , and not unsurprisingly , it seemed to work . one of the greatest twentieth - century thinkers , ludwig wittgenstein , showed that paying attention to ordinary language can help advance philosophy . he also showed that clinging dogmatically to a certain picture can lead to conceptual illness ( wittgenstein 1958 , sect . 115 ) . if we can thoroughly break with our unhealthy ( and inaccurate ) dualistic legacy and truly see that mind and body are inextricably connected , then we must agree with dan vento , joan didion , and many other sufferers that psychological pain exists and is just as important and worthy of our attention as physical pain . they are two sides of the same coin and should be spoken of and treated as such .

medicine regards pain as a signal of physical injury to the body despite evidence contradicting the linkage and despite the exclusion of vast numbers of sufferers who experience psychological pain . by broadening our concept of pain and making it more inclusive , we would not only better accommodate the basic science of pain but also would recognize what is already appreciated by the layperson that pain from diverse sources , physical and psychological , share an underlying felt structure .

in 2014 , it is estimated that there are 608,620 bladder cancer survivors living in the united states , and an additional 74,690 cases will be diagnosed . neoadjuvant chemotherapy ( nc ) followed by radical cystectomy ( rc ) is now considered the standard of care for muscle - invasive bladder cancer after numerous trials demonstrated a survival benefit , most notably in patients with advanced pathologic stage disease [ 24 ] . for highly selected patients , bladder - sparing surgery such as transurethral resection of bladder tumor ( turbt ) [ 5 , 6 ] or partial cystectomy ( pc ) [ 710 ] may provide similar oncologic outcomes to rc while maintaining bladder and sexual functions . of the two bladder - sparing options , pc has advantages over turbt as a third of patients are understaged with turbt and pc allows for full thickness examination of the bladder wall and concurrent lymphadenectomy resulting in more accurate staging and prognosis . our institution previously reported on 111 patients who received neoadjuvant methotrexate - vinblastine - doxorubicin - cisplatinum ( m - vac ) chemotherapy followed by turbt . of the 60 patients achieving ct0 , 15 subsequently underwent pc and 17 underwent rc . ten - year metastasis - free survival for the 15 patients who underwent pc was 73% with 53% of patients having their bladders intact , compared to the 65% 10-year metastasis - free survival for patients undergoing rc . similarly in a prospective trial conducted by sternberg et al . , 104 patients with muscle - invasive bladder cancer underwent turbt after 3 cycles of m - vac chemotherapy and 49% of the cohort achieved ct0 after nc . of these 104 patients , 13 patients with a solitary lesion underwent pc , while 39 patients underwent rc . for patients undergoing pc , 5-year survival was 69% with 4 patients alive after a median follow - up of 88 months ( range 16158 ) compared to 5-year survival of 46% with 15 patients alive after a median follow - up of 45 months ( range 4172 ) for patients undergoing rc . we herein report our contemporary experience with a highly select cohort of patients who received neoadjuvant chemotherapy followed by pc performed for curative intent at a single tertiary institution . in this institutional review board - approved retrospective study , we identified patients who underwent pc at memorial sloan kettering cancer center from 1995 to 2013 ( n = 331 ) . only patients who underwent nc for urothelial cell carcinoma of the bladder followed by pc with curative intent were included in our study ( n = 36 ) . these were not consecutive cases and all patients underwent restaging turbt at our institution prior to and after nc with cystoscopic mapping of bladder tumor / scar . all patients were followed up postoperatively with imaging and cystoscopy every 3 months for 2 years with widening of surveillance interval after . we recorded data for clinical and pathologic variables including age , gender , race , tumor size and focality , histology , presence of carcinoma in situ ( cis ) , cross - sectional imaging , type and duration of nc , clinical and pathologic stages according to american joint committee on cancer 2010 tnm staging 7th edition , surgical margin ( sm ) , disease status , and cause of death . survival outcomes included recurrence - free survival ( rfs ) which was defined as freedom from recurrence after pc , advanced recurrence - free survival ( arfs ) which was defined as freedom from recurrence after pc beyond salvage with intravesical therapy or rc , and overall survival ( os ) . kaplan - meier survival estimates were generated with time measured from the date of pc to the date of event ( recurrence , advanced recurrence , and death ) or last follow - up . using univariate cox regression for continuous and log - rank text for categorical variables , we analyzed variables for association with rfs , arfs , and os . all probabilities were two - sided , and a p value < 0.05 was considered significant for all analyses . all data were analyzed using stata version 12.0 ( statacorp , college station , tx , usa ) . median age for the cohort was 70 years old ( interquartile range ( iqr ) 58.876.8 ) . all tumors were solitary , less than 5 cm in diameter , and 22 ( 61% ) had a variant histology . six tumors were located in the anterior wall , 19 in the lateral wall , 5 in the posterior wall , 3 in the base / trigone , and 3 in a diverticulum . chemotherapy characteristics are described in table 1 ; most patients received platinum - based chemotherapy with 20 patients ( 56% ) having received gemcitabine and cisplatin combination . unilateral ureteral reimplantation was performed in 7 patients at the time of pc to achieve sm in all cases . margin status was evaluated by intraoperative frozen sections at pc in all patients . as shown in table 1 , prior to nc , 22 ( 61% ) patients had ct2 disease , 21 ( 58% ) all patients were clinically restaged after nc ( tables 1 and 2 ) with 21 ( 58% ) patients achieving ct0 , 3 ( 8% ) having ctis , and 4 ( 11% ) having cn+ . of the 4 patients with cn+ after nc , 3 had cn+ before nc , and information prior to nc was unavailable for 1 patient . as shown in tables 1 and 3 , pc pathologic findings were pt0 in 18 ( 50% ) patients , ptis in 6 ( 17% ) , pn+ in 4 ( 11% ) , and sm+ in 3 ( 8% ) . the sm+ was perivesical in 1 patient with pt3 disease and ptis at margin in 2 patients with pt3 disease and pt2 disease . all 3 patients with sm+ experienced recurrence and died of disease ( dod ) at 5 , 10 , and 43 months . of the 21 patients who were ct0 after nc , 7 ( 33% ) had residual bladder disease in the pc specimen ( table 3 ) . at last follow - up , 19 ( 53% ) patients had recurrence , 15 ( 42% ) had advanced recurrences , 10 ( 28% ) died of disease , and 1 died of another cause . twenty ( 56% ) patients were with no evidence of disease ( ned ) after median follow - up of 17 months ( iqr 9.438.2 ) , with 15 having had no recurrences . two of these 4 patients underwent intravesical bacillus calmette - guerin ( bcg ) treatment at 8 and 23 months after pc and the other 2 patients underwent rc at 23 and 43 months after pc . four were alive with disease ( awd ) : 2 patients having disease in the pelvis and 2 patients having disease in retroperitoneum . of the 19 ( 53% ) patients who experienced recurrence ( table 4 ) 9 had recurrence in the bladder with 6 in the bladder only . of the 9 patients with bladder cancer recurrences , 5 were at the resection site and 2 had sm+ at pc . of the 6 patients with bladder - only recurrences , 2 patients had ctis , received bcg therapy , and have been ned to date ; 2 patients had ct2 and ct1/ctis tumors , underwent rc , and remained disease free ; 1 patient had ctis and received bcg but developed distant metastases ( lung and adrenal ) without further bladder recurrences 7 years after pc and eventually died of disease . another patient developed persistent ct1/ctis disease that was managed with repeating turbt and bcg before a failed attempt at rc and later died of disease . as shown in table 5 , median time to recurrence was 23 months ( iqr 5.966.2 ) , median time to advanced recurrences was 66 months ( 7.2not reached ) , and median time to death was 79 months ( 20.6not reached ) . kaplan - meier survival estimates for 2- and 5-year rfs , arfs , and os were 37% and 28% , 58% and 51% , and 71% and 63% , respectively ( figure 1 ) . clinical stage > ct2 was associated with both worse rfs ( p = 0.03 ) and arfs ( p < 0.01 ) . after nc , the presence of cis was associated with worse os ( p = 0.04 ) and cn+ was associated with worse rfs ( p < 0.01 ) , arfs ( p < 0.01 ) , and os ( p < 0.01 ) . following pc , presence of pt2 disease was associated with worse rfs ( p = 0.02 ) and arfs ( p = 0.01 ) , pn+ was associated with worse rfs ( p = 0.04 ) , and sm+ on final pathology was associated with worse rfs ( p = 0.01 ) , arfs ( p = 0.04 ) , and os ( p < 0.01 ) . our findings have been consistent with those of the literature with regard to oncologic outcomes in patients undergoing pc after nc . in our series , 74% of patients were downstaged after nc with 58% having a complete clinical response . of the patients who achieved ct0 after nc , 7 ( 33% ) had evidence of residual disease within the resected specimen at pc , which is close to 30% of understaging with turbt alone reported by our institution 's herr and scher study . two- and 5-year overall survival were 71% and 63% , respectively , which were comparable to oncologic outcomes of other studies involving pc after nc [ 5 , 11 , 12 ] and rc . in our study , at last follow - up 19 patients ( 53% ) had experienced recurrence and 24 ( 67% ) were alive with 22 patients ( 61% ) having retained an intact bladder . nine patients had recurrence in the bladder with 5 at the suture line . in rc series by stein et al . , local pelvic recurrence only occurred in 6%13% of cases depending on radical cystectomy pathology and in our series 5 ( 14% ) patients had recurrence in the pelvis with only 2 having isolated pelvic recurrences . both of these patients with pelvic - only recurrences had negative surgical margins at pc with only 1 patient having recurrence on the ipsilateral side of the previous tumor . for our study , advanced recurrence was defined as presence of disease that can not be treated with salvage intravesical therapy or rc , which differs from previous reports of disease recurring in the bladder muscle and beyond [ 7 , 8 ] . we believe that , with the improved quality of surveillance cross - sectional imaging and follow - up cystoscopies , disease control can still be achieved despite recurrences . this was evident in the 6 patients with isolated bladder recurrence as only 1 patient experienced disease progression and died of disease . we also noted that 53% of patients who experienced recurrence had no disease in bladder and hence it is unclear whether a rc would have altered their disease course . in our previous report by holzbeierlein et al . , the authors noted that presence of cis preoperatively was associated with local recurrence and sm+ and pn+ were associated with advanced recurrence , as defined by muscle invasion and beyond . though this series differs for lack of association of cis with recurrence , we noted similar findings with sm+ and pn+ . all 3 patients with sm+ on final pathology experienced recurrence and died of diseasewith sm+ being associated with worse rfs , arfs , and os on univariable analysis . similarly , 4 patients had cn+ after nc with 2 eventually having pn+ at pc . of those 2 patients with pn+ , one was awd at 7.3 months of follow - up and the other died of disease at 16.8 months of follow - up . cn+ after nc was associated with worse rfs , arfs , and os on univariable analysis . unlike in kassouf anderson cancer center series , the need for ureteral reimplantation is not an exclusion criterion for pc at our institution . in this series , we performed 7 unilateral ureteral cases of reimplantation and we were able to achieve sm in 4 patients and the rest had pt0 disease . in terms of the concerning voiding dysfunction after pc , none of the patients in this series underwent any additional procedures for diminished bladder capacity . our study is limited by its small size , retrospective nature , surgical selection bias , and relatively short follow - up . additionally , patients did not receive a uniform nc regimen ; 4 ( 11% ) patients had evidence of clinical nodal involvement after nc , and 22 patients ( 61% ) had a variant histology including some with recognized aggressive nature such as small cell ( 25% ) and micropapillary ( 14% ) . also though none of our patients underwent any additional procedures for voiding dysfunction , we do not have long - term quality of life or medication usage data . in this contemporary institutional series , pc after nc in highly selected patients with muscle - invasive bladder cancer provides acceptable oncologic outcomes comparable to those in previously published reports .

objective . to report our contemporary experience with partial cystectomy after neoadjuvant chemotherapy . patients and methods . retrospective review of patients who underwent neoadjuvant chemotherapy and partial cystectomy for urothelial cell carcinoma of the bladder at memorial sloan kettering cancer center from 1995 to 2013 . log - rank test and cox regression models were used to analyze variables possibly associated with recurrence - free , advanced recurrence - free ( free from recurrence beyond salvage with intravesical therapy or radical cystectomy ) , and overall survival . results . all 36 patients had a solitary tumor < 5 cm in size . twenty - one patients ( 58% ) achieved ct0 following neoadjuvant chemotherapy with 7 ( 33% ) having residual disease at pc . at last follow - up , 19 ( 53% ) patients had recurrence , 15 ( 42% ) had advanced recurrence , 10 ( 28% ) died of disease , and 22 ( 61% ) maintained an intact bladder . median follow - up of those who were with no evidence of disease was 17 months . on univariable analysis , after neoadjuvant chemotherapy positive nodes on imaging and positive surgical margin at partial cystectomy were both associated with worse recurrence - free , advanced recurrence - free , and overall survival . five - year recurrence - free , advanced recurrence - free , and overall survival were 28% , 51% , and 63% , respectively . conclusion . partial cystectomy following neoadjuvant chemotherapy provides acceptable oncologic outcomes in highly selected patients with muscle - invasive bladder cancer .

selective serotonin reuptake inhibitors ( ssris ) , serotonin and norepinephrine reuptake inhibitors , and noradrenergic and specific serotonergic antidepressants are categorized as first - line treatments in many countries , including japan.1,2 thus , health care providers have the opportunity to choose an antidepressant from a variety of first - line treatment options . to select the most appropriate treatment for each patient , it is important for clinicians to be aware of the differences in efficacy between comparable antidepressants at the doses approved in their countries . one long - standing approach to comparing efficacy has been meta - analysis of several randomized controlled trials that compare widely used treatments.310 however , results have been inconsistent , perhaps partly due to the use of different methodologies . recent meta - analyses have used mixed treatment comparisons in which both direct and indirect comparisons were used.11,12 duloxetine , a second - generation norepinephrine reuptake inhibitor that has demonstrated efficacy , safety , and tolerability in patients with mdd,1316 has been the subject of several recent mixed treatment comparisons . a recent analysis by cipriani et al12 compared the effects of 12 new - generation antidepressants in adults with mdd , and gartlehner et al11 conducted an analysis of 234 studies that included placebo as a comparator . however , these results are not applicable to clinical practice in japan , because they included data from patients taking duloxetine up to 120 mg / day , which is twice the maximum dosage ( 60 mg / day ) approved in japan . another drawback was the inclusion of patients who took antidepressants not approved or not even available for use in japan . therefore , it is uncertain whether these results can be applied to daily clinical practice in countries ( eg , japan ) where the approved dosage for duloxetine is 4060 mg / day and ssris are limited to four compounds , ie , paroxetine , sertraline , escitalopram , and fluvoxamine . to the best of our knowledge , there are no meta - analyses that compare the efficacy of duloxetine 60 mg / day with these ssris . the primary purpose of the current study was to provide clinicians with efficacy results that would allow a comparison of duloxetine 60 mg / day with four approved ssris in japan . the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan . we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd . for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result , a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step , patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded . common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 . the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out . the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) . the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo . the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd . the study included 8 weeks of active treatment followed by a one - week dose - tapering period . the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day . this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram . the primary endpoint was mean change from baseline in hamd17 total score at week 8 . secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 . response was defined as a 50% reduction in hamd17 total score from baseline at week 8 . our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 . this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach . continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates . effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study . overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect . the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study . relative risk is presented with 95% confidence intervals ( ci ) and p - values . the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies . absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors . safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events . reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology . safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test . the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan . we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd . for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result , a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step , patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded . common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 . the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out . the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) . the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo . the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd . the study included 8 weeks of active treatment followed by a one - week dose - tapering period . the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day . this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram . the primary endpoint was mean change from baseline in hamd17 total score at week 8 . secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 . response was defined as a 50% reduction in hamd17 total score from baseline at week 8 . our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 . this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach . continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates . effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study . overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect . the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study . relative risk is presented with 95% confidence intervals ( ci ) and p - values . the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies . absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors . safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events . reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology . safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test . at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 . the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) . completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) . adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) . discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) . a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population , no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) . duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) . for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) . these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) . the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients . no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) . statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) . the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients . no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) . the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth . at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 . the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) . completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) . adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) . discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) . a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population , no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) . duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) . for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) . these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) . the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients . no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) . statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) . the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients . no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) . the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth . our results show that duloxetine and ssri treatment within their respective approved dosage in japan demonstrated comparable efficacy with regard to overall depressive symptoms . however , certain hamd17 subscale scores responded differently to duloxetine compared with ssris , and this may clarify the difference in clinical attributes among these antidepressants . first , when assessing overall efficacy using the mean changes in hamd17 total scores from baseline , both duloxetine and ssri demonstrated greater efficacy than placebo , but did not indicate any significant difference between each other for either the total population ( hamd17 total score 15 ) or the more severe population ( hamd17 total score 19 ) . on the one hand , this is consistent with a prior report by thase et al,4 where all randomized patients across any severity were analyzed and no significant difference in efficacy between duloxetine and ssri was found . however , on the other hand , this is inconsistent with prior reports that duloxetine 120 mg / day showed greater efficacy than ssris in patients with at least moderately severe depression.4,16 given this and the finding of a modest dose response trend for duloxetine efficacy by mallinckrodt et al16 this inconsistency may be explained by the upper limit on the dosage of duloxetine ( 60 mg / day ) in this study . second , when assessing energy and interest using retardation subscale scores , duloxetine showed greater efficacy regardless of baseline depression severity ( hamd17 total score 15 or 19 ) . this is consistent with previous reports by mallinckrodt et al16 and martinez et al.24 furthermore , katz et al25 reported that desipramine ( a norepinephrine reuptake inhibitor ) demonstrated better efficacy than paroxetine ( an ssri ) on psychomotor retardation symptoms . these results may be explained by the effect of duloxetine on norepinephrine , which has been shown to be associated with arousal and activity.26 the current results show some inconsistency with a report by cipriani et al in which some ssris were significantly more efficacious than duloxetine . this is partly due to the different methodologies used in that study and in our current study . in cipriani et al,12 approximately one third of the comparisons were direct comparisons from randomized controlled trials and many of the pairwise comparisons were based on only one study . degree of heterogeneity and statistical incoherence ( estimates based on the direct comparisons are not contained in the 95% ci for indirect comparisons ) in the study network were detected . study conclusions based on direct comparisons were much more conservative than those based on indirect ones . on the other hand , the strengths of this database analysis are that it analyzed individual patient data , used the same analysis method and endpoint across studies , and included similar patients . the adopted database analysis design allowed for a homogeneous patient population , with almost the same inclusion criteria and the exact same endpoint . although our analysis resulted in a comparatively smaller number of studies and patients , it achieved high internal validity and reproducibility . in addition , direct comparisons of head - to - head data enabled us to avoid the uncertainty of how much the two different evidence levels are weighted when pooling direct and indirect comparisons . the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications . similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population . another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan . furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan . further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole . in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis . lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed . the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications . similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population . another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan . furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan . further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole . in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis . lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed . comparison between duloxetine and ssri demonstrated comparable efficacy within the japan - approved dose range in patients with mdd . results of the hamd17 subscale analysis indicated that duloxetine might be superior to ssris in improving loss of energy and interest .

backgroundapproved doses of antidepressants in japan are usually lower than those in the usa and european union , but to date meta - analyses comparing antidepressants have all used the higher doses approved in the usa and european union and often have used indirect comparisons . the purpose of this study was to conduct an integrated database analysis of patient level data to compare the effects of duloxetine with those of selective serotonin reuptake inhibitors ( ssris ) at the doses approved in japan.methodspooled data were analyzed from four randomized , double - blind , placebo - controlled studies that compared duloxetine at the dose range approved in japan ( 4060 mg / day ) with other ssris ( paroxetine 20 mg / day or escitalopram 10 mg / day ) and placebo in patients with major depressive disorder . in total , 1,694 patients were included in the analysis ( duloxetine , n=688 ; selective serotonin reuptake inhibitors , n=690 ; placebo , n=316 ) . the primary outcome measure was the mean change from baseline at week 8 in 17-item hamilton rating scale for depression ( hamd17 ) total and subscale scores.resultsduloxetine and both selective serotonin reuptake inhibitors were superior to placebo in hamd17 total score at week 8 in both the all - randomized group and the more severe subgroup ( hamd17 total scores 19 ) . duloxetine was superior to ssris in improving the hamd17 retardation subscale score ( least squares mean difference [ 95% confidence interval ] ) : all - randomized group , 0.33 [ 0.60 , 0.07 ] , p=0.015 ; severe subgroup , 0.45 [ 0.83 , 0.07 ] , p=0.020).conclusionwithin the dose range approved in japan for patients with major depressive disorder , duloxetine and selective serotonin reuptake inhibitors demonstrated comparable overall efficacy , with a possible advantage for duloxetine in improving loss of energy and interest . to the best of our knowledge , this analysis is unique not only in evaluating dosages specific to japan , but also in using individual patient data and the same endpoint across studies to allow for strictly direct head - to - head data comparisons as opposed to pooling direct and indirect comparisons .

the prevalence rate in the general population ranges between 10% and 12% , making this disease one of the most common causes of pain , activity limitation , and restrictions in participation . knee osteoarthritis ( gonarthrosis ) is one of the most frequent forms of degenerative osteoarthritis . in knee osteoarthritis , pain is the primary symptom and probably the most important factor related to activity limitation and disability . although the pain in knee oa is multifactorial , acute exacerbations are usually closely related to synovial inflammation . various studies performed by using arthroscopy have shown synovitis in painful knees of patients with knee oa . on the basis of these reports , many investigators focused on noninvasive imaging techniques , such as magnetic resonance imaging and diagnostic ultrasonography . the term pes anserinus refers to the conjoined insertion of the sartorius , gracilis , and semitendinosus muscles along the proximal medial aspect of the tibia . these three muscles are primarily flexors of the knee ; they also influence internal rotation of the tibia and protect the knee against rotatory and valgus stress . in pes anserine bursitis , the fluid - filled bursa in the conjoined insertion of the three hamstring muscles becomes inflamed and causes pain . patients with this clinical picture may report vague medial knee pain or may present with tenderness and swelling along the proximal medial tibia . a diagnosis of pes anserine bursitis should be considered when there is spontaneous pain inferomedial to the knee joint and a history of trauma or diabetes . conditions associated with pes anserine bursitis include degenerative joint disease of the knee , obesity , valgus knee deformity , pes planus , and sporting activities . pes anserine bursitis affects the quality of life of patients with osteoarthritis , and the treatment methods are different . treatment includes nonsteroidal anti - inflammatory drugs ( nsaids ) , physiotherapy , and injections of corticosteroid , with highly variable responses ; recovery can take 10 days to 36 months . mesotherapy was introduced 50 years ago by michel pistor , a french physician who used this technique as a novel analgesic therapy for a variety of rheumatologic disorders . mesotherapy is a minimally invasive technique that consists of subcutaneous injections of drugs and , occasionally , plant extracts , homeopathic agents , or other bioactive substances ; for this reason , it has been often considered a form of complementary and alternative medicine rather than a conventional medical therapy . the objective of this type of administration is to modulate the pharmacokinetics of the injected substance and to prolong the pharmacologic effects at a local level . one of the main advantages of mesotherapy is that a local pharmacologic effect can be obtained without the need for high systemic concentrations . intradermal injections of small amounts of active substance where the injection site corresponds to the area of the pathologic condition for example , in low back pain may provide clinical benefits when other therapies are not available , are not effective , or can not be used for whatever reason . in addition , intradermal administration of active substances in combination with other systemic therapies can produce synergistic effects , and as a result mesotherapy may have dose - sparing effects . since its introduction , the use of mesotherapy has been expanded , and therapeutic indications have increased . although mesotherapy is primarily used for osteoarticular conditions , this technique has recently become popular in cosmetic medicine for treatment of edematous fibrosclerotic panniculopathy and local adiposity . the aim of the current study was to evaluate the effects of mesotherapy with diclofenac for anserine bursitis in knee osteoarthritis . the multicenter study was conducted at the d'annunzio university in chieti , italy , and at an affiliated rehabilitation center in florence . it was approved by the local ethics committee and was performed in accordance with the 1964 declaration of helsinki . one hundred and seventeen patients ( 59 men and 58 women ) age 1856 years ( average age , 36 years ) were evaluated and treated . all them had anserine bursitis , ascertained clinically and by ultrasonography , in association with grade ii kellgren - lawrence knee osteoarthritis ( fig . inclusion criteria were age between 10 and 60 years , grade ii kellgren - lawrence knee osteoarthritis , re - acutization of pain , and synovial inflammation . exclusion criteria were pregnancy , known hypersensitivity to products , infiltrative therapy with hyaluronic acid , polynucleotides or corticosteroids in progress , drug abuse or alcohol abuse , significant comorbidities ( such as the presence of neurologic abnormalities , concomitant severe rheumatic disease , and systemic abnormalities , such as diabetes ) , a surgical intervention within 3 months before the study , psychiatric conditions , or psychotherapy or physical therapy within 5 weeks before the study . the patients were randomly divided into two groups ( a , mesotherapy ; b , control ) after giving written consent and agreeing to the possible treatment ( local or oral ) . group a ( 60 patients ; 30 men and 30 women ) received nine sessions of mesotherapy with sodium diclofenac ( 25 mg/1 ml ; akis , ibsa , lugano , switzerland ) , 1 ml for each session , three times per week . group b ( 57 patients ; 29 men and 28 women ) received 21 oral administrations of sodium diclofenac ( 50 mg ; voltaren , novartis , parsippany , nj ) , once every day for 3 weeks . all patients reported pain during sleep and after a long period of rest , as well as reduction of pain after about a 200-m walk . all patients were asked to provide standard radiographs ( anterior - posterior and lateral ) of the lumbosacral tract and knee joints . primary outcome measures were pain intensity , quantified by using a 10-cm millimetric visual analogue scale ( vas ) , on which the patient is asked to mark the degree of pain intensity , ranging from 0 ( absence of pain ) to 10 ( the worst pain imaginable ) , along with abilities in activities of daily living ( adls ) , ability to participate in sports , level of pain , symptoms , and quality of life , as assessed by the knee injury and osteoarthritis outcome score ( koos ) . all these assessments , along with ultrasonography , were performed before ( t0 ) and after ( t1 ) the treatment period and at 30 days ' ( t2 ) and 90 days ' ( t3 ) follow - up in all patients . differences between mean values before and after the rehabilitation period were tested for significance by using two - way analysis of variance for repeated measures . graphpad prism ( version 6 ) software ( abacus concepts graphpad software , san diego , ca ) . in group a , the subjective pain levels assessed by vas were 71.05 at t0,20.35 at t1 , 3.80.85 at t2 , and 4.00.85 at t3 ( fig . disability during sports activity at t1 decreased significantly ( p<0.05 ) : t0 score , 7010.7 ; t1 score , 557.9 ; t2 score , 608.5 ; t3 score , 608.7 ( fig . perceived symptoms also were significantly reduced ( p<0.05 ) at t1 and at both follow - up times : t0 score , 71.411.9 ; t1 score , 46.49.4 ; t2 score , 509.7 ; t3 , score 53.69.0 ( fig . was seen in the effect of osteoarthritis on quality of life at t1 and at both follow - up times : t0 score , 68.813.2 ; t1 score , 43.810.9 ; t2 score , 509.7 ; t3 score , 509.8 ( fig . disability with regard to adls also decreased significantly ( p<0.05 ) at t1 and at both follow - up times : t0 score , 7214.2 ; t1 score , 478.7 ; t2 score , 478.7 ; t3 score , 509.2 ( fig . finally , pain perception was significantly reduced ( p<0.01 ) at t1 and at both follow - up times : t0 score , 7510.3 ; t1 score , 30.55.5 ; t2 score , 365.2 ; t3 score , 415.8 ( fig . error bars represent the standard deviation . a , group a ( mesotherapy treatment ) ; b , group b ( oral treatment ) . trend of disability during sports activity ( knee injury and osteoarthritis outcome score [ koos ] ) . trend of disabilities with regard to activities of daily living ( adls ) ( koos ) . b , the subjective pain levels assessed by vas were 7.51.85 at t0 , 31.10 at t1 , and 5 at t2 and t3 ( fig . the koos findings were as follows : disability during sports activity did not significantly decrease : t0 score , 7515.8 ; t1 score , 6515.5 ; t2 score , 6515.7 ; t3 score , 7017.2 ( fig . 3 ) . a significant reduction occurred ( p<0.05 ) in perceived symptoms at t1 : t0 score , 71.413.1 ; t1 score , 60.78.3 ; t2 score , 64.38.7 ; t3 score , 64.39.1 ( fig . the effect of osteoarthritis on quality of life decreased significantly ( p<0.05 ) at t1 : t0 score , 7513.2 ; t1 score , 5011.0 ; t2 score , 68.814.5 ; t3 score , 68.814.7 ( fig . disability with regard to adls was reduced significantly ( p<0.05 ) at t1 : t0 score , 70.612.7 ; t1 score , 61.79.4 ; t2 score , 66.210.9 ; t3 score , 67.612.5 ( fig . finally , a significant reduction ( p<0.05 ) was seen in pain perception at t1 and at both follow - up times : t0 score , 7510.4 ; t1 score , 52.78.3 ; t2 score , 55.68.8 ; t3 score , 58.39.5 ( fig . in particular , group a had pain remission after a 120-m walk at t1 and after a 160-m walk at t2 and t3 . ultrasonography showed a reduction of the hypoechoic area related to anserine bursitis only in group a ( ie , the mesotherapy group ) . the aim of this study was to evaluate the effectiveness of anti - inflammatory drugs administered via mesotherapy in patients with local inflammation in grade ii kellgren - lawrence knee osteoarthritis . present results showed that the administration of nsaids ( diclofenac ) via mesotherapy can provide the same therapeutic benefit as that induced by conventional ( oral ) drug administration in relation to pain after a 3-week treatment period . indeed , both treatments significantly reduced pain intensity and disability in daily life activity ; the effect was maintained up to 3 months ( 30 days ' and 90 days ' follow - up ) only in the group that received mesotherapy . these results are in accordance with previous studies showing that naproxen and diclofenac administered via mesotherapy were more effective than those given orally . the major finding of this study is the similar effectiveness of mesotherapy and conventional systemic therapy , despite the lower amount of drugs administered to patients undergoing mesotherapy ( 25 mg with mesotherapy vs 50 mg with oral administration ) and the lower short - term risk for adverse reactions in the mesotherapy group ( 3 weeks ) . the similar efficacy of mesotherapy and conventional therapy , despite different drug dosages , is difficult to explain . subcutaneous drug administration results in a very slow drug absorption in comparison with other systemic routes , such as oral and intramuscular . thus , it could be hypothesized that anti - inflammatory drugs administered via mesotherapy achieve a higher drug concentration in the subcutaneous tissue and exert local effects close to inflammatory cells , sensory fibers , and vascular mediators that orchestrate inflammation and pain . this hypothesis could be confirmed by the evidence of reduction of the anserine bursitis showed by ultrasonography in group a ( the mesotherapy group ) but not in group b ( the oral administration group ) . although this study did not measure drug plasma levels after the two routes of administration , it is possible to hypothesize that mesotherapy resulted in a lower systemic bioavailability of drugs , with consequent lower incidence of adverse reactions . this could offer a great therapeutic advantage given the high rates of adverse effects associated with nsaid or corticosteroid use in the elderly population . although mesotherapeutic techniques used in dermatologic surgery have been associated with many adverse effects at injection sites , including atypical mycobacterial infections , urticaria , lichenoid drug eruptions , and psoriasis , no evidence of local reactions was found in the present study . in conclusion , results of this study indicate that combined administration of conventional nsaids ( diclofenac ) by mesotherapy is an effective and well - tolerated method for managing anserine bursitis in ii grade knee osteoarthritis in the short term and mid - term compared with oral administration of the same drug with different concentrations . however , future studies could have a larger scale and thus also measure plasma bioavailability of the drug . these observations could be of potential interest for the pharmacologic treatment of anserine bursitis in order to reduce the adverse effects associated with high plasma levels of anti - inflammatory drugs .

abstractbackground : pes anserine bursitis strongly affects quality of life in patients with osteoarthritis . treatment includes nonsteroidal anti - inflammatory drugs ( nsaids ) , physiotherapy , and injections of corticosteroid , with highly variable responses ; recovery can take 10 days to 36 months . mesotherapy is a minimally invasive technique consisting of subcutaneous injections of bioactive substances . the goal is to modulate the pharmacokinetics of the injected substance and prolong the effects at a local level.objective : to evaluate the effects of mesotherapy with diclofenac for anserine bursitis associated with knee osteoarthritis.methods : one hundred and seventeen patients with anserine bursitis associated with grade ii kellgren - lawrence knee osteoarthritis , assessed by clinical , radiographic , and ultrasonographic examination , were evaluated and treated . they were randomly divided into two groups ( a , mesotherapy ; b , control ) . group a completed nine sessions of mesotherapy with sodium diclofenac ( 25 mg/1 ml ; akis , ibsa , lugano , switzerland ) , 1 ml for each session , three times per week . group b received 21 oral administrations of sodium diclofenac ( 50 mg ; voltaren , novartis , parsippany , nj ) , once a day for 3 weeks . primary outcome measures were pain intensity assessed by visual analogue scale ( vas ) , along with ability to perform activities of daily living , ability to participate in sports , level of pain , symptoms , and quality of life , as assessed by the knee injury and osteoarthritis outcome score . these measures were performed before and after the treatment period and at 30 and 90 days ' follow up.results : in both groups pain level decreased significantly after the treatment period . ultrasonography showed a reduction of the hypoechoic area related to anserine bursitis only in group a.conclusion : administration of conventional nsaids ( diclofenac ) by mesotherapy is effective in managing anserine bursitis in knee osteoarthritis in the short term and mid - term . these observations could be of interest for efforts to reduce the adverse effects of oral administration of anti - inflammatory drugs .

neurological soft signs ( nss ) have long been considered one of the functional features ( tsuang and faraone , 1999 ; tsuang et al . , 1991 ) and endophenotypes ( chan and gottesman , 2008 ; chan et al . , 2010a ) of schizophrenia spectrum disorders . however , most of the studies of nss have been limited to the use of clinical ratings . instead , recent findings from both structural and functional imaging studies have shown that nss may be associated with specific brain alterations . specifically , in individuals with psychosis , more nss have been associated with smaller volumes of the inferior frontal lobes ( thomann et al . , 2009a ) , the pre - central gyrus ( heuser et al . , 2011 ; mouchet - mages et al . , 2011 ) , the global cortical sulci ( dazzan et al . , 2004 ; gay et al . , 2013 ) as well as the cerebellum ( ho et al . , 2004 ; thomann et al . , nss may be responsible for some of the observed clinical manifestations in schizophrenia ( keshavan et al . , 2003 ; mouchet - mages et al . , 2011 ; schroder et al . , 1999 ) . a recent meta - analysis of structural and functional imaging findings suggests that nss are associated with volume reductions of the pre - central gyrus , the cerebellum , the inferior frontal gyrus and the thalamus . furthermore , the same meta - analysis also suggests that functional imaging studies support an association between nss and altered neural activation in the inferior frontal gyrus , the bilateral putamen , the cerebellum and the superior temporal gyrus in patients with schizophrenia ( zhao et al . , , these findings support the presence of a dysfunction of neural circuitry that underlies the presence of these minor neurological abnormalities , which are already present at illness onset . on the other hand , empirical findings have also shown that non - psychotic first - degree relatives of schizophrenia patients exhibit a higher prevalence of nss compared to healthy controls ( egan et al . a meta - analysis comparing the prevalence of nss between patients with schizophrenia , non - psychotic first - degree relatives and healthy controls indicated a mean effect size of 0.8 and 0.97 for patients and their non - psychotic first - degree relatives and for non - psychotic first - degree relatives and controls respectively ( chan et al . , 2010b ) . these results are consistent with the argument that nss are familial in nature and segregate with the illness . however , no studies had examined the brain correlates of nss in healthy relatives of patients with schizophrenia . furthermore , all the aforementioned neuroimaging studies in schizophrenia patients used conventional subtraction analysis and did not examine the underlying connectivity between the brain regions identified . given that some nss , such as the fist palm ( fep ) task ( first described by luria , see heuser et al . , 2011 ) , involve the functional integration or regulation of areas involved in motor sequencing rather than being directly activated , a connectivity approach is more appropriate to identify the specific network involved . only one study ( rao et al . , 2008 ) adopted a psychophysiological interaction ( ppi ) ( friston et al . , 1997 ) method to re - analyze their previous findings on the fep task in healthy volunteers ( chan et al . , 2006 ) . this new analysis showed enhanced functional connectivities between the left- and right sensorimotor cortices ( smc ) and the right inferior and middle prefrontal cortices during fep task performance compared to a simple palming task . these findings suggest a regulatory role of the prefrontal cortex on fep task execution . in this study , we examined whether prefrontal regions are involved in the integration or regulation of neural activity underlying motor sequencing in patients with first - episode schizophrenia and their non - psychotic first degree relatives ( fdr ) . more specifically , we aimed to identify any frontal regions where coupling in these areas and the sensorimotor cortex ( smc ) significantly differed between the complex fep task and a simple control motor task using ppi analysis . we hypothesized that patients with first - episode schizophrenia and their non - psychotic fdrs would show a reduced dysfunction of the prefrontal cortex and smc while performing the fep task . thirteen right - handed first - episode schizophrenia patients were recruited from the mental health center , peking university , beijing , for the study . participants were recruited to the study if they met the following inclusion criteria : a ) diagnosis of schizophrenia ascertained by experienced psychiatrists according to the dsm - iv ( apa , 1994 ) ; b ) aged 1840 years ; and c ) illness duration within 2 years . the exclusion criteria were : a ) a history of neurological disorders ; b ) a lifetime history of substance abuse and c ) an estimated iq lower than 70 . current symptom severity was assessed with the positive and negative syndrome scale ( kay et al . , 1987 ) . fourteen non - psychotic fdrs of the patients taking part in the study were also invited to participate . potential participants were excluded if they a ) met the dsm - iv criteria for substance abuse ; b ) were suffering from any clinically unstable medical disorder ; c ) had a history of head injury ( past or present ) ; and d ) had an estimated iq lower than 70 . fourteen healthy controls matched with the patients in age , gender and handedness were recruited from local universities through advertisements . the institutional review board of the institute of psychology , the chinese academy of sciences , approved the study protocol . behavioral neurological soft signs were examined with the abridged version of the cambridge neurological inventory ( cni ) ( chan et al . , 2009 ) . this abridged version offers instructions for eliciting and rating a comprehensive range of nss in motor coordination , sensory integration and disinhibition . rao et al . , 2008 ) ( for the earliest description , please see luria , 1966 ; in heuser et al . , 2011 ) . in brief , the task consisted of three right - hand motor tasks which varied in complexity . in the simple palm tapping ( pt ) task , participants were required to repeat only one right palm tapping in the prone position . in the intermediate complex pronation / supination ( ps ) task , participants were required to perform right palm tapping in the prone and supine positions alternatively . in the complex fep task , participants were required to successivley place their right hand in a fist resting position vertically ( fist ) , a palm resting position vertically ( edge ) , and a palm resting position horizontally ( palm ) . a resting condition ( a ) without any hand movement when participants were asked to focus on the screen was used as the control baseline of the pt task , and the pt task was in turn used as the control baseline of the ps and fep tasks . participants were asked to practice the motor actions correctly at a constant rate before entering the scanner . during scanning , their performance was monitored by the experimenter through the window of the scanner room . in the formal imaging task , participants were instructed to perform the three tasks at a similar pace throughout the entire experiment . pt and ps tasks were executed in 1 s and the fep task was executed in 1.5 s. the experimenter monitored the task performance outside the scanner room to ensure that the participants performed the correct hand movements . first , a resting condition lasted for 20 s and then 6 s of counting backward reminded the participants to get ready for their hand movement . after backward counting , the pt or ps or fep task lasted for 40 s. one of these three hand movements would be presented on the screen . participants were required to conduct the hand movements according to the demonstration on the screen . the sequence of the three hand movement tasks was optimized and counterbalanced within the three runs . the functional imaging data was originally acquired in a ge 3 t sigma scanner ( general electric , waukesha , wi , usa ) with a standard ge birdcage - type rf coil using a standard t2 * -weighted epi sequence . the epi parameters were : tr = 2 s te = 60 ms , fov = 24 24 cm , matrix = 64 64 , flip angle = 60 , 22 axial slices ( 5 mm thick/1.2 mm sp , from superior to inferior ) . the spatial resolution for the functional images was 3.75 3.75 6.2 mm . high - resolution anatomical images were also obtained using the standard t1-weighted sequence ( 66 axial slices , 2.0 mm thick / interleaved , fov = 24 24 cm , matrix = 256 256 ) . images were analyzed with statistical parametric mapping software ( spm8 , wellcome department of imaging neuroscience , london , uk ) implemented in matlab 2009b ( mathworks inc . , sherborn , ma , usa ) . three dummy scans in the beginning of the experiment were removed automatically from the dataset . images were registered to the first icbm 152 , which was based on 152 brains and was created by the montreal neurological institute ( mni ) with a 2 2 2 mm resolution . in the final step of pre - processing , the images were spatially smoothed by an isotropic gaussian kernel with fwhm of 8 mm . conventional analyses were first conducted at the individual - level using voxel - wise general linear modeling ( glm ) and four t - contrasts were defined between the tasks and the corresponding baselines , i.e. , pt vs. rest , ps vs. rest , fep vs. rest and fep vs. pt . data from the contrasts of the first level model in the healthy control group , the schizophrenia group and the fdr group were entered into this model . a threshold of alphasim corrected p < 0.01 and cluster size larger than 15 voxels were used to identify the activations associated with each contrast . furthermore , we used a regression model to find the regions with group difference . we set up an f contrast to find the regions with main effect of group difference . signal change percentage was retrieved from the rois with group difference for post - hoc analysis . ppi analysis was used to estimate functional integration during task execution under different motor complexity conditions . the left smc was determined a - priori as the reference region for the ppi analysis because the motor tasks in the present study were only completed with the right hand . this region was defined by using a sphere with a radius of 8 mm and a center at the peak activation in the left smc activation ( mni coordinates = [ 36 28 52 ] , from the conventional analysis of the contrast of pt vs. rest ) . we performed voxel - wise ppi analysis at individual - level for the left smc to see if any other brain areas connected to the smc showed a significant increase in functional coupling ( the slope of regression ) during the fep task compared with a control task ( e.g. , the pt or ps task ) . for each participant , the activation time course signal in the reference region ( i.e. , the first eigenvariate time series , adjusted by effect of interest ) was extracted from the conventional glm and entered into the ppi analysis as the first regressor representing the physiological variable . a second regressor representing the motor tasks with different complexity was entered into the ppi analysis as the psychological variable . the psychophysiological interaction between task complexity and activation signal in the reference region was designated as the regressor of interest in the ppi analysis . thereafter we performed group - level random effect analysis on the individual results using one sample t - tests for the contrast fep vs. pt . group - level paired t - tests were conducted for the contrast fep vs. ps . areas of significant activation were identified at a threshold of uncorrected p < 0.001 and cluster size larger than 15 voxels . there was no significant difference between patients with schizophrenia , their fdrs and healthy controls in the three subscales and total score of cni . conventional activation of the pt , ps and fep tasks in the three groups , after co - varying for age , is shown in table 3 . the left frontal parietal region was significantly activated when participants performed the pt , ps and fep tasks with their right hand . moreover , in the pt rest contrast , activation in the left frontal parietal region , together with the left medial frontal region , were lowest in patients with schizophrenia , intermediate in the fdrs and highest in healthy controls ( scz < rel < hc ) . we further used a regression model and conducted a post - hoc analysis to identify the regions that were different across groups both linearly and non - linearly . in the pt rest contrast , we confirmed that the left frontal precentral gyrus ( 30 34 64 , k = 100 , f(2,37 ) = 20.23 ) was linearly activated in all three groups ( scz < rel < hc ) . the left medial frontal region ( hc = rel > scz , 8 18 64 , k = 136 , f(2,37 ) = 15.59 ) and the left middle temporal gyrus ( hc > scz = rel , 48 68 10 , k = 124 , f(2,37 ) = 11.7 ) were non - linearly activated in all three groups . the activations associated with task complexity were examined in the fep ps and the fep pt contrasts . in the fep pt contrast , the bilateral middle frontal regions were activated in the healthy control group . the left middle frontal region was activated in the fdr group , but there was no frontal region activation in first episode schizophrenia patients . 2 illustrates the frontal activation in both healthy controls and fdrs . in the fep ps contrast , the left middle frontal region was also activated in the fdr group , but not in the healthy control group or the schizophrenia group . the ppi analysis identified right frontal regions in which the activity showed significant activation coupling to activity in the left smc during performance of the more complex fep task relative to the simple pt or ps tasks in the healthy control group , but not in the schizophrenia group . in this study , we report for the first time the neural activation and connectivity elicited by execution of the fep task in patients with first episode schizophrenia and their healthy first degree relatives . our main finding is that patients with first episode schizophrenia do not show , in comparison with healthy controls and their fdrs , activation of the left middle frontal gyrus in the execution of the fep versus a simpler motor task like the pt . this provides evidence of a frontal dysfunction in these patients when performing a complex motor task . the right inferior frontal gyrus was found to modulate the activation of the sensorimotor cortex with the increase in motor complexity in healthy controls . a frontal dysfunction was also demonstrated by our second main finding , suggesting that with increase in task difficulty , patients with first episode schizophrenia do not show functional connectivity between the sensorimotor cortex and the right frontal gyrus , in contrast to healthy controls . the presence of a prefrontal dysfunction in schizophrenia is supported by extensive evidence from both structural and functional neuroimaging studies . prefrontal areas have been consistently reported as reduced in volume in patients with schizophrenia ( chan et al . , 2011 ; dazzan et al . , furthermore , fmri studies have shown altered activation of frontal areas during executive and working memory tasks , particularly with increasing capacity demand ( barch et al . , 2012 ; callicott et al . , 2000 ; perry et al . , 2001 ) . at a functional level , patients with schizophrenia also show an excess of signs reflecting frontal release , such as abnormalities in eye movements and short - term memory deficits , which again point to a frontal cortical alteration ( hyde et al . , 2007 ) . in this study , we tested the activation of frontal areas during a complex motor task , the fep task , which has long been considered in neurological and neurocognitive studies as indicative of frontal lobe lesions ( luria , 1966 ) . two previous fmri studies have reported that in healthy individuals , the execution of this task did not induce activation of prefrontal areas , but induced activation of other parts of the cortex , including the sensorimotor areas ( chan et al . , 2006 ; umetsu et al . , 2002 ) . a subsequent study , using the same ppi approach that we used in this study , showed that this finding could reflect an indirect involvement of the prefrontal cortex , which could exert an integrative and regulatory function on neural circuitry involved in complex motor sequences . we found here that in healthy individuals , the execution of the fep task , compared to a simpler motor act , was indeed associated with both direct activation of the prefrontal cortex , and greater coupling of prefrontal areas and sensorimotor cortex . interestingly , a recent meta - analysis of the brain connectome suggests that the frontal lobe represents a hub ( a region highly interconnected with other brain regions and valuable for integrative information processing and adaptive behavior ) particularly affected in patients with schizophrenia ( crossley et al . , 2014 ) . there is a paucity of studies on brain activation during complex motor tasks in these patients . however , neuroimaging studies that examined gross motor coordination signs such as finger - to - thumb operation tests in these patients have reported reduced activation in the smc and the supplementary motor area ( sma ) in comparison to healthy controls ( schrder et al . , a later similar study used pronation / supination tests and found significant reduced activation in the smc in patients with schizophrenia compared to healthy controls ( schroder et al . , 1999 ) . the lack of activation we found with a task like the fep , which requires fine motor coordination and requires executive processes like inhibition , planning and updating , could be interpreted as part of the hypofrontality hypothesis of schizophrenia reflecting a reduced function of the prefrontal cortex ( callicott et al . , 2000 ) . response relationship of the left frontal parietal activation across patients , their fdrs and healthy controls . here , in the pt vs. rest contrast , activation was lowest in the schizophrenia group , intermediate in the fdr group and highest in the healthy control group . although these differences were highlighted with a motor sequence less complex than the fep , the finding suggests that at least part of the pathophysiological substrate that underlies motor difficulties in psychosis may be linked to a genetic susceptibility to schizophrenia . indeed , deficits in motor skills have been reported extensively in individuals at risk of schizophrenia because of either genetic loading or prodromal features ( blanchard et al . , 2010 ; first , the clinical sample size was relatively small . however , the merit of the present sample is that it included both first - episode schizophrenia patients and their non - psychotic siblings . secondly , the use of ppi analysis may not fully detect the connectivity changes related to the performance of the fep task . however , ppi is an appropriate method to test the relationship between two simple mental activities ( friston et al . , 1997 ) . other more sophisticated approaches , such as dynamic casual modeling , may help identify further subtle connectivity changes in the performance of the fep task . thirdly , the use of the screen to synchronize the motor actions might induce the activation of mirror neurons . however , the contrasts between any two of the three motor actions ( fep , pt , and ps ) should minimize this effect by subtraction methods . finally , we should also consider the validity of our task , since previous subtraction analyses of the fep task did not elicit activation of the frontal cortex . however , the fact that the left frontal parietal region was significantly activated when participants performed the pt , ps and fep tasks with their right hand suggests that this hand movement imaging task was valid . notwithstanding these limitations , our findings suggest that the fep task may be a useful endophenotype of schizophrenia , as it fulfills the criteria of being familial and shows a dose previous study had demonstrated that patients with schizophrenia showed aberrant brain activation in the premotor area after a week of motor training ( kodama et al . , 2001 ) . given this temporal stability of nss , the fact that this is an imaging endophenotype rather than a simple behavioral rating arguably enhances its precision . this task is also simpler to carry out when compared to a full nss scale . we believe that our findings add valuable information to the understanding of the origin and underlying neural mechanism of motor coordination signs . future studies should recruit a larger sample of first - episode , preferably medication - nave schizophrenia patients for further validation of our findings .

neurological soft signs have been considered one of the promising neurological endophenotypes for schizophrenia . however , most previous studies have employed clinical rating data only . the present study aimed to examine the neurobiological basis of one of the typical motor coordination signs , the fist edge palm ( fep ) task , in patients with first - episode schizophrenia and their non - psychotic first degree relatives . thirteen patients with first - episode schizophrenia , 14 non - psychotic first - degree relatives and 14 healthy controls were recruited . all of them were instructed to perform the fep task in a 3 t ge machine . psychophysiological interaction ( ppi ) analysis was used to evaluate the functional connectivity between the sensorimotor cortex and frontal regions when participants performed the fep task compared to simple motor tasks . in the contrast of palm - tapping ( pt ) vs. rest , activation of the left frontal parietal region was lowest in the schizophrenia group , intermediate in the relative group and highest in the healthy control group . in the contrast of fep vs. pt , patients with schizophrenia did not show areas of significant activation , while relatives and healthy controls showed significant activation of the left middle frontal gyrus . moreover , with the increase in task complexity , significant functional connectivity was observed between the sensorimotor cortex and the right frontal gyrus in healthy controls but not in patients with first episode schizophrenia . these findings suggest that activity of the left frontal parietal and frontal regions may be neurofunctional correlates of neurological soft signs , which in turn may be a potential endophenotype of schizophrenia . moreover , the right frontal gyrus may play a specific role in the execution of the fep task in schizophrenia spectrum disorders .

colonic necrosis has been described as a rare complication after the administration of kayexalate [ 1 , 2 ] . in this case study , we present a case of calcium polystyrene sulfonate - induced colonic necrosis and perforation to remind clinicians of this rare , but dangerous , toxicity associated with this commonly used medication . a 78-year - old woman with stage 4 chronic kidney disease ( ckd ) due to chronic pyelonephritis , and a right hypoplastic kidney was presented to our emergency department with a 2-day history of abdominal pain . her medical history included epilepsy that was treated with carbamazepine , hypertension and hyperlipidaemia , for which she received carvedilol and atorvastatin . because of the persistent hyperkalaemia , she was treated with calcium polystyrene sulfonate at 30 g / day ( anti - potassium granule ; assos drug , istanbul , turkey ) . as a result of the ckd , she was prescribed calcitriol for a mineral and bone disorder , darbepoetin alpha for anaemia and sodium hydrogen carbonate for acidosis . upon initial physical examination , her abdomen was non - distended with normoactive bowel sounds , but there was diffuse tenderness in her abdomen . significant laboratory values at the time of admission included sodium 138 meq / l , potassium 4.6 meq / l , ph 7.20 , bicarbonate 13 meq / l , blood urea nitrogen 71 mg / dl , creatinine 2.6 mg / dl , leukocytes 15 500/mm , eosinophils 110/mm , haematocrit 32.7% , platelets 221 000/mm and c - reactive protein 10.9 mg / dl . on the second day of hospitalization , the abdominal pain worsened and free air under the diaphragm was found on abdominal radiography . her sigmoid colon was found to be necrotic and perforated . a biopsy was performed and the perforated segment was repaired by primary closure . the surface of the deep ulcer contained necroinflammatory debris and various sized fragments of basophilic crystalloid material with angulated margins on microscopic examination ( figure 1a and b ) . also , there were no features of chronic colitis , including inflammatory bowel disease or chronic ischaemic colitis . ( a ) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface ( haematoxylin and eosin , original magnification , 100 ) . ( b ) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface ( haematoxylin and eosin , original magnification , 400 ) . sodium polystyrene sulfonate can also bind intraluminal calcium , leading to constipation , fecal impaction and subsequent bowel obstruction or perforation . gerstman et al . reported a 0.27% overall incidence , with a higher incidence ( 1.8% ) during the postoperative period . sorbitol enemas , along with experimental evidence suggesting that the necrosis was due to sorbitol rather than the kayexalate in presence of uraemia . extensive transmural necrosis was noted in rats receiving enemas of sorbitol or kayexalate in sorbitol in both the uraemic and non - uraemic groups . as in this case report , renal failure may be an important facilitating factor in the pathogenesis of the necrosis . in contrast to the experimental data , our case was treated with calcium polystyrene sulfonate orally , not rectally . rashid et al . noted that kayexalate in sorbitol given as an enema or orally to treat hyperkalaemia has been reported to induce intestinal necrosis in uraemic patients . they studied clinical and pathologic features of 15 patients and observed kayexalate crystals in tissue specimens from surgical resections and endoscopic biopsies . one possibility is elevated renin levels , commonly seen in renal insufficiency , that predispose the patient to non - occlusive mesenteric ischaemia via angiotensin - mediated vasoconstriction . one gram of kayexalate possesses a theoretical in vitro exchange capacity of 23.1 meq of potassium and in vivo capacity of 1 meq . emmett et al . reported that in vivo potassium - binding capacity may be lower than previously estimated , more on the order of 0.40.8 meq / g of kayexalate resin . in contrast to other minor digestive complications associated with kayexalate treatment , colonic perforation results in significant morbidity and mortality . as a result , potassium exchange resins may , although rarely , induce a colonic perforation , and this diagnosis should be considered in a patient treated as such in case of acute abdomen . the clinicians must be aware of the possible rare and serious complications of potassium exchange resins .

sodium or calcium polystyrene sulfonate ( kayexalate or analog ) is an ion - exchange resin commonly used to treat hyperkalaemia in patients with chronic kidney disease . it is known to cause digestive complications , such as nausea , vomiting and constipation . although rare , colonic necrosis and perforation are very severe complications associated with the medication . in this case report , we present a case of calcium polystyrene sulfonate - induced colonic necrosis and perforation to remind clinicians of this rare , but dangerous , toxicity associated with this commonly used medication .

it was recently suggested that endometriosis may commence as an endometrial disease provoked by the aberrant expression of aromatase in this tissue , as this appears to play a pivotal role in its survival in ectopic locations.4 gene array studies have shown that the expression profiles of endometriosis lesions and the eutopic endometrium are similar , which supports the hypothesis that endometriosis is an endometrial disease.6 it is biologically plausible that the presence of aromatase activity in the endometrium is pivotal for the development of endometriosis by blocking phagocytosis by activated macrophages and may also be responsible for the exacerbation of menstrual symptoms whose onset predates the diagnosis of this pathology.1,4,7 in this hypothesis , endometriosis would be a late consequence of enzymatic changes that occur in the endometrium prior to its establishment outside the uterus , which could explain the lag that is often reported between the onset of symptoms and diagnosis of the disease . this hypothesis could be tested by examining patients with severe dysmenorrhea or infertility to determine whether laparoscopy reveals endometriosis despite aromatase expression already being positive in the endometrium . in the present study , aromatase expression was determined in the eutopic endometrium of patients with severe menstrual symptoms and/or infertility who underwent laparoscopy and hysteroscopy as a part of their diagnostic work - up . a total of 106 patients of reproductive age ( range = 1847 years ) received a laparoscopy and hysteroscopy with endometrial biopsy that was carried out using a 4 mm karman curette attached to a 10 ml syringe to produce a vacuum , which is the standard procedure for the evaluation of menorrhagia , severe dysmenorrhea , or infertility in the day hospital where the current study was carried out . all patients included in this group had reported at least two of these three symptoms for periods ranging from 2 to 8 years . the control group consisted of sixteen patients who received a laparoscopy for reasons unrelated to infertility or severe dysmenorrhea and who were found to be endometriosis - free during the examination . the indications for laparoscopy in this group were either benign non - endometriotic ovarian cysts ( seven patients ) or tubal ligation ( nine patients ) . in these patients , an endometrial biopsy was performed at the time of the laparoscopy using a 4 mm karman curette . the patients enrolled in the study gave written informed consent for immunohistochemical studies to be carried out on the endometrial biopsy specimens obtained as part of their standard medical care . laparoscopies were performed either during the proliferative phase ( n = 72 ) or in the secretory or menstrual phase ( n = 34 ) , since the timing of the procedure depended on the availability of the surgical theater and the specifics of the institution s waiting list . the laparoscopy reports and color images were retained in the patients files for further evaluation if necessary . during the laparoscopy procedure , the surgical team ascertained the presence of endometriosis and graded the stage of the disease in accordance with the american society of reproductive medicine ( asrm ) classification . 8 using laparoscopic classification criteria , the patients with endometriosis were divided into four groups according to the severity of their lesions , using the asrm classification of minimal ( stage i ) , mild ( stage ii ) , moderate ( stage iii ) , or severe ( stage iv ) . the severity of menstrual symptoms such as dysmenorrhea , the presence or absence of infertility , and the reason for laparoscopy were recorded in the patients files . aromatase expression was investigated using the commercially available monoclonal antibody mca2077 clone h4 ( serotech , raleigh , nc ) . antigen retrieval was performed using tris - edta buffer ( ph 8.0 ; sigma - aldrich , so paulo , brazil ) . the presence of aromatase expression was rated as positive if there was any detectable staining reaction in the glandular epithelium or negative when no reaction was observed . placental tissue and an atrophic endometrial sample were used as positive and negative controls , respectively , in all immunostaining reactions for aromatase p450 . statistical analysis was performed using the one - tailed chi - square test for differences in percentages , using an unnamed online interactive calculation tool for chi - square tests of goodness of fit and independence , which is hosted at http://quantpsy.org . in the group of patients with severe dysmenorrhea , endometriosis was diagnosed in 90/106 patients . in the remaining 16 cases , aromatase expression in the eutopic endometrium was detected by immunohistochemistry mainly in the glandular epithelium ( figure 1 ) . the staining reaction was positive in 66/92 patients ( 72% ) with endometriosis and symptoms of dysmenorrhea and/or infertility , and there were no significant differences between the phases of the patient s menstrual cycle . in symptomatic patients who were found to have normal pelvic results during the laparoscopy , aromatase expression was still positive in the endometrium in 13/14 cases ( 95% ) , though this difference was not statistically significant ( p = 0.09 ) . in the asymptomatic control group without endometriosis , however , aromatase expression was negative in the eutopic endometrium in all cases . in the subset of symptomatic patients with a laparoscopic diagnosis of endometriosis , the percentage of endometrial samples positively expressing aromatase in the glandular epithelium showed a trend toward greater positivity as the severity of the disease increased in accordance with the asrm criteria . aromatase expression was detected in the eutopic endometrium of 28/45 patients with stage i endometriosis ( 62% ) , 11/14 patients with stage ii ( 78% ) , 14/20 patients with stage iii ( 70% ) and 12/13 patients with stage iv endometriosis ( 92% ) . however , only the difference between stages i and iv of the disease was statistically significant ( p = 0.04 ) ( table 1 ) . the present study not only showed an association between the presence of aromatase expression in the eutopic endometrium and the severity of endometriosis lesions at laparoscopy but also suggests that this expression was already present in symptomatic patients in whom pelvic endometriosis was not found . these findings agree with the hypothesis that endometriosis may commence as an endometrial disease resulting from functional changes in the endometrium before the disease establishes outside the uterus . the presence of similar epigenetic changes in the eutopic endometrium and the endometriotic lesions further corroborates the hypothesis that endometriosis is an endometrial disease.6 the positive correlation between mrna levels in the eutopic endometrium and the severity of endometriosis and adenomyosis also supports this hypothesis . 9 whether endometriosis starts as an endometrial disease before establishing itself elsewhere in the pelvis6,7 can not be proven by the present study , but previous work shows that the onset of symptoms associated with this pathology may commence many years prior to its diagnosis , which support this hypothesis . 1 the occurrence of severe dysmenorrhea for many years prior to a diagnosis of endometriosis may reflect these initial endometrial changes favoring the upregulation of both estradiol and prostaglandin production in this tissue.4,6,7 the occurrence of such symptoms in patients with a normal pelvis at laparoscopy , as shown in the present study , may suggest that these functional changes in the endometrium are not only associated with the onset of endometriosis - related symptoms but that they may occur prior to the diagnosis of any pelvic pathology by laparoscopy . functional endometrial changes leading to an increase in aromatase expression may therefore predate the development of endometriosis , initially provoking dysmenorrhea and possibly infertility before triggering the development of the disease outside the uterus.4 it still remains to be determined whether this group of symptomatic patients represents a high - risk population for the development of endometriosis . in patients with an established diagnosis of endometriosis , however , endometrial positivity for aromatase tends to increase with the severity of the disease . this may be due to the greater possibility of spontaneous regression of early stage endometriosis , which may occur because of reduced aromatase activity in the eutopic endometrium . although aromatase expression in the eutopic endometrium may be pivotal for the onset and progression of endometriosis , it has yet to be clarified how this enzyme affects the development of endometriosis . the role played by estrogens in inhibiting immune surveillance by activated macrophages has been suggested as one of the likely mechanisms by which aromatase affects the course of endometriosis.4,7,10 this local estrogen production in the endometrial cells shed to the pelvis in retrograde menstruation may favor their survival in these ectopic locations by directly preventing their destruction by activated immune cells through the inhibition of the phagocytosis mechanism.1013 the presence of aromatase in the eutopic endometrium may therefore represent the initial step in the development of endometriosis and could be necessary for the development of lesions outside the uterus.7 the similar genetic alterations found between endometriotic lesions and the respective eutopic endometrium also indicate a common histological origin.7 the current study s finding that aromatase expression is detectable by immunohistochemistry in the endometrium of symptomatic , endometriosis - free patients agrees with the hypothesis that this enzyme initially exacerbates dysmenorrhea or causes infertility prior to facilitating the development of endometriosis elsewhere in the pelvis . a previous study has shown that aromatase expression in the endometrium reduces implantation rates in patients undergoing in vitro fertilization , however , which may help to explain the lower fertility rate of women with endometriosis.14 the presence of aromatase and other enzymes related to estrogen metabolism in the eutopic endometrium will ultimately create a hyperestrogenic milieu , resulting in increased prostaglandin production , particularly because cox-2 expression is upregulated by estradiol in this tissue.69,12,13 this would explain why the pelvis of a patient with severe dysmenorrhea or infertility may be normal , as was reported in the current study aromatase expression in the eutopic endometrium may be sufficient to provoke these symptoms by enhancing the production of proinflammatory prostaglandins in this tissue . however , the possibility that some of these endometriosis - free patients have adenomyosis can not be completely excluded in this study , despite the fact that myometrial findings were normal at transvaginal sonography . estrogens produced locally in the endometriotic lesions and eutopic endometrium result in the mechanism of decreased immune surveillance and enhanced tolerance found in endometriosis,14 which may explain the inverse relationship between estrogen levels and impaired nk - cell function found as a function of the severity of the disease . 15 local estrogen production would also stimulate the production of vegf , growth factors , and inflammatory cytokines by immune cells , thus favoring the progression of endometriosis.12,15,16 the blockade of phagocytosis by the local production of estrogens and the concomitant stimulation of growth and inflammatory factors would explain the paradoxical effects of macrophages in endometriosis , which seem to interact with rather than destroy these ectopic endometrial cells , thus enhancing their growth and angiogenesis.12,17 in conclusion , a vicious cycle of enhanced inflammation and local estrogen production mediated by cox-2 and aromatase expression in the eutopic endometrium of endometriosis patients may play a pivotal role not only in determining the clinical course of this disease but also in defining the intensity of its debilitating symptoms . in the pelvic cavity , the ensuing inflammatory reaction provoked by the arrival of endometrial cells will further exacerbate their preexisting aromatase activity , thus augmenting local estrogen production and halting phagocytosis by activated macrophages.6,7

objectiveto investigate whether aromatase expression in the eutopic endometrium correlates with the presence and severity of endometriosis in patients with infertility and/or dysmenorrhea undergoing laparoscopy and hysteroscopy.patientsthe study involved 106 patients of reproductive age with symptoms of dysmenorrhea and infertility . sixteen endometriosis - free asymptomatic patients were used as a control group.methodsconcomitant laparoscopy and hysteroscopy was carried out in all cases . an endometrial biopsy was taken to determine aromatase p450 expression by immunohistochemistry . endometriosis was staged according to the american society of reproductive medicine classification.resultsendometriosis was diagnosed by laparoscopy in 92/106 symptomatic patients . in this group , aromatase expression was detected in the eutopic endometrium of 66/92 patients with endometriosis ( 72% ) and in 13/14 ( 95% ) patients in the symptomatic , endometriosis - free group ( p = 0.09 ) . aromatase expression was not detected in any patients from the control group . in the endometriosis group , aromatase expression was detected in the eutopic endometrium of 28/45 patients ( 62% ) with american society of reproductive medicine classification stage 1 of the disease , in 11/14 patients ( 78% ) with stage ii , 14/20 patients ( 70% ) with stage iii , and in 12/13 patients ( 92% ) with stage iv ; however , the difference was only statistically significant between stages i and iv ( p = 0.04).conclusionaromatase expression in the endometrium was associated with the presence of dysmenorrhea and infertility irrespective of the presence of endometriosis . when endometriosis was present , however , there was a tendency for aromatase expression to be positively correlated with dysmenorrhea severity .

we also classified patients based on age , sex , performance status ( according to the eastern cooperative oncology group ) , international prognostic index ( ipi ) score , presence of b symptoms , clinical stage , serum lactate dehydrogenase ( ldh ) level , primary site and treatment protocol . clinical staging was determined according to the revised american joint committee on cancer ( ajcc ) for lymphoid neoplasm.9 the patients had the following laboratory work ups : complete blood count ( cbc ) , serum lactate de - hydrogenase ( ldh ) level , alkaline phosphatase ( alp ) , liver function tests , renal function tests , and x - ray or computed tomography ( ct ) of the bone lesion , chest x - ray ( cxr ) , abdominopelvic ultrasonography ( us ) or ct , and bone marrow biopsies ( bmb ) in 9 patients . all patients were evaluated for presence of b symptoms ( fever38c , night sweating , and weight loss10 kg in 6 months ) . overall survival ( os ) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . disease free survival ( dfs ) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . differences were considered significant if the p value was 0.05 ( two tailed ) . all survival analyses were performed using the spss ( ibm company , united states ) , version 14 . we retrospectively analyzed all patients ( 28 ones ) who were diagnosed with pbl and referred to our center , ( omid hospital ) , between march 2001 and february 2009 . all pathological paraffin blocks were reviewed by an experienced pathologist in our center and the pbl diagnosis was confirmed . we also classified patients based on age , sex , performance status ( according to the eastern cooperative oncology group ) , international prognostic index ( ipi ) score , presence of b symptoms , clinical stage , serum lactate dehydrogenase ( ldh ) level , primary site and treatment protocol . clinical staging was determined according to the revised american joint committee on cancer ( ajcc ) for lymphoid neoplasm.9 the patients had the following laboratory work ups : complete blood count ( cbc ) , serum lactate de - hydrogenase ( ldh ) level , alkaline phosphatase ( alp ) , liver function tests , renal function tests , and x - ray or computed tomography ( ct ) of the bone lesion , chest x - ray ( cxr ) , abdominopelvic ultrasonography ( us ) or ct , and bone marrow biopsies ( bmb ) in 9 patients . all patients were evaluated for presence of b symptoms ( fever38c , night sweating , and weight loss10 kg in 6 months ) . overall survival ( os ) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . disease free survival ( dfs ) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . differences were considered significant if the p value was 0.05 ( two tailed ) . all survival analyses were performed using the spss ( ibm company , united states ) , version 14 . we retrospectively analyzed all patients ( 28 ones ) who were diagnosed with pbl and referred to our center , ( omid hospital ) , between march 2001 and february 2009 . all pathological paraffin blocks were reviewed by an experienced pathologist in our center and the pbl diagnosis was confirmed . we also classified patients based on age , sex , performance status ( according to the eastern cooperative oncology group ) , international prognostic index ( ipi ) score , presence of b symptoms , clinical stage , serum lactate dehydrogenase ( ldh ) level , primary site and treatment protocol . clinical staging was determined according to the revised american joint committee on cancer ( ajcc ) for lymphoid neoplasm.9 the patients had the following laboratory work ups : complete blood count ( cbc ) , serum lactate de - hydrogenase ( ldh ) level , alkaline phosphatase ( alp ) , liver function tests , renal function tests , and x - ray or computed tomography ( ct ) of the bone lesion , chest x - ray ( cxr ) , abdominopelvic ultrasonography ( us ) or ct , and bone marrow biopsies ( bmb ) in 9 patients . all patients were evaluated for presence of b symptoms ( fever38c , night sweating , and weight loss10 kg in 6 months ) . overall survival ( os ) was calculated from the date of pathological diagnosis to the date of the last follow up or death from any cause . disease free survival ( dfs ) survival curves were constructed according to the method of kaplan - meier10 and compared using the log rank test . differences were considered significant if the p value was 0.05 ( two tailed ) . all survival analyses were performed using the spss ( ibm company , united states ) , version 14 . patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement ( 71% ) including humerus 7 ( 25% ) , femur 7 ( 25% ) , and tibia 4 ( 14.3% ) . the pelvis was the second most common site of involvement in 7 ( 25% ) cases . mandible was involved in 1 ( 3.5% ) , radius in 1 ( 3.5% ) and clavicle in 1 ( 3.5% ) patient . 26 ( 93% ) patients had dlbcl and 2 ( 7% ) patients had small lymphoblastic lymphoma . in 15 ( 53.6% ) , patients b symptoms were reported . serum ldh level was recorded in 20 patients ; in 6 ( 30% ) patients was 500 and in 14 ( 70% ) > 500 . according to eastern cooperative oncology group ( ecog ) performance status scoring , 5 patients ( 17.9% ) had ps of -1 , 9 ( 32.1% ) had ps of -2 , 12 ( 42.9% ) had ps of -3 and 2 ( 7.1% ) ones had ps of -4 . we did nt have any patient with stage 3 ( based on ajcc staging system ) . a stage 4 was defined as multiple bone sites lesions ( 2 cases ) , diffuses involvement of single bone ( 3 cases ) or bm involvement ( 1 case ) . because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma , we excluded all patients with bone lymphoma and non - regional lymph node involvement . chemotherapy with cyclophosphamide ( ctx ) , vincristin ( vcr ) , doxorubicin ( adr ) and prednisolone ( chop regimen ) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 ( range : 1 - 8 ) . one patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy , on cobalt 60 conventional protocol . we excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . at the end of follow up , 16 ( 61.5% ) dfs did nt reach the median because of small number of events ( death or recurrence ) but the mean dfs was 51 months ( range : 37 - 66 ) . overall survival was 54 months ( range : 40 - 68 , figure 2 ) . overall survival curve 4 ( 15% ) cases recurred . the mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies , 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients ( 29% ) died because of disease progression in 5 cases , recurrence in 3 cases and treatment complication in one . we also compared chemotherapy alone with chemoradiotherapy based on dfs and os ( figures 3 and 4 ) . dfs curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) os curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) 19 ( 73% ) patients received chemoradiotherapy and 7(27% ) patients received chemotherapy alone . os was significantly better in the chemoradiotherapy group compared with other two groups ( 64 vs. 27 months , respectively , p=0.014 ) . dfs was also significantly better in combined modality arm compared with other two groups ( 64 versus 21 months , respectively , p=0.003 ) . patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement ( 71% ) including humerus 7 ( 25% ) , femur 7 ( 25% ) , and tibia 4 ( 14.3% ) . the pelvis was the second most common site of involvement in 7 ( 25% ) cases . mandible was involved in 1 ( 3.5% ) , radius in 1 ( 3.5% ) and clavicle in 1 ( 3.5% ) patient . 26 ( 93% ) patients had dlbcl and 2 ( 7% ) patients had small lymphoblastic lymphoma . in 15 ( 53.6% ) , patients b symptoms were reported . serum ldh level was recorded in 20 patients ; in 6 ( 30% ) patients was 500 and in 14 ( 70% ) > 500 . according to eastern cooperative oncology group ( ecog ) performance status scoring , 5 patients ( 17.9% ) had ps of -1 , 9 ( 32.1% ) had ps of -2 , 12 ( 42.9% ) had ps of -3 and 2 ( 7.1% ) ones had ps of -4 . we did nt have any patient with stage 3 ( based on ajcc staging system ) . a stage 4 was defined as multiple bone sites lesions ( 2 cases ) , diffuses involvement of single bone ( 3 cases ) or bm involvement ( 1 case ) . because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma , we excluded all patients with bone lymphoma and non - regional lymph node involvement . chemotherapy with cyclophosphamide ( ctx ) , vincristin ( vcr ) , doxorubicin ( adr ) and prednisolone ( chop regimen ) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 ( range : 1 - 8 ) . one patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy , on cobalt 60 conventional protocol . we excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . at the end of follow up , 16 ( 61.5% ) dfs did nt reach the median because of small number of events ( death or recurrence ) but the mean dfs was 51 months ( range : 37 - 66 ) . overall survival was 54 months ( range : 40 - 68 , figure 2 ) . overall survival curve 4 ( 15% ) cases recurred . the mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies , 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients ( 29% ) died because of disease progression in 5 cases , recurrence in 3 cases and treatment complication in one . we also compared chemotherapy alone with chemoradiotherapy based on dfs and os ( figures 3 and 4 ) . dfs curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) os curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) 19 ( 73% ) patients received chemoradiotherapy and 7(27% ) patients received chemotherapy alone . os was significantly better in the chemoradiotherapy group compared with other two groups ( 64 vs. 27 months , respectively , p=0.014 ) . dfs was also significantly better in combined modality arm compared with other two groups ( 64 versus 21 months , respectively , p=0.003 ) . patients demographic characteristics patients clinical characteristics long bones were the most primarily site of involvement ( 71% ) including humerus 7 ( 25% ) , femur 7 ( 25% ) , and tibia 4 ( 14.3% ) . the pelvis was the second most common site of involvement in 7 ( 25% ) cases . mandible was involved in 1 ( 3.5% ) , radius in 1 ( 3.5% ) and clavicle in 1 ( 3.5% ) patient . 26 ( 93% ) patients had dlbcl and 2 ( 7% ) patients had small lymphoblastic lymphoma . in 15 ( 53.6% ) , patients b symptoms were reported . serum ldh level was recorded in 20 patients ; in 6 ( 30% ) patients was 500 and in 14 ( 70% ) > 500 . according to eastern cooperative oncology group ( ecog ) performance status scoring , 5 patients ( 17.9% ) had ps of -1 , 9 ( 32.1% ) had ps of -2 , 12 ( 42.9% ) had ps of -3 and 2 ( 7.1% ) ones had ps of -4 . we did nt have any patient with stage 3 ( based on ajcc staging system ) . a stage 4 was defined as multiple bone sites lesions ( 2 cases ) , diffuses involvement of single bone ( 3 cases ) or bm involvement ( 1 case ) . because it was impossible to distinguish pbl from bone involvement due to nodal lymphoma , we excluded all patients with bone lymphoma and non - regional lymph node involvement . chemotherapy with cyclophosphamide ( ctx ) , vincristin ( vcr ) , doxorubicin ( adr ) and prednisolone ( chop regimen ) used for 26 patients with standard doses.3 the mean chemotherapy cycles were 5 ( range : 1 - 8 ) . one patient did not receive any adjuvant treatment and one patient received radiotherapy only with a total dose of 5000 cgy , on cobalt 60 conventional protocol . we excluded two patients from survival analysis because one did not take any treatment and another did not finish his treatment program . at the end of follow up , 16 ( 61.5% ) dfs did nt reach the median because of small number of events ( death or recurrence ) but the mean dfs was 51 months ( range : 37 - 66 ) . overall survival was 54 months ( range : 40 - 68 , figure 2 ) . overall survival curve 4 ( 15% ) cases recurred . the mean time to progression was 16 months . 2 cases recurred with non regional lymphadenopathies , 1 patient had brain recurrence and in 1 case recurrence was in humerus . 9 patients ( 29% ) died because of disease progression in 5 cases , recurrence in 3 cases and treatment complication in one . we also compared chemotherapy alone with chemoradiotherapy based on dfs and os ( figures 3 and 4 ) . dfs curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) os curves comparing chemoradiotherapy ( crt ) ( 1 ) versus chemotherapy alone ( 2 ) 19 ( 73% ) patients received chemoradiotherapy and 7(27% ) patients received chemotherapy alone . os was significantly better in the chemoradiotherapy group compared with other two groups ( 64 vs. 27 months , respectively , p=0.014 ) . dfs was also significantly better in combined modality arm compared with other two groups ( 64 versus 21 months , respectively , p=0.003 ) . it was first described as a distinct clinicopathological entity in 1939 by parker and jackson.11 the real prevalence of pbl is unclear because of the considerable difficulty distinguishing primary from secondary bone lymphoma . in our study pbl did constitute 5.3% of all dlbcls like many previous study results.312 long bone was the primarily affected site in our case series , the same as western country reports in which femur was the most affected site.36713 but in ramadan et al . results , spine involvement had the same prevalence as the long bones ( 33%).4 in dai maruyama et al . report , the pelvis was the common involved site ( 54%).5 horsman et al . reported that the most commonly presented site was the pelvis , although they described that the femur was the most frequently involved bone.13 most of our patients were younger than 60 years which was similar to other reports.3 according to the who classification , lymphoma involving the bone can be classified in four groups : group 1 , lymphoma in a single bone site with or without regional lymph node involvement ; group 2 , lymphoma in multiple bones , but no visceral or lymph node involvement ; group 3 , bone tumor with involvement of other visceral sites or lymph nodes at multiple sites ; and group 4 , lymphoma involving any other site and found by bone biopsy which was done to rule out possible involvement . we defined pbl in our study according to who classification groups 1 and 2 . who classification and some previous reports have indicated that groups 1 and 2 should be considered as pbl , but group 3 should be excluded from pbl and be considered as systemic lymphoma regardless of the bone lesion . histopathologically , the previous studies reported that the majority of patients with pbl had dlbcl.3615 in our study , 26 ( 93% ) patients had dlbcl and 2 ( 7% ) patients had small lymphoblastic lymphoma . several studies have suggested that the combined modality ( chemotherapy and radiotherapy ) was the best treatment for patients with pbl.36 beal et al . concluded that pbl patients treated with combination chemotherapy and irradiation had significantly better survival than the patients treated with single modality ( chemotherapy or radiotherapy alone ) , but the 5-year os rate between the two groups was not significantly different.16 in our study , os and dfs was significantly better in combined modality group ( os was 64 vs. 27 months , respectively , p=0.014 , and dfs was 64 vs. 21 months , p=0.003 ) . ramadan et al . reported patients with advanced - stage disease who received chemotherapy plus irradiation with a poor outcome when compared with those who received chemotherapy alone ( 10-years os were 25% and 56% , respectively).4 however , this difference must be very cautiously interpreted because the decision to use radiotherapy was individualized . because of small number of patients in our study , further studies are necessary to clarify the characteristics of pbl and its optimal treatment strategy . we will continue our study on pbl and report our results with more patients and longer follow ups . all the authors have carried out the study , participated in the design of the study and acquisition of data performed the statistical analysis and wrote the manuscript .

background : primary bone lymphoma ( pbl ) is a rare disease and distinct clinicopathological entity . the optimal treatment strategy is still unclear . because of rarity of pbl , we report our institute experience in pbl clinicopathological feature and treatment results.methods:28 patients diagnosed with pbl were referred to omid hospital , cancer research center ( crc ) , between march 2001 and february 2009 . immunophenotype studies on 16 out of 28 pathological blocks were performed . we analyzed disease free survival ( dfs ) and overall survival ( os ) rates.results:14 patients with pbl were analyzed retrospectively . 17 patients ( 60.7% ) were male and 11 ( 39.3% ) were female with a median age of 41 years ( range : 11 - 79 ) . long bones were the most primarily site of involvement ( 71% ) . 26 ( 93% ) patients had diffuse large b cell lymphoma and 2 ( 7% ) had small lymphoblastic lymphoma . one ( 3% ) patient received radiation alone , 18 ( 66% ) cases received combined modality ( chemotherapy + radiotherapy ) and 8 ( 30% ) received only chemotherapy during their treatment period . the median follow up was 18 months ( range : 1 - 82 ) . mean dfs was 51 months ( range : 37 - 66 ) . overall survival ( os ) was 54 months ( range : 40 - 68 ) . os was significantly better in the chemoradiotherapy group compared with other two groups ( 64 versus 27 months , respectively , p=0.014 ) . dfs was also significantly better in combined modality arm compared with other two groups ( 64 versus 21 months , respectively , p=0.003).conclusions : in spite of small number of patients reported in this study , combined modality treatment ( chemotherapy and radiotherapy ) was shown to be useful as an effective treatment strategy in pbl .

the dental industry has a long history in the development of dental prostheses to recover a patient 's tooth function.1 as a substitute for teeth , a dental prosthesis must show stable durability , aesthetic value , precise function , and convenient use , as well as biocompatibility in order to perform the desired function properly . in addition , these factors must be applied to a wide range of manufacturing methods used in the construction of dental prostheses.2 metal ceramic is a very common material used worldwide , and it has been successfully used as the gold standard for long - term clinical use ; it provides excellent results in stability , aesthetic value , and marginal adaptation.345 in recovery using a dental prosthesis , marginal adaptation is an important factor.3 an inappropriate margin could cause a minute gap between the abutment tooth and prosthesis , which may lead to a periodontal lesion , plaque accumulation , secondary caries , microleakage , inflammation after endodontic treatment , or periodontal disease.5678 in addition , according to previous research , a defective margin may cause a failure of the long - term preservation of the prosthesis , resulting in an increase in the failure rate.9 conventional fabrication methods for a prosthesis is a series of processes that includes taking an impression of the patient 's oral cavity , pouring stone , producing a wax pattern , and performing the investing , casting , and polishing . however , during this process , the risk of inaccuracy may increase due to the properties of the material used and the worker 's ability . in addition , temporal labor and cost could increase as well.1345 therefore , to address these problems , an automated cad / cam system was introduced to the dental field.15 the cad / cam system is a type of subtractive manufacturing that cuts the materials to the desired shape and size . it enables a larger quantity of production than traditional methods , is easy to use , and saves the time . because of these advantages , the cad / cam system is widely used.5 however , bornemann et al.10 showed that this system tends to reduce accuracy through the scanning process , software design , milling , and a number of other related processes . this results in too much consumption of raw material , and the waste of bur was increased . accordingly , the additive manufacturing ( am ) method , which supplements labor - intensive conventional manufacturing methods and subtractive manufacturing methods with high raw material consumption , is being considered a technology - intensive alternative in the field . multi - jet modeling , an additive manufacturing process used in the dental field , is the 3d printer , which is very advantageous in terms of manufacture speed and applicability with various materials compared with other 3d printings , as it has a number of jet nozzles.11 as a very professional 3d printer , a newly - launched additive manufacturing process " micro - sla " is characterized by high accuracy , and thanks to its minute ability in realization it is more appropriate for manufacturing the dental prosthesis than any other printers . also , compared to the other 3d printings , it is cheaper and speedy printing ( 14 mm / hour on the basis of the vertical standard ) is possible to shorten the time required . as a disruptive technology , am has the potential to revolutionize our lives , work , and international economy.12 only a few companies are applying am to dentistry , and , therefore , there are a limited number of studies done in this field . identifying the limits and advantages of this manufacturing method is an important task in the prosthesis and dentistry fields . thus , the purpose of this study was to verify whether the marginal and internal gap of a prosthesis made according to the am method is within the clinically allowable range by conducting a comparative evaluation of the conventional lost wax technique ( clwt ) , the subtractive manufacturing system with wax blank milling ( wbm ) , and am with multi jet modeling ( mjm ) and micro - stereolithography ( micro - sla ) . the null hypothesis was that there is no difference in the marginal and internal gap among the 4 groups . an acrylic model ( standard working model ag-3 zpvk 13 , 14 , 16 , frasaco gmbh , tettnang , germany ) with abutment teeth was used . therefore , the maxillary right canine , first pre - molar , first molar were provided with a 360 1.0 mm chamfer preparation.13 the incisal and occlusal reductions were 1.5 - 2.0 mm . the maxillary right canine , first pre - molar , first molar were reproduced using duplication silicone ( deguform , degudent gmbh , hanau , germany ) . in the reproduced area , the epoxy model ( master model ) was reproduced by pouring the epoxy ( modralit 3k , dreve dentamid gmbh , unna , germany ) . for the reproduced epoxy model , 10 plaster molds for each tooth were produced using duplication silicone , and , as a result , a total of 30 molds were produced . after filling the plaster molds with type iv stone ( dentona esthetic - base gold ; dentona ag , dortmund , germany ) , a total of 30 study models were produced . the study models were scanned by a non - contact blue light scanner ( identica ; medit co. , ltd . , based on the scanned files , a metal framework was designed by delcam power shape pro ( delcam plc , birmingham , uk ) according to the manufacturer 's instruction , with the following parameters ( thickness ) : 30 m for the cement film , 0.3 mm for the maxillary right canine , and 0.5 mm for the maxillary right first pre - molar and first molar . from this design , a standard template library ( stl ) file was created . for the clwt method , the lost wax technique was applied . after applying separating medium onto the study models and passing through the wax dipping process , each of the 10 abutment teeth , 30 in total , for the wbm method , the 30 wax patterns were produced , based on the stl files , using the cad / cam system ( dwx-50 , roland dg corporation , shizuoka , japan ) and milling the wax blank ( dmax co. , ltd . , , the 30 resin patterns were produced using the mjm printer ( projet - dp3000 , 3d system , rock hill , sc , usa ) and the stl files by jetting the light curing resin ( build material visijet dp200 , visijet , 3d system , rock hill , sc , usa ) and wax ( support material visijet s100 , visijet , 3d system , rock hill , sc , usa ) simultaneously through the inkjet print heads . finally , for the micro - sla method , the 30 resin patterns were produced using a micro - sla printer ( projet1200 , 3d systems , rock hill , sc , usa ) and the stl files , by projecting the desired metal framework via the beam projector onto the liquid uv curable plastic ( visijet ftx green material , 3d systems , rock hill , sc , usa ) . the test piece was mounted on the uv curing station and photopolymerized for 10 minutes . the patterns were placed in the crucible former , covered with the metal ring , and invested in accordance with the proper water / powder ratio through phosphate - bonded investment ( deguvest - impact - degussa - hls , hanau , germany ) . after passing through the burnout furnace ( ring furnace , seki dental co. , seoul , korea ) , the nickel - chromium ( ni - cr ) alloy ( verabond 2v ; aalbadent inc . , fairfield , ca , usa ) was cast in the casting machine ( seki dental co. , seoul , korea ) , and each test piece was produced ( fig . , skilled dental technician has used a silicone replica method . after mixing the light - body silicone ( aquasil ultra xlv regular set , dentsply caulk , milford , de , usa ) , it was injected between the metal framework and model , and 50 n of finger pressure14151617 was applied . after hardening , the metal framework was separated carefully from the model , and heavy - body silicone ( aquasil ultra rigid regular set , dentsply caulk , milford , de , usa ) was injected into the circular tray so that the light - body and heavy - body silicone could combine through an even pressure , and embed the light - body silicone . in order to cut the equal part , the zig ( modralit 3k , dreve dentamid gmbh , unna , germany ) was made by duplicating each two epoxy models , cutting one in a bucco - lingual direction , and cutting the other in a mesio - distal direction . the silicone replica reproduced by using this method was cut in a bucco - lingual direction and mesio - distal direction , respectively , using a razor blade ( fig . the thickness of the light - body silicone replica , which corresponds to the gap between model and the metal framework , was measured using a digital microscope ( kh-7700 ; hirox , tokyo , japan ) at 140 magnification . the margin gaps ( mgs ) , which corresponded to the absolute marginal discrepancy and internal gap , rounded chamfer ( rc ) , axial wall ( aw ) , incisal area ( ia ) , and occlusal area ( oa ) were measured . to confirm an accurate measurement , the measurement points ( 16 in total ) 3 ) . the total gap and the mean and standard deviation of the 16 points were determined , and this data met the hypothesis of a normal distribution ( p>.01 ) . the 16 points were divided into 4 regions as follows : mg , points 1 , 8 , 9 , 16 ; rc , points 2 , 7 , 10 , 15 ; aw , points 3 , 6 , 11 , 14 ; and ia or oa , points 4 , 5 , 12 , 13 . after each region 's mean and standard deviation were determined , two - way analysis of variance ( anova ) was conducted to evaluate the difference in average values according to tooth type and fabrication method . as the reciprocal action between the tooth variable and fabrication method variable was significant ( p<.05 ) , this analysis allowed verification of the significance of the difference between groups as a full factorial model . the level of the type i - error for statistical significance was fixed at 0.05 , and the statistical analysis was conducted by using ibm spss statistics 21.0 ( ibm co. , armonk , ny , usa ) . results of the analysis of the marginal and internal gap according to tooth type and fabrication methods are listed as the mean and standard deviation ( table 1 ) . the mean value of mg , rc , aw , ia or oa according to the four fabrication methods and the three tooth types are shown in figures 4 and 5 . as a result of the two - way anova analysis of the means , there was a significant difference in mg according to tooth type ( p<.001 ) and fabrication method ( p<.037 ) . in addition , there was a significant interaction ( p<.001 ) between tooth type and fabrication method . further , there were significant differences in rc , aw , and ia or oa according to tooth type ( p<.001 ) and fabrication method ( p<.001 ) , with significant interaction ( p<.001 , table 2 ) between tooth type and fabrication method . the results of the post - hoc test using the tukey hsd method ( table 2 , fig . 5 ) indicate that mg was the lowest for the molar type fabricated using the clwt method , and was the highest for the canine type fabricated using the mjm method . in the case of rc , the canine type manufactured using the clwt method had the lowest value , while the molar type manufactured using the mjm method had the highest value . in aw , the premolar type fabricated using the wbm method showed had the lowest value , while the molar type fabricated using the clwt method had the highest value . lastly in the case of ia or oa , the canine type fabricated using the clwt method had the lowest value , while the molar type fabricated using the mjm method had the highest value . to assess the results measured at individual points ( 16 points ) , results were classified into four regions : mg , rc , aw , and ia or oa . in mg , the canine and molar types showed the lowest value when fabricated using the clwt method , and the premolar type showed the lowest value using the micro - sla method . in the case of rc , the canine type showed the lowest value using the clwt method , while premolar and molar types showed the lowest value using the micro - sla method . in case of aw , the lowest value of the canine type was shown using the clwt method , while the lowest values for the premolar type and molar type were shown using the wbm method and the mjm method , respectively . in the case of ia or oa , the canine type showed the lowest value using the clwt method , while the premolar and molar type showed the lowest value using the micro - sla method ( table 2 , fig . 4 , fig . 5 ) . in addition , a whole am , mg , ia or oa , and rc showed a lower value ( fig . this study evaluated the marginal and internal gaps according to four fabrication methods , in order to verify the applicability of am in dentistry . clwt and micro - sla did not show a significant difference in the marginal gap , but , since these fabrication methods are significantly different from the other two methods , the null hypothesis was rejected . as shown in figures 4 and 5 , the values increased in the following order : aw , mg , ia or oa , and rc . because the abutment tooth was parallel to prosthetic appliance , aw showed a low value . however , because the occlusal surface has an irregular curve , ia or oa showed a relatively higher value compared to mg . rc had the highest value because the shape of the margin forms a rounded chamber . according to the table 2 , the marginal gap using either clwt or micro - sla was better than that using the other two methods . in other studies , clwt has been shown to achieve the most suitable marginal - adaptation value , and , therefore , clwt has been designated as the gold standard.5 however , considering that the micro - sla method shows no significant difference from the clwt method in the marginal gap , we infer that the micro - sla method has a better fitness value than the wbm and mjm methods . in the wbm method , it is difficult to reproduce the projection part , undercut part , and sharp edge accurately due to the positive error , as well as negative error resulting from the limits of the currently available bur diameters.216 in spite of the advantages of the mjm method in delicacy and precision , it combines wax and thermoset material in the fabrication process . thus , the mjm method has several drawbacks such as weak solidity among the 3d printers and its deformation at high temperatures.12 as a modified method of digital light processing , the newly released micro - sla method projects a shaped light beam on the liquid photopolymer resin , hardens the resin as projected , builds the model layer by layer , and then , hardens the built shape by exposure to light again in the built - in uv curing station . the advantage of this method is that the manufacturing speed is even as the model forms and is comparatively fast . the micro - sla method shows high precision and surface roughness since the layers are applied at a thickness of 30 m . the sls method and sla method are currently the most widely used am method.17 according to the research relating to the stereolithography ( sla ) method , the mean ( sd ) of margin , axial wall , occlusal are 96.9 m ( 17.6 m ) , 84.7 m ( 16.8 m ) , 114.2 m ( 16.7 m ) respectively.17 and as a result of the research , mjm method and micro - sla method showed better fitness value , except the occlusal part in the current study . according to the report of rtorp et al.,16 the mean ( sd ) value of the sls showed the best fitness value , with the measured value 133 m ( 89 m ) , 117 m ( 89 m ) , 166 m ( 135 m ) , and 84 m ( 60 m ) , at all measurement points in the conventional lost wax method , milled wax method , milled co - cr , and sls method . however , according to the earlier research , the mean ( sd ) value of the mg ( absolute marginal discrepancy ) part of premolar and molar made in the sls method showed 132.1 m ( 60.5 m ) and 128.0 m ( 68.8 m ) each . and it was found that the sls method has worse fitness than the mjm method and micro - sla method , showing the higher values than those of both methods used in the current study.15 also , kim et al.15 reported that the efforts to improve the sls method will be required since the current sls method is highly inferior to the conventional lost wax technique method and even has the gaps beyond the clinically allowable range . according to previous studies , no difference is observed in the marginal and internal gap between anterior , premolar , and molar teeth.1819 however , nakamura et al.20 showed that there are differences according to the tooth type . our results on the canine , premolar , and molar teeth ( table 2 , fig . 4 ) indicate a difference in the marginal and internal gap according to tooth type . this is significant because each tooth type has a different morsal surface condition and appearance , although there is the uniformity in the tamper degree and chamfer margin . there are several ways to measure the fitness of a prosthesis , including a direct measurement after the cementation process of the prosthesis on the tooth model21 or observing the inside of the prosthesis using x - ray micro - computed tomography ( micro ct).2223 in the present study , we used the cross - sectioning replica technique with silicone , which is considered the most suitable method to measure prosthesis prior to cementation.24 according to previous studies , there is much controversy as to the clinical validity of the size of the margin adaptation5 . for example , fransson et al.25 reported that the clinically allowable range is 100 m , and the value suggested by mclean and von fraunhofer26 and belser et al.27 is 120 m . beuer et al.28 stated that the size of the marginal adaptation ranges from 100 to 150 m and boening et al.29 suggested the range is from 100 to 200 m based on the long - term preserved prosthesis . in this study , all of the mg results showed values within the clinically allowable range suggested by the preceding studies ( table 2 ) . in addition , the values for rc , aw , ia or oa were also within the clinically allowable range ( table 1 and table 2 ) . therefore , all the four methods can be used clinically . a limitation to this study is that there could be an error in the resin pattern and wax pattern due to the characteristic contraction of the material itself . thus , the development of suitable materials should be included in future studies , and , especially in the case of the am methods , further clinical studies should be conducted . all of the four fabrication methods have sufficient marginal adaptation , since the marginal and internal gaps were within the allowable clinical range . the results of the micro - sla method showed a statistically significant difference in outcome compared to the wbm and mjm methods and a significant difference from the gold standard clwt method in the internal gap , but no statistically significant difference in the marginal gap from the clwt method . together , our results demonstrate that additive manufacturing can be used clinically as an alternative to the conventional lost wax - technique or subtractive manufacturing in the creation of dental prostheses .

purposethe purpose of this study was to verify the clinical - feasibility of additive manufacturing by comparing the accuracy of four different manufacturing methods for metal coping : the conventional lost wax technique ( clwt ) ; subtractive methods with wax blank milling ( wbm ) ; and two additive methods , multi jet modeling ( mjm ) , and micro - stereolithography ( micro - sla).materials and methodsthirty study models were created using an acrylic model with the maxillary upper right canine , first premolar , and first molar teeth . based on the scan files from a non - contact blue light scanner ( identica ; medit co. ltd . , seoul , korea ) , thirty cores were produced using the wbm , mjm , and micro - sla methods , respectively , and another thirty frameworks were produced using the clwt method . to measure the marginal and internal gap , the silicone replica method was adopted , and the silicone images obtained were evaluated using a digital microscope ( kh-7700 ; hirox , tokyo , japan ) at 140x magnification . analyses were performed using two - way analysis of variance ( anova ) and tukey post hoc test ( =.05).resultsthe mean marginal gaps and internal gaps showed significant differences according to tooth type ( p<.001 and p<.001 , respectively ) and manufacturing method ( p<.037 and p<.001 , respectively ) . micro - sla did not show any significant difference from clwt regarding mean marginal gap compared to the wbm and mjm methods.conclusionthe mean values of gaps resulting from the four different manufacturing methods were within a clinically allowable range , and , thus , the clinical use of additive manufacturing methods is acceptable as an alternative to the traditional lost wax - technique and subtractive manufacturing .

geriatric population is on rise globally because of increasing life span . as per the us census , people above 60 years constituted 6.4% of the total population in 1900 , which increased to 18.4% in 2010 and predicted to go up to 25.5% by 2050.1 spinal problems and spine surgeries in geriatric population are also showing a similar trend . lumbar fusion surgeries in people aged 60 years and above have increased by 230% in a decade from 1991 to 2001.2 desire to lead a more active life in advanced age , improved diagnostic techniques , and better operative results are some of the reasons for increasing spine surgeries in the elderly . in general , spine surgery in the elderly in the presence of comorbidities is feared among both patients and surgeon , as it is presumed to have higher perioperative complications and increased cost . many articles can be found in literature supporting this.345 a study by daubs et al . involving adult spinal deformity in people over 60 years of age has reported that age and complication rates do not affect the surgical outcome.6 similar studies have reported 91%96% good to excellent results following surgical treatment of lumbar canal stenosis ( lcs ) by decompression and decompression with fusion on people aged above 6570 years.789 this indicates that in the absence of complications , spinal decompression and fusion surgeries would result in a satisfied patient even in the elderly with comorbidities . therefore , measures to reduced complications in such patients should be looked at rather than denying surgical management in symptomatic patients due to their old age or comorbidities . this study evaluates the perioperative complications and the contributing factors in patients over 60 years of age undergoing lumbar fusion surgeries . patients aged 60 years and above with one or more comorbidities undergoing lumbar decompression and instrumented fusion at our institute between january 2012 and december 2013 ( 2-year period ) were included in the study . in all these patients , perioperative complications ( intraoperative and complications occurring within 3 weeks postoperative period ) and their incidence were recorded . the technique was a standard open technique of pedicle screw instrumentation and fusion , either interbody by transforaminal approach or posterolateral using morcellized bone from the posterior elements or rarely with iliac crest . age , number of levels instrumented and fused , operative time , blood loss , comorbidities , and the duration of stay were correlated with the incidence of perioperative complications using spss software ( ibm , spss statistics v 23.0 , new york , united states ) . factors contributing to perioperative complications were noted and measures to reduce them were suggested by these results and compared with the available literature in discussion . analysis of our medical records revealed a total of 52 patients operated by lumbar fusions in the 2-year study period , who were aged 60 years and above and had one or more comorbidities . most common indication for surgery was spondylolisthesis in 17 ( 32.7% ) followed by lcs in 15 ( 28.8% ) patients . hypertension ( htn ) was the most common comorbidity found in 39 patients ( 75% ) , followed by diabetes mellitus ( type 2 ) in thirty patients ( 56.4% ) . twenty patients had single comorbidity while 18 patients had two comorbidities and 13 patients were found to have three comorbidities [ table 1 ] . preoperative mri t2w , midsagittal ( a ) and axial ( b ) images showing multilevel listhesis and canal stenosis . postoperative x - ray lumbosacral spine anteroposterior ( c ) and lateral ( d ) views showing implant ( pedicle screws ) in situ following instrumented fusion diagnosis and comorbidites of patients forty six patients were operated under general anesthesia ( ga ) while the remaining six patients were operated in regional or spinal anesthesia . 3.8 levels were the average levels instrumented per patient while one patient underwent 9 levels instrumentation . interbody fusions were performed at single level in 24 , 2 levels in 22 , and 3 levels in 6 patients . average operative time and blood loss were 150 min ( range 60270 min ) and 369 ml ( range 901050 ml ) , respectively . both operative time and blood loss the levels of instrumentation and fusions were decided on segmental instability observed on dynamic radiographs . the interbody fusions were based on degree of segmental stenosis , disc degeneration , and instability . clinical details of patients operative time and blood loss with respect to the number of interbody fusion levels a total of 11 complications were noted , 3 systemic and 8 local . among the systemic complications , 2 were hypostatic pneumonia with secondary infection and one was a psychiatric illness called ganser 's syndrome . all the three patients required transfer to icu and one patient with pneumonia expired due to septicemia and shock . the average total duration of stay in the hospital was 6.2 days ( range 4 - 14 days ) . on comparing the complication rates with other variables , we found that the patients with complications had higher blood loss , operative time , number of instrumented levels , and number of interbody fusion levels [ tables 4 and 5 ] . similarly , the duration of stay was longer in these patients . on analyzing these results statistically by anova , the association of blood loss with complications was found to be statistically significant with p = 0.002 . the duration of stay , operative time , and number of interbody fusion levels were close to significance with p = 0.63 , 0.58 , and 0.61 , respectively , while number instrumented levels and the number of associated comorbidities showed no significance [ tables 4 and 5 ] . the complications and their incidence comparisons of different variables in patients with and without complications on analyzing the correlations between different variables , we found that there was a strong positive correlation of blood loss with operative time , number of instrumented levels , and number of interbody fusions which was statistically significant . similarly , operative time showed a strong positive correlation with number of interbody fusions and a significant but a weaker positive correlation with number of instrumented levels [ table 6 ] . the perioperative complication rates in the present study occurred in 11 of 52 patients with an incidence of 21% . increased blood loss strongly correlated with the incidence of complications . age , operative time , number of levels of fusion , and the duration of stay were also more in patients with complications and were close to statistical significance while number of instrumented levels and number of associated comorbidities were unrelated to the complication rates . perioperative complication rates in instrumented lumbar fusions in patients above 60 years of age described in literature range from 29% to 62% [ table 7 ] . reported increased complications in patients with advanced age and surgeries with increased blood loss and number of levels of fusions.1112 carreon et al.11 found increased complication rates with increased operative time while cho et al.12 found no such association . guigui et al.10 found comorbidities to influence the complication rates and a similar study by acosta et al.4 found ten times higher complication rates in patients with htn while others found no association between comorbidities and the perioperative complications.612 the incidence of complications and factors affecting it described in the literature considering the group of population included , the complication rate in our series was within the acceptable limits compared to literature [ table 7].4611121314 our patients were 60 years and above with the average age of 69 years , all of them had one or more comorbidities , and the average number of levels fused was 3.8 , making this group more vulnerable for complications . despite this , the complications in our series were about 21% with most of them being minor reversible complications such as dural tear , transient root deficits , and postoperative persistent radicular pain . similarly , the operative time and blood loss , in our series , was lesser compared to that described in literature for multilevel fusions [ table 7].4611121314 the blood loss in literature ranged from 206 ml in single level to 2056 ml in 9-level fusions and the operative time ranged from 145 min in single level to 415 min in 10.5-level fusions [ table 7].4611121314 in comparison in our series with an average of 3.8 levels of fusion , the average operative time and blood loss were 150 min ( range 60270 min ) and 367.45 ml ( range 901050 ml ) , respectively . this could possibly explain the lesser complication rates in our study as the complications were strongly related to the operative time and blood loss . on reviewing literature and analyzing our surgical technique , we found several strategies that helped in reducing the blood loss and operative time , and hence the complications . injection tranexamic acid 1 g intravenous was given routinely preoperatively , immediately before skin incision in all cases . literature describes that a single dose of tranexamic acid 15 mg / kg can effectively reduce blood loss without increasing the risk of deep vein thrombosis.15 the other technique employed in our surgeries was simultaneous exposure and instrumentation on either side by two trained spine surgeons [ figure 2 ] . this reduced the surgery time and also the blood loss as compared to a single surgeon exposing and instrumenting one side after the other . we employed a laminectomy technique described by okuda et al.16 in which lamina was removed as a single fragment using osteotome and making cuts at pars on either side . this further reduced the operative time as compared to the classical technique of removing the lamina piecemeal by rongeurs . ( a and b ) intraoperative images showing the technique of bilateral exposure and bilateral instrumentation operative time and blood loss were strongly related to the number of levels of vertebra instrumented and fused . even though operative time and blood loss could be reduced by reducing the number of instrumented and fused levels , not instrumenting or fusing the levels when indicated would compromise the principles of surgery and therefore affect the clinical outcome . hence , the number of vertebrae fused or instrumented should be restricted to the minimum indicated levels , without compromising on indications . we also found that the blood loss increased steeply with number of interbody fusion levels . the average blood loss in single level interbody fusion was 307 ml which increased to 362 ml in 2 levels and almost doubled in 3-level interbody fusions [ table 3 ] . the reason for this exponential increase being continued bleeding from the bed of prepared interbody levels while performing the next level . performing interbody fusions at selected levels such as the most stenotic or unstable levels or at the bottom of the construct and posterolateral fusions at other levels also could reduce the blood loss and hence the complications [ figures 3 and 4 ] . bar diagram showing the relation between the intraoperative blood loss and number of interbody fusion levels postoperative x - rays anteroposterior and lateral views of lumbosacral spine showing interbody fusions at l2l3 , l4l5 , l5s1 with posterolateral fusion at l3l4 images apart from reduction in operative time and blood loss , a thorough preoperative workup with concerned specialist consultations such as pulmonologist , cardiologist , and optimization of the medical conditions helped in reducing the anesthetic risks during surgery . six of our patients in the series underwent surgery under spinal or combined spinal epidural anesthesia , due to poor pulmonary or cardiac status . studies have shown regional anesthesia ( ra ) in spine surgery to have many advantages over ga in high - risk patients , like lesser anesthetic intraoperative complications , lesser postoperative htn , respiratory and cardiovascular complications , lesser postoperative vomiting , longer postoperative analgesia , and shorter hospital stay.1718 as surgeons we found spinal anesthesia to be satisfactory with reduced blood loss due to stable blood pressure one of these patients underwent surgery in sitting position which has shown in literature to be more convenient for the patient under ra , with the blood draining by gravity , resulting in clearer operative field and also reduce anesthesia complications by creating a hemodynamic status similar to that in othostasis.19 early mobilization postoperatively with optimal control of medical comorbidities also helped in reducing the early postoperative complications . lumbar fusion surgeries in the elderly with comorbidities have higher complications rates . increased intraoperative blood loss significantly correlated with the complication rates . spinal decompression and fusion surgery when indicated should not be denied merely considering the age and comorbidities of the patients , fearing complications . causes and measures to reduce complications should be considered as the outcome of surgeries in these patients in the absence of complications is good . the authors propose some of the measures to reduce the operative time , blood loss , and hence the complication rates in these patients . mahesh - research grant by ulrich india and consultancy from medtronic india ltd.upendra - research grant from medtronic india . mahesh - research grant by ulrich india and consultancy from medtronic india ltd.upendra - research grant from medtronic india .

background : spine surgery in elderly with comorbidities is reported to have higher complication rates and increased cost . however , the surgical outcome is good irrespective of the complications . hence , it is essential to identify the factors affecting the complication rates in such patients and the measures to reduce them . this retrospective observational study determines the perioperative complications , their incidence and the measures to reduce complications in the elderly with comorbidities , operated by instrumented multilevel lumbar fusion.materials and methods : patients aged 60 years and above with one or more comorbidities operated by multilevel instrumented lumbar fusion in our center between january 2012 and december 2013 were included in the study . perioperative complications and their incidence were calculated . age , number of levels fused , operative time , blood loss , and complication rates were correlated with the duration of stay and the incidence of perioperative complications using spss software . measures to reduce complications are determined by these results and by review of literature.results:fifty two patients were included in the study ( 28 females and 24 males ) with an average age of 69 years ( range 60 - 84 years ) . hypertension was the most common comorbidity followed by diabetes . spondylolisthesis was the most common indication . eleven complications were noted with an incidence of 21% . three were systemic complications which required transfer to intensive care unit . local complications were incidental durotomy ( three ) , transient root deficits ( two ) , wound infections ( one ) , and persistent radicular pain ( two ) . operative time and blood loss were significantly higher in patients with complications.conclusion:complication rates strongly correlate with the blood loss and operative time . reducing the operative time and blood loss by intraoperative tranexamic acid , laminectomy using osteotome , simultaneous bilateral exposure and instrumentation and reducing the number of interbody fusions can help in reducing the complications .

molecular profiling ( mp ) encompasses the testing of multiple biomarkers to evaluate the underlying genetic alterations present in a tumor at any one point in time [ 13 ] . to date molecular profiles may consist of multiple gene mutational analyses , gene copy number changes by fluorescence in situ hybridization ( fish ) , gene expression profiles measured by microarrays ( ma ) and protein expression by immunohistochemistry ( ihc ) . this approach is superior to the testing of one biomarker target which does not take into account the complexity of multiple signaling pathways and cross talk . the cancer literature is replete with studies exploring single biomarkers in clinical trials designed to test single agents or biomarker analyses performed as a post hoc analysis . although there are a plethora of biomarkers that have emerged that may positively prognosticate or predict response to various therapies , the clinical utility and adoption of this approach has been slow due to validation concerns , reproducibility and translation into clinical care [ 57 ] . since comprehensive mp uses a multi - dimensional approach to testing , it is inherently more complex and requires extensive validation . there is significant investment in high cost , high throughput technologies , trained laboratory work force , and laboratory informatics to achieve the level of validation required by clia or cap to offer the test for patient - care . laboratory developed test - validations in a clia mandated environment typically requires the following : specimen type and specimen handling protocols : since this variable can hugely affect reproducibility of the test , standardization of specimen handling is imperative . more and more , the formalin fixed paraffin embedded tissue is becoming the sample of choice as it is readily available . this sample type has been shown to perform adequately for mutational analysis ; gene expression profiles measured by rt - pcr or oligonucleotide arrays , fish , and ihc . however , the quality of analysis on this preferred sample type has to be closely monitored based on time to fixation , time in fixative and age of the samples . additionally for newer molecular techniques , internal and external quality checks such as the amount or percentage of tumor nuclei , the quantity and quality of dna and rna , measurement of internal housekeeping genes are all important determinants in the overall quality of results [ 915 ] . validation of each component of the mp assay , whether being performed in a non - profit hospital - based / academic laboratory or for - profit reference labs , has to follow the strict clia guidelines as well as guidelines provided by laboratory associations such as college of american pathologists ( cap ) or cls1 . for each test offered , per validation guidelines , the laboratory must document certain performance characteristics which include : accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . from these accuracy studies the analytical sensitivity , specificity and accuracy of the assay can be determinedprecision studies are performed to determine intra - run and inter - run reproducibility.the assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . ongoing quality assurance and proficiency testing are some other additional requirements by clia specimen type and specimen handling protocols : since this variable can hugely affect reproducibility of the test , standardization of specimen handling is imperative . more and more , the formalin fixed paraffin embedded tissue is becoming the sample of choice as it is readily available . this sample type has been shown to perform adequately for mutational analysis ; gene expression profiles measured by rt - pcr or oligonucleotide arrays , fish , and ihc . however , the quality of analysis on this preferred sample type has to be closely monitored based on time to fixation , time in fixative and age of the samples . additionally for newer molecular techniques , internal and external quality checks such as the amount or percentage of tumor nuclei , the quantity and quality of dna and rna , measurement of internal housekeeping genes are all important determinants in the overall quality of results [ 915 ] . validation of each component of the mp assay , whether being performed in a non - profit hospital - based / academic laboratory or for - profit reference labs , has to follow the strict clia guidelines as well as guidelines provided by laboratory associations such as college of american pathologists ( cap ) or cls1 . for each test offered , per validation guidelines , the laboratory must document certain performance characteristics which include : accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . from these accuracy studies the analytical sensitivity , specificity and accuracy of the assay can be determinedprecision studies are performed to determine intra - run and inter - run reproducibility.the assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . ongoing quality assurance and proficiency testing are some other additional requirements by clia accuracy to document that the test produces expected result by appropriate testing of known positive and negative samples . from these accuracy studies the analytical sensitivity , specificity and accuracy of the assay can be determined precision studies are performed to determine intra - run and inter - run reproducibility . the assay will also have to determine appropriate reference ranges and limit of detection for appropriate reporting of results . ongoing quality assurance and proficiency testing are some other additional requirements by clia given the resource investment requirements for conducting these multi - dimensional , labor intense assays , it is easily conceivable that these assays are increasingly being offered by large centralized laboratories . ( examples include : genomic health inc . , pathwork diagnostics and caris life sciences ) . the rapidly developing genomic information is leading to the proliferation of mp services and assays and their subsequent introduction into clinical care . this service offers a new approach in which an evidence rated review of the literature based on the the us preventive task force rating is utilized to identify targets in tumor tissue associated with current therapies . using a technology and platform agnostic approach , various targets are analyzed using a combination of assays such as gene sequencing , oligonucleotide microarray , mutational analyses , copy number changes using fish analysis and protein expression by ihc . this particular approach for mp to measure molecular targets was studied in a feasibility study in 2006 and most recently in a multi - center clinical trial , across nine different cancer centers in the us . using the caris life sciences caris target now mp service , von hoff et al . reported a longer pfs for patients on mp - directed therapy than physician choice for 27 % of patients ( 95 % ci , 1738 % p = 0.007 ) . this study used a novel study design in which the patient served as their own controls and pfs ratio was determined by actual comparison of pfs on mp therapy versus pfs on patient s last prior therapy . for the participants ( 18/66 ) who had a pfs 1.3 overall survival was 9.7 months compared to 5 months on physician directed therapy . interestingly , mp of tumors yielded actionable targets in 98 % by this assay indicating that such an approach is feasible . however , it is to be noted that the targets identified may involve off - label use of therapies . . study was restricted to advanced stage patients with metastases and refractory tumors , the approach may have significant benefits when used earlier . , reported two patients with advanced stage colon cancers in which identification of a target mgmt by ihc with this assay , led to measurable response to temozolomide treatment with decrease in serum markers and tumor shrinkage on ct . using the same assay , discovery of targets was also reported in a interdigitating reticulum cell sarcoma , an exceedingly rare tumor . median time to treatment failure ( ttf ) in 161 patients with one aberration treated with matched targeted therapy was 5.3 months ( 95 % ci : 4.1 , 6.6 ) versus 3.2 months ( 95 % ci : 2.94.0 ) for their prior systemic antitumor therapy ( prior to referral to phase i ) ( p = 0.0003 ) . for patients with one aberration , the cr - pr rate was 29 % with matched targeted therapy versus 8 % without matching ( p = 0.0001 ) . the cr + pr rate was 6 % in 438 patients without molecular testing treated on the same studies . interestingly , these rates compare favorably with those reported by von hoff et al . for the caris target now service . these preliminary results suggest that in early clinical trials , matching patients with targeted drugs based on their molecular profile results in ( a ) longer ttf compared to their prior therapy and ( b ) higher rates of response , survival and ttf compared to those seen in patients treated without molecular matching . the battle trial for personalizing therapy for lung cancer identified targets of high interest in treatment of lung cancer and using an adaptive randomized trial utilized real time biomarker analyses to predict sensitivity or resistance to targeted agents . a similar trial i - spy 2 also employs this groundbreaking clinical trial model that uses genetic or biological markers ( biomarkers ) from individual patient s tumors to screen promising new treatments , identifying which treatments are most effective in specific types of patients . in addition , this innovative adaptive trial design similar to battle trial will enable researchers to use early data from one set of patients to guide decisions about which treatments might be more useful for patients later in the trial , and eliminate ineffective treatments more quickly . with more focus on the mp of tumors and greater realization of the limitations of one - biomarker one target approach , cancer treatment in the us is about to experience a major revolution . upfront mp of tumors at the time of diagnosis and subsequently at all points of tumor recurrence , whether local or distant , will change the treatment of oncology care forever . this will hopefully lead to better control of cancer , improved outcomes for patients , and a more rational and less expensive oncology care .

the pace of genomic discoveries in the field of cancer is revolutionizing our understanding of the biological dynamics of cancerous growth and , at the same time , fueling research for newer and smarter cancer therapies to reverse the effects of these alterations . these dynamics are driving a tremendous paradigm shift in cancer diagnostics , drug development and clinical trial design with the hope of eliminating the current structure and approach of cancer care , to one which is driven by the underlying biology of the tumor and , thus highly personalized . much of this paradigm shift has been fueled by the current availability of novel technologies , platforms and bioinformatic tools . today , therapies are being rationally designed to target the precise genetic alterations with better clinical outcomes with reduced morbidity . therefore , molecular profiling of tumors to identify the multiplicity of alterations in a tumor is an essential and necessary companion for targeted therapies .

microfluidics is the science and technology that deals with fluids usually in the range of microliters ( 10 ) to picoliters ( 10 ) . since microfluidics typically deals with fluids in the microliter scale , it has several advantages , including low consumption of samples , short analysis time , and high sensitivity [ 1 - 4 ] . the portability and fast processing speed of microfluidic devices also allows for in situ and real - time analysis . polydimethylsiloxane ( pdms ) is commonly used for molding microfluidic chips because it is a transparent and biocompatible elastomer . the fabricating process of pdms - based microfluidic devices , which is based on soft lithography , consists of the following steps : ( 1 ) master fabrication steps , including spin coating of a photoresist film , exposure , and development to form the mold on a silicon substrate ; ( 2 ) device fabrication steps , including pouring the pdms on the master , punching holes with a biopsy punch , and bonding the pdms structure to glass . recently , paper - based microfluidic devices have been proposed as cheap , portable , and disposable devices [ 5 - 7 ] . ever since this emerging discipline was introduced in the early 1990s , microfluidics has grown rapidly in the field of biomedical applications . microfluidic devices are very useful tools for molecular separation , biochemical assays , drug screening , chromatography , and migration assays [ 10 - 14 ] . lap - on - chip and organ - on - chip based on microfluidic devices have also been widely used for high - throughput screening applications . microfluidic devices have not been widely used in urological research yet . however , some pioneering studies have been reported for bladder and prostate cancer detection , urine analysis , and sperm characterization . in this review , we will summarize research works that use microfluidics and discuss its application in urology . microfluidics has been considered a promising tool to analyze cancer cells and tumor function owing to its high sensitivity , high throughput , and less material consumption . microfluidic devices offer various microenvironments for cell culture , from 2-dimensional to complex 3-dimensional systems , and the complex coculture system . various microfluidic platforms have been developed to understand cancer cell migration and metastasis under various conditions , such as varying chemical gradients and mechanical constraints ( fig . 1 ) . a microfluidic device to detect metastatic cancer cells based on their size and deformability was also fabricated . a microfluidic device has been developed for prostate and bladder cancer research for various procedures , including biomarker detection , characterization of cancer cells in various microenvironments , and circulating tumor detection . prostate cancer is the most common type of cancer among males and the sixth most common cause of death in males . a prostatic - specific antigen ( psa ) , which is one of the available biomarkers of prostate cancer , is widely used for the screening of prostate cancer . however , the conventional psa test has drawbacks , such as excessive sample consumption , restrictive control of the analytes in the reaction chamber , and lack of multiplexing capabilities . therefore , the use of a microfluidic device has been considered an efficient approach to overcome these drawbacks . the microfluidic enzyme - linked immunosorbent assay was integrated with photodetectors to sense the free isoform of the psa with labeled antibodies by using all 3 detection modes . this integrated system showed enhanced reliability in signal acquisition and refined sensitivity even for low detection limits . this platform allowed for quick screening and contemporary detection of free and total psa using 2 different antibodies that are immobilized on a single chip . bladder cancer is a common urologic cancer that has the highest recurrence rate among all other malignancies . the microfluidic - based microenvironment is a good model to investigate the tumor growth , metastasis , and dynamic interplay between the cells . the mitochondrial - related protein expression in the bladder cancer cells was investigated in coculture conditions using a microfluidic chip . a chip for the coculture of bladder tumor cells and fibroblasts was composed of 2 cell culture pools for human skin fibroblasts and the human bladder tumor cells . the pools were connected using microchannels and peripheral perfusion channels . using this coculture system , it was observed that the high - energy metabolites transferred to adjacent tumor cells in a specified direction . to study the effects of the microenvironment on bladder cancer , the microfluidic device based on 4 types of cells coculture system was suggested . this system consisted of perfusion equipment , matrigel channel units , a medium channel , and four indirect contact culture chambers in which the 4 types of cells ( stromal cells , fibroblasts , endothelial cells , and macrophages ) could be cultured simultaneously . the changes in urine composition , such as the changes in the levels of glucose , albumin , creatinine , and uric acid , may provide information about organ dysfunctions , drug abuse , and exposure to chemicals and toxins . conventional methods to examine urine samples are physical ( color , odor , etc . ) , biochemical , and microscopic examinations . microfluidic devices are typically used in the manipulation of fluids with a small quantity of sample , under a precisely controlled flow rate . therefore , they are very useful devices for urine sample pretreatments and sensing urinary molecules . liquid extraction as a sample pretreatment device prior to carrying out gas chromatography analysis of the amphetamine - type stimulants in the urine ( fig . the interface between the organic solvent and alkalized urine was stabilized by using the microchannels modified by a capillary - restricted modification method . microfluidic devices have been used for the detection of urinary macromolecules , including protein , virus , and bacteria . demonstrated the cost - effective detection of the bk virus ( viral load ) from nonpretreated urine samples using microfluidic devices . a detection sensitivity of as low as 12 viral copies was obtained by the integration of a small volume genetic amplification ( polymerase chain reaction ) . microfluidic devices for urinary micromolecules , including creatinine , galactose , and inorganic ions have also been demonstrated by several groups . for analyzing urinary creatinine , a microfluidic system based on a 2-point alkaline picrate kinetic reaction the microchip was connected to 2 syringes for delivering the samples , and it was connected to a miniature fiber optic spectrophotometer for measuring the absorption light at 510 nm . the integration of microfluidic devices with different approaches , including electrophoresis , optics , and immunoassays , was successfully used for detecting drugs and metabolites in urine samples . caffeine and theophylline could be effectively separated using the microchannel electrophoresis integrated with electrochemical detection , and illicit drugs were detected with a high accuracy using the microfluidic device with an ultraviolet detection function . many microfluidic systems have been proposed for quantifying sperm quality and sorting sperm by their motility . according to the principle , the microfluidic device for sperm sorting can be categorized into the following 3 groups : passively driven devices , flow - driven devices , and combined devices for thermotaxis and chemotaxis . the principle of passively driven devices is that motile sperm can be collected within a specific period of time in the outlet of the microfluidic channels , while less - motile sperm are left behind in the microchannels . in flow - driven devices the sperm are segregated into different channels depending on their motility and are counted thereafter . the combined microfluidic device with a chemoattractant was fabricated with a straight channel connected to a bi - branch channel . the sperm motility under the chemical gradient was monitored , and the chemotaxis was analyzed by using the ratio of the sperm collected in different branches . microfluidics has been considered a promising tool to analyze cancer cells and tumor function owing to its high sensitivity , high throughput , and less material consumption . microfluidic devices offer various microenvironments for cell culture , from 2-dimensional to complex 3-dimensional systems , and the complex coculture system . various microfluidic platforms have been developed to understand cancer cell migration and metastasis under various conditions , such as varying chemical gradients and mechanical constraints ( fig . 1 ) . a microfluidic device to detect metastatic cancer cells based on their size and deformability was also fabricated . a microfluidic device has been developed for prostate and bladder cancer research for various procedures , including biomarker detection , characterization of cancer cells in various microenvironments , and circulating tumor detection . prostate cancer is the most common type of cancer among males and the sixth most common cause of death in males . a prostatic - specific antigen ( psa ) , which is one of the available biomarkers of prostate cancer , is widely used for the screening of prostate cancer . however , the conventional psa test has drawbacks , such as excessive sample consumption , restrictive control of the analytes in the reaction chamber , and lack of multiplexing capabilities . therefore , the use of a microfluidic device has been considered an efficient approach to overcome these drawbacks . the microfluidic enzyme - linked immunosorbent assay was integrated with photodetectors to sense the free isoform of the psa with labeled antibodies by using all 3 detection modes . this integrated system showed enhanced reliability in signal acquisition and refined sensitivity even for low detection limits . this platform allowed for quick screening and contemporary detection of free and total psa using 2 different antibodies that are immobilized on a single chip . bladder cancer is a common urologic cancer that has the highest recurrence rate among all other malignancies . the microfluidic - based microenvironment is a good model to investigate the tumor growth , metastasis , and dynamic interplay between the cells . the mitochondrial - related protein expression in the bladder cancer cells was investigated in coculture conditions using a microfluidic chip . a chip for the coculture of bladder tumor cells and fibroblasts was composed of 2 cell culture pools for human skin fibroblasts and the human bladder tumor cells . the pools were connected using microchannels and peripheral perfusion channels . using this coculture system , it was observed that the high - energy metabolites transferred to adjacent tumor cells in a specified direction . to study the effects of the microenvironment on bladder cancer , the microfluidic device based on 4 types of cells coculture system was suggested . this system consisted of perfusion equipment , matrigel channel units , a medium channel , and four indirect contact culture chambers in which the 4 types of cells ( stromal cells , fibroblasts , endothelial cells , and macrophages ) could be cultured simultaneously . the changes in urine composition , such as the changes in the levels of glucose , albumin , creatinine , and uric acid , may provide information about organ dysfunctions , drug abuse , and exposure to chemicals and toxins . conventional methods to examine urine samples are physical ( color , odor , etc . ) , biochemical , and microscopic examinations . microfluidic devices are typically used in the manipulation of fluids with a small quantity of sample , under a precisely controlled flow rate . therefore , they are very useful devices for urine sample pretreatments and sensing urinary molecules . liquid extraction as a sample pretreatment device prior to carrying out gas chromatography analysis of the amphetamine - type stimulants in the urine ( fig . the interface between the organic solvent and alkalized urine was stabilized by using the microchannels modified by a capillary - restricted modification method . microfluidic devices have been used for the detection of urinary macromolecules , including protein , virus , and bacteria . demonstrated the cost - effective detection of the bk virus ( viral load ) from nonpretreated urine samples using microfluidic devices . a detection sensitivity of as low as 12 viral copies was obtained by the integration of a small volume genetic amplification ( polymerase chain reaction ) . microfluidic devices for urinary micromolecules , including creatinine , galactose , and inorganic ions have also been demonstrated by several groups . for analyzing urinary creatinine , a microfluidic system based on a 2-point alkaline picrate kinetic reaction the microchip was connected to 2 syringes for delivering the samples , and it was connected to a miniature fiber optic spectrophotometer for measuring the absorption light at 510 nm . the integration of microfluidic devices with different approaches , including electrophoresis , optics , and immunoassays , was successfully used for detecting drugs and metabolites in urine samples . caffeine and theophylline could be effectively separated using the microchannel electrophoresis integrated with electrochemical detection , and illicit drugs were detected with a high accuracy using the microfluidic device with an ultraviolet detection function . many microfluidic systems have been proposed for quantifying sperm quality and sorting sperm by their motility . according to the principle , the microfluidic device for sperm sorting can be categorized into the following 3 groups : passively driven devices , flow - driven devices , and combined devices for thermotaxis and chemotaxis . the principle of passively driven devices is that motile sperm can be collected within a specific period of time in the outlet of the microfluidic channels , while less - motile sperm are left behind in the microchannels . in flow - driven devices the sperm are segregated into different channels depending on their motility and are counted thereafter . the combined microfluidic device with a chemoattractant was fabricated with a straight channel connected to a bi - branch channel . the sperm motility under the chemical gradient was monitored , and the chemotaxis was analyzed by using the ratio of the sperm collected in different branches . although a microfluidic system has many advantages , it is still a relatively new technology in the field of urology research . recently , some successful implementations of microfluidic devices have been demonstrated for cancer diagnosis , urine analysis , and sperm sorting . however , more promising microfluidic systems and research works are required for such systems to be applied in the clinic . therefore , it is important to continue the development of this new technology in cooperation with the relevant users .

microfluidics is considered an important technology that is suitable for numerous biomedical applications , including cancer diagnosis , metastasis , drug delivery , and tissue engineering . although microfluidics is still considered to be a new approach in urological research , several pioneering studies have been reported in recent years . in this paper , we reviewed urological research works using microfluidic devices . microfluidic devices were used for the detection of prostate and bladder cancer and the characterization of cancer microenvironments . the potential applications of microfluidics in urinary analysis and sperm sorting were demonstrated . the use of microfluidic devices in urology research can provide high - throughput , high - precision , and low - cost analyzing platforms .

the prevalence of overweight and obesity among children and adolescents has widely increased worldwide [ 1 , 2 ] , making it one of the most common chronic disorders in this age group and in adulthood . the use of body mass index ( bmi ) for age to define being overweight and obese in children and adolescents is well established for both clinical and public health applications , because of their feasibility under clinical settings and in epidemiological studies [ 3 , 4 ] . in children and adolescents , the natural increases in bmi that occur with age necessitate the use of age - sex - specific thresholds . the most widely used growth charts are the centers for disease control and prevention ( cdc-2000 ) , the international task force ( iotf ) , and the 2007 growth references for 5 to 19 year olds produced by the world health organization ( who-2007 ) . the cdc-2000 growth charts were developed to evaluate the nutritional status of us children and were originated from five cross - sectional representative surveys carried out in the us between 1963 and 1994 . these growth charts are routinely applied to identify children and adolescents with a bmi greater than the 85th or 95th percentiles following the advice of the us expert committee on childhood obesity . however , the appropriateness of an american dataset for defining overweight in young people from other countries is questionable . the iotf reference also uses age - sex - specific bmi percentiles , and overweight and obesity definition corresponds to an adult bmi of 25 and 30 kg / m , respectively , and reflects values in children tracking to overweight and obesity in adults . this reference is based on six large international cross - sectional representative datasets , identifying the bmi values that extrapolate to childhood . the who-2007 growth references were created to replace the national center for health statistics ( nchs ) references [ 10 , 11 ] . this reference was constructed using data from the 1977 nchs / who growth reference ( 1 to 24 years old ) merged with data from the 2006 who child growth standards for preschool children ( under 5 years of age ) using state - of - the - art statistical methods . although valuable information has been appearing in the literature or online , such as works from the health behaviour of school - aged children study which is mainly related to social determinants of health and well - being among young people , no systematic review has been conducted to understand the worldwide magnitude of the overweight and obesity problem among the adolescent population . thus , the objective of this study was to systematically review the literature regarding the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes published in the past 12 academic years ( 19992011 ) . a systematic literature search was performed which ended on june 7 , 2012 ( see figure 1 ) . the literature search was conducted in medline and scopus using the following mesh terms : overweight ; obesity ; prevalence ; adolescent . in total , 2537 articles were selected . we also reviewed the data on the prevalence of childhood overweight and obesity on the international obesity task force website at http://www.iaso.org / iotf/. to find the articles included in this review , the following inclusion criteria were used : ( 1 ) cross - sectional studies conducted in the last 12 years ( 19992011)when the original study did not report the survey year , it was not included ; ( 2 ) national and regional representative samples , but articles published on the prevalence of overweight in towns , urban , or rural areas in a country were excluded ; ( 3 ) weight and height objectively measured ; ( 4 ) results presented by sex ; ( 5 ) and for both overweight and obesity prevalence ; ( 6 ) the definition of overweight and obesity using the ( i ) cdc-2000 , ( ii ) iotf , and ( iii ) who-2007 growth references ; and ( 7 ) studies written in english , spanish , italian , or portuguese . moreover , if there were more than one national or regional study in the same country , the most recent one was included in the prevalence tables ( except for usa and canada , countries in which the most recent data was not included in the tables due to differences in the representativeness of the samples and the impossibility to calculate a single prevalence of overweight and obesity for adolescents ' boys and girls ; however , no differences in prevalence were observed between studies as it has been indicated in the discussion ) . the final number of articles included in this review was 39 articles related to overweight and obesity and also a study on the latest statistics on the prevalence of overweight and obesity in south africa . potentially relevant papers were selected by ( 1 ) screening the titles ; ( 2 ) screening the abstracts , and ( 3 ) if abstracts were not available or did not provide sufficient data , the entire article was retrieved and screened to determine whether it met the inclusion criteria . full - text articles were assessed by 2 authors ( m. m. bibiloni and j. a. tur ) . any matter of doubt was discussed by at least two of the reviewers ( m. m. bibiloni , a. pons , and j. a. tur ) . a total of thirty - nine articles and a national health report were eligible according to the inclusion criteria established for this review . table 1 presents a description of the forty studies selected for this review including the continent and the country where it was performed ( and region for not national studies ) , year of publishing , total number of participants in the study , number of adolescents , age range , proportion of girls , and number and definition for overweight and obesity classification used . nationally representative data were obtained in twenty - five countries ( including northern ireland ) [ 1539 ] , and ten countries were represented only by regional data [ 40 , 42 , 44 , 45 , 47 , 5054 ] . table 2 shows overweight and obesity prevalence from the twenty - five national studies ( one of them including data from northern ireland ) that were included in this review according to the continent and the country where it was performed , year of survey , study population , age range , criteria used for classifying overweight and obesity used , and along with total data by sex . there were thirty - two different prevalence levels described in the included articles , because five countries presented data using at least two different criteria for overweight and obesity classification [ 18 , 23 , 27 , 36 , 39 ] . the iotf cut - off was used to classify overweight and obesity in twenty - three of the twenty - five national studies considered in the present review . in general , the prevalence of overweight plus obesity was higher in america [ 1820 ] , oceania [ 38 , 39 ] and europe [ 3037 ] and lower in africa [ 1517 ] and certain parts of asia [ 2129 ] ( in china and iran the total prevalence was less than 10% by the iotf cut - offs ) . overall , about 30% of american adolescents and 22%25% of european adolescents ( excepting the czech republic and italian adolescents ' which showed a prevalence of 13.7% and 17.9% , resp . ) were overweight or obese . among oceanian adolescents the prevalence ranged from 23.2% in australia in 2004 to 34.2% in new zealand in 2007 . in africa , the overall prevalence of overweight and obesity was lower than 20% . among asian adolescents there was a broad range of overweight plus obesity . using iotf cut - off , the prevalence of being overweight or obese for asian boys and girls ranged from 5.2% in china in 2002 to 36.4% in bahrain in 2000 . specific prevalence from all the geographic regions was included in this review from three countries : south africa ( nine provinces ) , usa ( fifty two states ) , and italy ( five regions ) . in europe , data from islands of greece ( crete ) and italy ( sicily and sardinia ) [ 48 , 49 ] and spain ( balearic islands ' archipelago ; and the grand canary island ) were also included . on the other hand , regional but not national data was found for eleven countries ( italy , brazil , india , jordan , denmark , france , hungary , poland , spain [ 51 , 52 ] , switzerland , and turkey ) . the iotf cut - off was used to classify overweight and obesity in fourteen of the eighteen selected studies that included regional data . in one study , data was presented using only the who-2007 growth charts and in two studies using only the cdc-2000 growth reference [ 20 , 43 ] . in south africa and usa , substantial geographic variations in adolescent overweight and obesity prevalence varied in south africa from 13.5% in limpopo to 25.5% in kwazulu - natal . in 2007 , overweight and obesity varied in usa from 23.1% in utah and minnesota to 44.5% in mississippi . in 2002 , the prevalence of overweight and obesity in southern italy and italian islands was higher among boys . in southern italy , the overweight prevalence among girls also was higher than in the other geographic regions . comparison between the islands from greece ( crete ) , italy ( sicily and sardinia ) , and spain ( balearic islands and grand canary island ) which were included in this review showed that crete had the highest prevalence of overweight and obesity despite data were presented using different definition . in spain , using the iotf cut - off ( data not shown for balearic islands but given by authors ) , the prevalence of overweight plus obesity was higher in the grand canary island ( 29.1% ) than in the balearic islands ( 24.7% ) . according to national data , the prevalence of overweight among boys was 10% higher than girls in nine countries ( canada , qatar , taiwan , cyprus , czech republic , germany , greece , italy , australia , denmark , and hungary ) and among girls 10% higher than boys in seven of the twenty - five countries ( south africa , seychelles , tunisia , mexico , bahrain , saudi arabia , and sweden ) . the obesity prevalence was 10% higher among boys in seventeen countries ( canada , usa , china , iran , israel , qatar , saudi arabia , taiwan , cyprus , czech republic , germany , greece , italy , portugal , sweden , australia , new zealand , denmark , and hungary ) and 10% higher among girls in four of the twenty - five countries ( south africa , seychelles , tunisia , and bahrain ) . the aim of this study was to review systematically the literature on overweight and obesity prevalence among adolescents worldwide . thirty - nine articles and one national health report that met the inclusion criteria were considered . the overweight and obesity prevalence in the included studies ranged widely . in sixteen of the twenty - three countries with national representative data using the iotf cut - off , overweight and obesity prevalence higher than 20% were found , five countries showed prevalence above 30% , and just in two countries prevalence was lower than 10% . regarding national data , when prevalence was analysed according to sex , it was observed that boys showed a higher prevalence of overweight in almost half of the countries and a higher prevalence of obesity in almost all countries . these results are consistent with previous studies that pointed out a high prevalence of abdominal obesity among boys . differences of prevalence of overweight and obesity between genders have been related to geopolitical and cultural conditions . eight articles compared data between 1980s and/or 1990s with 2000s [ 16 , 19 , 20 , 22 , 28 , 32 , 37 , 50 ] and pointed out an increased prevalence of overweight and obesity in both sexes over this period . however , among australian adolescents the overweight and obesity prevalence increased significantly among boys but not among girls over the period 19972004 . in the australian national children 's nutrition and physical activity survey 2007 ( ncnpas07 ) , 25% of boys and 30% of girls aged 9- to 13-year - olds and 25% of boys and 23% of girls aged 14- to 16-year - olds were overweight or obese using the iotf criteria . a comparison of the 1985 , 1995 , and 2007 australian national surveys of 7- to 15-year - olds indicated that australian children are changing body shape to a more central fat distribution . in usa , overweight and obesity prevalence increased by 3% and 18% among usa girls over this period . however , a cross - sectional analyses of a representative sample ( n = 4111 ) of the usa child and adolescent population ( birth through 19 years of age ) with data from the national health and nutrition examination survey 2009 - 10 ( nhanes ) indicated a prevalence of overweight and obesity among adolescents aged 12 through 19 years of 15.2% and 18.4% , respectively . analyses of trends in obesity prevalence for the last two nhanes surveys ( 2007 - 08 and 2009 - 10 ) indicated that the prevalence of obesity in children and adolescents has not changed in 2009 - 10 compared with 2007 - 08 . on the other hand , since 2004 the overweight and obesity trends were stabilized or decreased among german adolescents . in usa , substantial geographic disparities in adolescent overweight and obesity were found , with an apparent shift toward higher prevalence in 2007 for several states . reported that children in northern europe countries generally tended to have lower overweight and obesity prevalence ( 1020% ) than in southern europe ( 2035% ) . also within the same country , the prevalence and trends of overweight and obesity may not be homogeneous according to different geographic regions . in italy , a north - south gradient in overweight and obesity prevalence among boys but also in overweight prevalence among girls was also reported . a higher prevalence of overweight and obesity has been reported in southern spain in both children and adults . it is important to note that the choice of a reference and a cut - off point will determine the absolute prevalence of overweight and obesity and its trends , and hence different information will be obtained from the papers . argued that the reference they published , supported by the iotf , is less arbitrary and more international than others and recommended its use in international comparisons . lately , monasta et al . suggested that the iotf reference and cut - offs could be preferable to identify overweight and obesity both at individual and population levels because they are at least based on a crude association with ill and health later in life , namely , the definition of overweight and obesity at age 18 years . however , the iotf cut - offs have been not recommended for clinical use when assessing an individual child 's growth [ 9 , 6264 ] . furthermore , recent findings suggested that a universal bmi classification system for childhood and adolescent overweight and obesity may not correspond to a comparable level of body fatness in all populations . the prevalence estimates may not accurately characterize the population groups most at risk of health disadvantages because the correlation of bmi with adiposity is highly variable and dependent on ethnic group [ 9 , 60 , 65 , 66 ] . the comparisons of overweight and obesity prevalence need interpretation with caution due to the difference in survey sampling methods , sample sizes , age range of subjects , quality of data in terms of height and weight measurement , and whether national programmes or strategies to tackle overweight and obesity are in place . even within the same country , the prevalence and trends of overweight and obesity may not be homogenous in view of different ethnicities , geographic regions , and socioeconomic status . only articles in english , spanish , italian , and portuguese were included in this review . the results of this review allow the following conclusions : ( 1 ) overweight and obesity prevalence is high ; ( 2 ) obesity is higher among boys , although it is not clear which sex has a higher proportion of adolescents with overweight ; ( 3 ) despite that there is no consensus about criteria to be used to classify adolescents as overweighed or obese , the most frequently used was the iotf reference . however , the international reference charts for monitoring the secular trends in childhood obesity need to be continually refined and evaluated . the results of this study would contribute to guiding health planners and administrators to develop proper tools for adolescent obesity management .

objective . to review the extant literature on the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes . design . the search was carried out using medline and scopus considering articles published from the establishment of the databanks until june 7 , 2012 . data on the prevalence of children being overweight and obese from the international obesity task force ( iotf ) website was also reviewed . only original articles and one national health report were considered . forty studies met the inclusion criteria . results . twenty - five of these studies were nationally representative , and ten countries were represented only by regional data . conclusions . the prevalence of overweight and obesity among adolescents worldwide is high , and obesity is higher among boys . the iotf criterion is the most frequently used method to classify adolescents as overweighed or obese in public health research .

an institutional review board approved this retrospective analysis , which was performed at the department of general surgery , xinhua hospital , school of medicine , shanghai jiao tong university . from january 2009 to september 2014 , 220 consecutive patients with bile duct stones ( 152 ) or a common bile duct ( cbd ) stricture ( 68 ) , who had previously undergone gastrectomy and were referred for ercp , were analyzed . the surgical approaches applied to these patients included proximal gastrectomy with esophagogastrostomy ( 10 patients ) , distal subtotal gastrectomy with billroth i ( 42 patients ) or billroth ii ( 160 patients , including 25 patients with braun anastomosis ) , and total gastrectomy combined with roux - en - y reconstruction ( 8 patients ) ( table 1 ) . characteristics of the 220 patients who had previously undergone gastrectomy the procedures were performed in all patients under pharyngeal anesthesia , sedation ( intramuscular 10 mg diazepam injection ) , and pethidine ( 50 mg ) . all patients received oxygen administered by nasal prong and were monitored by pulse oximetry and electrocardiography . ercp was performed under fluoroscopic control using a conventional side - viewing duodenoscope with a total length of 120 cm and a working channel of 3.7 mm in diameter , which allows the use of a wide range of catheters according to the diagnostic or therapeutic objective ( olympus v260 ; olympus medical systems , tokyo , japan ) . alternatively , we used a triple - lumen retrieval balloon catheter ( the extractor pro rx retrieval balloon catheter ) , which is capable of accepting a 0.035-inche ( 0.089 mm ) guidewire in the open - channel guidewire lumen , while allowing simultaneous injection and inflation of the balloon in the other 2 lumens . review of the surgical procedure notes before initiating ercp in postgastrectomy patients provided the necessary details , such as the type of reconstruction and the length of the limbs . in addition , review of the available postoperative gastrointestinal imaging studies , including upper gastrointestinal series , abdominal computed tomography examinations , and magnetic resonance imaging , also provided helpful information . ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy ( fig . 1 ) and distal subtotal billroth i gastrectomy ( fig . 2 ) was similar to routine ercp , because endoscopically the postsurgical anatomy appears mostly similar to the normal digestive tract . for ercp in patients who underwent distal subtotal billroth ii gastrectomy ( figs . 3 , 4 ) or total gastrectomy combined with roux - en - y reconstruction ( figs . 5 , 6 ) , we facilitated successful enteroscopy with endoscope insertion using a triple - lumen retrieval balloon catheter . 7 ) , and then a retrieval balloon was inserted over the guidewire ( fig . 8) . following the injection of contrast agent , we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images ( fig . the balloon was then hooked to the limb , not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall ( fig . as the balloon catheter was strongly retracted into the working channel to allow the scope to advance , the endoscope was propelled slightly forward . it should be emphasized that in ercp postgastrectomy , we relied not only on what was visible through the endoscope but also on the x - ray dynamic images from enterology . we termed the procedure retrieval balloon assisted enterography.1114 for patients with a billroth ii gastroenterostomy and braun anastomosis ( fig . 11 ) , we also used the procedure of retrieval balloon assisted enterography along the optimal route as previously reported15 ( fig . conventional side - viewing duodenoscope for ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy . conventional side - viewing duodenoscope for ercp in patients who had undergone distal subtotal billroth i gastrectomy . the procedure of retrieval balloon assisted enterography for patients who had undergone billroth ii gastroenterostomy . conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy . the procedure of retrieval balloon assisted enterography for patients who had undergone total gastrectomy with roux - en - y reconstruction . conventional side - viewing duodenoscope for ercp in patients who had undergone total gastrectomy with roux - en - y reconstruction . following the injection of contrast agent , we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images . the balloon was then hooked to the limb , not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall . conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy with braun anastomosis . the procedure of retrieval balloon assisted enterography along the optimal enterography route for patients who had undergone billroth ii gastroenterostomy with braun anastomosis . all procedures were performed by an experienced pancreaticobiliary endoscopist ( x .- f.w . ) who routinely performs > 300 to 600 ercps each year . successful enteroscopy ( endoscopic success ) was defined as enterography along the correct limb and the ability to identify the papilla of vater . diagnostic success was defined as successful duct cannulation and a successful cholangiogram leading to a diagnosis . ercp success was defined as a successful enteroscopy with successful diagnostic and therapeutic interventions.16 post - ercp pancreatitis was defined according to cotton s criteria.17 hemorrhage was defined as bleeding requiring the local injection of hemostatic agents or clipping at the time of the procedure or a few days later . all statistical analyses were performed using the spss software , version 18.0 ( spss inc . , logistic regression models were constructed by including variables that had significant univariate associations with post - ercp complications . anova testing was constructed by including independent variables that had significant univariate associations with success rate . the procedures were performed in all patients under pharyngeal anesthesia , sedation ( intramuscular 10 mg diazepam injection ) , and pethidine ( 50 mg ) . all patients received oxygen administered by nasal prong and were monitored by pulse oximetry and electrocardiography . ercp was performed under fluoroscopic control using a conventional side - viewing duodenoscope with a total length of 120 cm and a working channel of 3.7 mm in diameter , which allows the use of a wide range of catheters according to the diagnostic or therapeutic objective ( olympus v260 ; olympus medical systems , tokyo , japan ) . alternatively , we used a triple - lumen retrieval balloon catheter ( the extractor pro rx retrieval balloon catheter ) , which is capable of accepting a 0.035-inche ( 0.089 mm ) guidewire in the open - channel guidewire lumen , while allowing simultaneous injection and inflation of the balloon in the other 2 lumens . review of the surgical procedure notes before initiating ercp in postgastrectomy patients provided the necessary details , such as the type of reconstruction and the length of the limbs . in addition , review of the available postoperative gastrointestinal imaging studies , including upper gastrointestinal series , abdominal computed tomography examinations , and magnetic resonance imaging , also provided helpful information . ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy ( fig . 1 ) and distal subtotal billroth i gastrectomy ( fig . 2 ) was similar to routine ercp , because endoscopically the postsurgical anatomy appears mostly similar to the normal digestive tract . for ercp in patients who underwent distal subtotal billroth ii gastrectomy ( figs . 3 , 4 ) or total gastrectomy combined with roux - en - y reconstruction ( figs . 5 , 6 ) , we facilitated successful enteroscopy with endoscope insertion using a triple - lumen retrieval balloon catheter . 7 ) , and then a retrieval balloon was inserted over the guidewire ( fig . 8) . following the injection of contrast agent , we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images ( fig . the balloon was then hooked to the limb , not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall ( fig . as the balloon catheter was strongly retracted into the working channel to allow the scope to advance , the endoscope was propelled slightly forward . it should be emphasized that in ercp postgastrectomy , we relied not only on what was visible through the endoscope but also on the x - ray dynamic images from enterology . 11 ) , we also used the procedure of retrieval balloon assisted enterography along the optimal route as previously reported15 ( fig . conventional side - viewing duodenoscope for ercp in patients who had undergone proximal gastrectomy with esophagogastrostomy . conventional side - viewing duodenoscope for ercp in patients who had undergone distal subtotal billroth i gastrectomy . the procedure of retrieval balloon assisted enterography for patients who had undergone billroth ii gastroenterostomy . conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy . the procedure of retrieval balloon assisted enterography for patients who had undergone total gastrectomy with roux - en - y reconstruction . conventional side - viewing duodenoscope for ercp in patients who had undergone total gastrectomy with roux - en - y reconstruction . the retrieval balloon was inserted over the guidewire . following the injection of contrast agent , we used this retrieval balloon to explore the tract of the target limb on dynamic radiographic images . the balloon was then hooked to the limb , not only to indicate the direction of the tract to guide the endoscope forward but also to facilitate the forward movement of the endoscope with fewer injuries to the intestinal wall . conventional side - viewing duodenoscope for ercp in patients who had undergone billroth ii gastroenterostomy with braun anastomosis . the procedure of retrieval balloon assisted enterography along the optimal enterography route for patients who had undergone billroth ii gastroenterostomy with braun anastomosis . all procedures were performed by an experienced pancreaticobiliary endoscopist ( x .- f.w . ) who routinely performs > 300 to 600 ercps each year . successful enteroscopy ( endoscopic success ) was defined as enterography along the correct limb and the ability to identify the papilla of vater . diagnostic success was defined as successful duct cannulation and a successful cholangiogram leading to a diagnosis . ercp success was defined as a successful enteroscopy with successful diagnostic and therapeutic interventions.16 post - ercp pancreatitis was defined according to cotton s criteria.17 hemorrhage was defined as bleeding requiring the local injection of hemostatic agents or clipping at the time of the procedure or a few days later . all statistical analyses were performed using the spss software , version 18.0 ( spss inc . , logistic regression models were constructed by including variables that had significant univariate associations with post - ercp complications . anova testing was constructed by including independent variables that had significant univariate associations with success rate . the study group included 220 patients with altered gastrointestinal anatomy ( 77 women and 143 men ; mean age , 72.2 y ; range , 11 to 93 y ) . the indications for ercp included cbd stones ( 152 patients ) and cbd stricture because of tumor recurrence ( 68 patients ) . the overall enterography success rate was 90.5% ( 199/220 ) , and the diagnostic success and ercp success rates were both 88.6% ( 195/220 ) . among patients who underwent billroth i gastroenterostomy and proximal gastrectomy with esophagogastrostomy , endoscopic success rate was 100% ( 42/42 ) , and the diagnostic success and ercp success rates were both 100% ( 42/42 ) . for billroth ii gastroenterostomy , the endoscopic success rates without or with braun anastomosis were 88.9% ( 120/135 ) and 88.0% ( 22/25 ) , respectively . the diagnostic success and ercp success rates for billroth ii gastroenterostomy with the duodenoscope without or with braun anastomosis were 86.7% ( 117/135 ) and 84.0% ( 21/25 ) , respectively . for patients who underwent total gastrectomy with roux - en - y reconstruction , the endoscopic success rate was 62.5% ( 5/8 ) , and the diagnostic success and ercp success rates were both 62.5% ( 5/8 ) . endoscopy was unsuccessful in 21 patients with a billroth ii gastroenterostomy and roux - en - y reconstruction because of failure to access the papilla due to the presence of a long afferent loop and tumor infiltration of the afferent loop . unsuccessful diagnostic and ercp outcomes after endoscopic success occurred only in patients with billroth ii gastroenterostomy without or with braun anastomosis because of cannulation failure in 4 patients due to tumor infiltration . factors that increased the rates of enterography success , diagnostic success , and ercp success were cbd stone , proximal gastrectomy , esophagogastrostomy , and billroth i reconstruction ( table 2).the procedure - related complication rate was 5.5% ( 12/220 ) , including hemorrhage ( 0.9% , 2/220 ) , pancreatitis ( 4.1% , 9/220 ) , and perforation ( 0.5% , 1/220 ) ( table 3 ) . one patient with a billroth ii gastroenterostomy developed afferent loop perforation , underwent laparotomy , and was discharged 2 weeks later . two patients experienced hemorrhage at the time of ercp , which was successfully treated by the local injection of epinephrine and clipping . factor that increased the risk of any procedure - related complication was type of previous surgery ( table 4 ) . factors that affected the rates of enterography success , diagnostic success , and ercp success details of the ercp treatment procedures risk factors for post - ercp complication in 220 patients who had previously undergone gastrectomy ercp in patients after gastrectomy remains a challenging technique for ercp endoscopists . as in patients with normal anatomy , anterior oblique - viewing endoscopes , side - viewing endoscopes , forward - viewing gastroscopes , and multibending endoscopes have been reported in previous studies of ercp for postgastrectomy patients.1822 however , there are 3 major obstacles to overcome to successfully perform ercp when using these enteroscope variants : ( 1 ) the approach to the ampulla of vater , ( 2 ) selective bile duct cannulation , and ( 3 ) procedural reliability , including skillful technique and dedicated devices . the forward - viewing endoscope has a long - working length and permits the operator to enter the afferent loop easily and safely because of the ability to see the lumen en face . however , this approach is particularly difficult through a native ampulla because an en face view of the papilla is difficult to obtain using forward - viewing endoscopes . moreover , optimal access to the papilla is restricted without an elevator function , and compatible devices for these enteroscopes are difficult to obtain . this lack of a cannula elevator makes it difficult to cannulate the native papilla , and the lack of dedicated devices makes it difficult to achieve therapeutic success.23,24 thus , the enterography success rate of the forward - viewing endoscope is relatively higher , but the therapeutic success rate is lower . in contrast , the side - viewing endoscope with a shorter working length has a larger working channel and a cannula elevator . however , the fact that it is impossible to see the lumen en face makes it difficult to enter the limb safely , and there are some reports of small bowel perforation associated with ercp using a side - viewing endoscope . however , the cannula elevator makes it easy to cannulate the desired duct selectively , and the larger working channel together makes it easy to achieve therapeutic success . although the enterography success rate of the side - viewing endoscope is relatively lower , the therapeutic success rate is higher after successful enterography . in this study , therefore , 2 strategies that can improve the ercp success rate in patients after gastrectomy include improved therapeutic success with the forward - viewing endoscope25,26 and improved enterography success with the side - viewing endoscope.27,28 from our experience , the working length of the side - viewing duodenoscope is sufficiently long for almost all patients after gastrectomy . our study also demonstrated a significantly higher therapeutic success rate after successful enterography using the side - viewing duodenoscope . thus , our strategy for successful ercp was to improve the enterography success rate of the side - viewing duodenoscope . among the patients who underwent billroth i gastroenterostomy and proximal gastrectomy with esophagogastrostomy , the endoscopic procedures were similar to those performed in normal digestive tracts , and the ercp success rate was 100% using the side - viewing duodenoscope in this study . compared with patients with billroth i gastroenterostomy and esophagogastrostomy , ercp in patients with billroth ii gastrectomy and roux - en - y reconstruction is more difficult and hazardous due to the markedly altered anatomy , with the direction of approach shifted to the 6-oclock position . these alterations together with abdominal adhesions create more difficulties in the intubation of the afferent loop and the approach to the papilla along the afferent loop when using the side - viewing duodenoscope . in our technique , the guidewire of the retrieval balloon was advanced to the appropriate limb , and then a retrieval balloon was inserted over the guidewire . we used this retrieval balloon to explore the correct limb with contrast enhancement to observe the tract of the limb on the radiographic images . the balloon was then hooked to the correct limb and inflated , which not only indicated the direction of the tract to guide the endoscope forward but also facilitated the forward movement of the endoscope with fewer injuries to the intestinal wall . as the balloon catheter was strongly retracted into the working channel to allow the scope to advance , the endoscope was propelled slightly forward . by placing it within the correct limb , the retrieval balloon catheter may also be used as a guide to prevent the duodenoscope from sliding out of the correct limb and into another limb upon forward motion . after successful access of the appropriate limb is achieved , the retrieval balloon becomes visible within the tract ahead , instead of emerging from it . this is particularly important at the anastomosis site , where the correct limb must be identified . it should be emphasized that the x - ray dynamic images we observed with balloon - assisted enterography proved more helpful for ercp in patients with altered gastrointestinal anatomy . such visualization aids the endoscopist in viewing the altered structure clearly and allows the endoscope to move more smoothly along the digestive duct , minimizing accidental injury to the intestinal wall . we termed this procedure retrieval balloon assisted enterography,11,1315 which may ensure the success of ercp using the side - viewing duodenoscope . in patients who have undergone billroth ii gastrectomy and braun anastomosis , we recommend extending the duodenoscope along the greater curvature of the stomach to the gastrojejunal anastomosis , then advancing the endoscope through the efferent loop and along this efferent loop to the braun anastomosis , whereby the middle entrance is the correct entrance to reach the papilla of vater . for patients with billroth ii gastroenterostomy and braun anastomosis , we believe that this is the optimal ercp enterography route.14,29 using these strategies , we obtained encouraging results . for billroth ii gastroenterostomy , the endoscopic success rates without or with braun anastomosis were 88.9% ( 120/135 ) and 88.0% ( 22/25 ) , respectively . the diagnostic success and the ercp success rates for billroth ii gastroenterostomy with the duodenoscope without or with braun anastomosis were 86.7% ( 117/135 ) and 84.0% ( 21/25 ) , respectively . for patients who underwent total gastrectomy with roux - en - y reconstruction , the endoscopic success rate was 62.5% ( 5/8 ) , and the diagnostic success and ercp success rates were both 62.5% ( 5/8 ) . moreover , retrieval balloon assisted enterography using the side - viewing duodenoscope was safe for ercp in postgastrectomy patients . only 1 patient with a billroth ii gastroenterostomy developed afferent loop perforation and underwent laparotomy . two patients experienced hemorrhage at the time of ercp , which was successfully treated by local injection of epinephrine and clipping . thus , we believe that retrieval balloon assisted enterography is an effective and safe method , which can improve the enterography success rate in patients with billroth ii and roux - en - y reconstruction postgastrectomy . in conclusion , the side - viewing duodenoscope is a useful instrument for performing successful ercp in patients postgastrectomy . procedure may improve the enterography success rate in patients with billroth ii and roux - en - y reconstruction postgastrectomy . however , this study was retrospective and reflects the experience of a single center , suggesting that the reproducibility of this technique should be assessed in future prospective studies .

objectives : the aim of this study was to evaluate the usefulness of the conventional side - viewing duodenoscope for successful endoscopic retrograde cholangiopancreatography ( ercp ) in postgastrectomy patients.methods:a total of 220 consecutive patients with bile duct stones or a distal common bile duct stricture who had previously undergone gastrectomy and were referred for ercp were analyzed for the outcome of their ercp . all ercp procedures were performed using a conventional side - viewing duodenoscope . in patients who had undergone a billroth ii gastroenterostomy and total gastrectomy with roux - en - y reconstruction , we also used the procedure of retrieval balloon assisted enterography.results:the study group included 220 patients who had previously undergone gastrectomy ( 77 women and 143 men ; mean age , 72.2 y ; range , 11 to 93 y ) . the overall enterography success rate was 90.5% ( 199/220 ) , and the diagnostic and ercp success rates were both 88.6% ( 195/220 ) . endoscopy was unsuccessful in 21 patients who received billroth ii gastroenterostomy and roux - en - y reconstruction . after successful endoscopy , diagnostic and ercp success was not achieved in 4 patients with billroth ii gastroenterostomy , with or without braun anastomosis , due to cannulation failure . the procedure - related complication rate was 5.5% ( 12/220 ) , including immediate bleeding ( 0.9% , 2/220 ) , pancreatitis ( 4.1% , 9/220 ) , and perforation ( 0.5% , 1/220 ) . there were no procedure - related deaths.conclusions:the side - viewing duodenoscope is a useful instrument for performing successful ercp in patients postgastrectomy . in addition , retrieval balloon assisted enterography may improve the enterography success rate in postgastrectomy patients with billroth ii and roux - en - y reconstruction .

the treatment of intracranial aneurysms has been revolutionized by the introduction of detachable coils for endovascular therapy , and it has been demonstrated that it is a safe and effective method for treating aneurysms . it is not unusual , however , to encounter either transient or permanent ischemic events after embolization procedure of acoa aneurysms . its prevalence of thromboembolic complications , including transient ischemic and stroke , has been reported to be 10.3% . at present , reliable early warning methods are lacking , so anterior circulation cerebral infarction ( aci ) is often diagnosed after clinical deterioration of neurological function , resulting in passive treatment . the risk factors relevant to aci after embolization are poorly understood , making preventative treatment difficult . the current study is the first to elucidate the risk factors which lead to aci after endovascular coiling of acoa aneurysms . during the past 2 decades , the emphasis on ischemic stroke pathogenesis has largely switched from hypoperfusion and hemodynamic - related explanations to thromboembolism as the predominant mechanism . nevertheless , the close relationship between severity of extracranial arterial stenosis and brain infarction , the correlation between impaired functional blood flow reserve and subsequent brain infarction , and the correlation of reduced collateral blood flow with a poor prognosis indicate that ischemic stroke pathogenesis is a multifactorial process . the purpose of the present study was to investigate the inpatient database in acoa aneurysms using endovascular treatment . in particular , we wished to elucidate the risk factors relevant to aci after embolization using univariate and logistic regression analysis . case files from patients that had undergone coil embolization of cerebral anterior communication artery aneurysms from october 2008 to april 2012 at tangshan gongren hospital were retrospectively reviewed . the patency of 54 patients aca and acoa during embolization was evaluated in the study . the stroke rate was 8.7% in ruptured patients for one time before embolization finished and 37.5% in ruptured patients for twice before embolization finished . the mean age of the study group was 53.6 years with a 1:1 female - to - male ratio . hospital records including clinical charts , operative reports , and radiological studies were used to obtain patient characteristics including : age , coil type , rupture status , sex , aneurysm location , aneurysm size , embolization results , complication , and necessity of retreatment . the timing of the infarction following treatment occurred at 3 postoperative hours to 3 days . this study was conducted with approval from the ethics committee and intuitional review board of hebei medical university . the technique for endovascular coiling alone and stent - assisted coiling ( boston scientific / target , california , america ) has been previously described in the literature . all procedures were performed using a biplane angiographic system ( philippe , amsterdam , holland ) with three - dimensional rotational and digital subtraction capabilities and with the patients under general anesthesia and a systemic anticoagulant . anticoagulation was aimed at keeping the activated clotting time at two to three times above the normal value ( approximately 100 s ) during catheterization , stent , and coil placement . our protocol includes : 1 ) pretreatment with aspirin ( 300 mg ) and clopidogrel bisulfate ( 300 mg ) 30 min before coiling for stented patients and no pretreatment before coiling for non - stented patients , 2 ) systemic intravenous heparinization after placement of the femoral sheath for a goal activated clotting time of 250 - 300 s , 3 ) systemic intravenous heparinization 12 h after the procedure , 4 ) continuation of clopidogrel bisulfate ( 75 mg daily ) for 6 months for stented patients and discontinuation of clopidogrel bisulfate for non - stented patients , and 5 ) aspirin ( 100 mg daily ) for all patients indefinitely . the risk factors considered in the study were sex , age , smoking , alcohol consumption , hypertension , diabetes mellitus , hyperfibrinogenemia , hyperlipidemia , ischemic changes on electrocardiogram ( ecg ) , fever , fluid volume balance , modified fisher grade , hunt - hess grade , intracerebral hematoma , ventricular hemorrhage , cerebral swelling , hydrocephalus , unilateral dysplasia of cerebral anterior circulation , direction of aneurysm body , aneurysm blood supply from left aca , aneurysm rupture times , time from aneurysm recent rupture before admission to embolization , stent use , packing degree , leukocyte count , platelet count , hematocrit , comorbidity score , aneurysm neck width , aneurysm parent artery width , aneurysm size , and embolization time . we defined the potential risk factors as follows : smoking ( currently smoking more than one cigarette per day)alcohol consumption ( drinking more than twice per week)hypertension ( systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , and/or presence of antihypertensive drug treatment)diabetes mellitus was defined as a high - fasting plasma glucose level ( 126 mg / dl ) , or a current treatment with an oral hypoglycemic agent or insulinhyperfibrinogenemia was defined as a high plasma fibrinogen level ( 400 mg / dl)hyperlipidemia was defined as a fasting serum total cholesterol level of 240 mg / dl , or a current treatment with an antihyperlipidemic agentischemic changes on electrocardiogram ( ecg ; st - t change , flat t , and negative t)fever ( normal body temperature range 36.0 - 37.4c , low grade fever range 37.5 - 38.0c , and high grade fever 38.1c)fluid volume balance was defined as intake and output balance from admission to the end of operationpacking degree was classified as defined by raymond et al . class 1 complete occlusion , class 2 residual neck , and class 3 residual aneurysm sac . smoking ( currently smoking more than one cigarette per day ) alcohol consumption ( drinking more than twice per week ) hypertension ( systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , and/or presence of antihypertensive drug treatment ) diabetes mellitus was defined as a high - fasting plasma glucose level ( 126 mg / dl ) , or a current treatment with an oral hypoglycemic agent or insulin hyperfibrinogenemia was defined as a high plasma fibrinogen level ( 400 mg / dl ) hyperlipidemia was defined as a fasting serum total cholesterol level of 240 mg / dl , or a current treatment with an antihyperlipidemic agent ischemic changes on electrocardiogram ( ecg ; st - t change , flat t , and negative t ) fever ( normal body temperature range 36.0 - 37.4c , low grade fever range 37.5 - 38.0c , and high grade fever 38.1c ) fluid volume balance was defined as intake and output balance from admission to the end of operation packing degree was classified as defined by raymond et al . class 1 complete occlusion , class 2 residual neck , and class 3 residual aneurysm sac . for all 54 patients , cerebral angiography showed patency of the cerebral artery during the process of treatment . non - contrast brain ct ( siemens , munich , germany ) was performed immediately after the procedure . for the 47 patients demonstrating no neurological deficit , repeat cts were requested at days 1 and 7 or sooner at the physician 's discretion and no cerebral infarction occurred . with seven patients in the aci group and 47 patients in the no aci group , the study was designed to detect the risk factors of aci after embolization and a two - sided a level of 0.05 . descriptive statistics , stratified by cerebral anterior circulation state , were calculated for baseline demographic and clinical characteristics . student 's t - test was used to compare continuous variables , the test to compare categorical variables , and the rank sum test to compare multicategorical variables according to cerebral infarction or no cerebral infarction of anterior circulation after embolization . multiple logistic regression analysis was performed to identify independent risk factors for aci in the two groups . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated after adjusting for possible confounders ( aneurysm size , modified fisher grade , and embolization time ) . all p - values are two - sided with statistical significance evaluated at the 0.05 a level . ninety - five percent cis were calculated to assess the precision of the obtained adjusted odds ratio estimates . all analyses were performed using statistical package for social sciences ( spss ) 17.0 ( spss , chicago , il ) . case files from patients that had undergone coil embolization of cerebral anterior communication artery aneurysms from october 2008 to april 2012 at tangshan gongren hospital were retrospectively reviewed . the patency of 54 patients aca and acoa during embolization was evaluated in the study . the stroke rate was 8.7% in ruptured patients for one time before embolization finished and 37.5% in ruptured patients for twice before embolization finished . the mean age of the study group was 53.6 years with a 1:1 female - to - male ratio . hospital records including clinical charts , operative reports , and radiological studies were used to obtain patient characteristics including : age , coil type , rupture status , sex , aneurysm location , aneurysm size , embolization results , complication , and necessity of retreatment . the timing of the infarction following treatment occurred at 3 postoperative hours to 3 days . this study was conducted with approval from the ethics committee and intuitional review board of hebei medical university . the technique for endovascular coiling alone and stent - assisted coiling ( boston scientific / target , california , america ) has been previously described in the literature . all procedures were performed using a biplane angiographic system ( philippe , amsterdam , holland ) with three - dimensional rotational and digital subtraction capabilities and with the patients under general anesthesia and a systemic anticoagulant . anticoagulation was aimed at keeping the activated clotting time at two to three times above the normal value ( approximately 100 s ) during catheterization , stent , and coil placement . our protocol includes : 1 ) pretreatment with aspirin ( 300 mg ) and clopidogrel bisulfate ( 300 mg ) 30 min before coiling for stented patients and no pretreatment before coiling for non - stented patients , 2 ) systemic intravenous heparinization after placement of the femoral sheath for a goal activated clotting time of 250 - 300 s , 3 ) systemic intravenous heparinization 12 h after the procedure , 4 ) continuation of clopidogrel bisulfate ( 75 mg daily ) for 6 months for stented patients and discontinuation of clopidogrel bisulfate for non - stented patients , and 5 ) aspirin ( 100 mg daily ) for all patients indefinitely . the risk factors considered in the study were sex , age , smoking , alcohol consumption , hypertension , diabetes mellitus , hyperfibrinogenemia , hyperlipidemia , ischemic changes on electrocardiogram ( ecg ) , fever , fluid volume balance , modified fisher grade , hunt - hess grade , intracerebral hematoma , ventricular hemorrhage , cerebral swelling , hydrocephalus , unilateral dysplasia of cerebral anterior circulation , direction of aneurysm body , aneurysm blood supply from left aca , aneurysm rupture times , time from aneurysm recent rupture before admission to embolization , stent use , packing degree , leukocyte count , platelet count , hematocrit , comorbidity score , aneurysm neck width , aneurysm parent artery width , aneurysm size , and embolization time . we defined the potential risk factors as follows : smoking ( currently smoking more than one cigarette per day)alcohol consumption ( drinking more than twice per week)hypertension ( systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , and/or presence of antihypertensive drug treatment)diabetes mellitus was defined as a high - fasting plasma glucose level ( 126 mg / dl ) , or a current treatment with an oral hypoglycemic agent or insulinhyperfibrinogenemia was defined as a high plasma fibrinogen level ( 400 mg / dl)hyperlipidemia was defined as a fasting serum total cholesterol level of 240 mg / dl , or a current treatment with an antihyperlipidemic agentischemic changes on electrocardiogram ( ecg ; st - t change , flat t , and negative t)fever ( normal body temperature range 36.0 - 37.4c , low grade fever range 37.5 - 38.0c , and high grade fever 38.1c)fluid volume balance was defined as intake and output balance from admission to the end of operationpacking degree was classified as defined by raymond et al . class 1 complete occlusion , class 2 residual neck , and class 3 residual aneurysm sac . smoking ( currently smoking more than one cigarette per day ) alcohol consumption ( drinking more than twice per week ) hypertension ( systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , and/or presence of antihypertensive drug treatment ) diabetes mellitus was defined as a high - fasting plasma glucose level ( 126 mg / dl ) , or a current treatment with an oral hypoglycemic agent or insulin hyperfibrinogenemia was defined as a high plasma fibrinogen level ( 400 mg / dl ) hyperlipidemia was defined as a fasting serum total cholesterol level of 240 mg / dl , or a current treatment with an antihyperlipidemic agent ischemic changes on electrocardiogram ( ecg ; st - t change , flat t , and negative t ) fever ( normal body temperature range 36.0 - 37.4c , low grade fever range 37.5 - 38.0c , and high grade fever 38.1c ) fluid volume balance was defined as intake and output balance from admission to the end of operation packing degree was classified as defined by raymond et al . class 1 complete occlusion , class 2 residual neck , and class 3 residual aneurysm sac . for all 54 patients , cerebral angiography showed patency of the cerebral artery during the process of treatment . non - contrast brain ct ( siemens , munich , germany ) was performed immediately after the procedure . for the 47 patients demonstrating no neurological deficit , repeat cts were requested at days 1 and 7 or sooner at the physician 's discretion and no cerebral infarction occurred . with seven patients in the aci group and 47 patients in the no aci group , the study was designed to detect the risk factors of aci after embolization and a two - sided a level of 0.05 . descriptive statistics , stratified by cerebral anterior circulation state , were calculated for baseline demographic and clinical characteristics . student 's t - test was used to compare continuous variables , the test to compare categorical variables , and the rank sum test to compare multicategorical variables according to cerebral infarction or no cerebral infarction of anterior circulation after embolization . multiple logistic regression analysis was performed to identify independent risk factors for aci in the two groups . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated after adjusting for possible confounders ( aneurysm size , modified fisher grade , and embolization time ) . all p - values are two - sided with statistical significance evaluated at the 0.05 a level . ninety - five percent cis were calculated to assess the precision of the obtained adjusted odds ratio estimates . all analyses were performed using statistical package for social sciences ( spss ) 17.0 ( spss , chicago , il ) . after exclusion criteria were met , 54 acoa aneurysms from 54 patients were analyzed . of these , seven ( 13.0% ) were categorized into group i ( aci after embolization ) and 47 ( 87.0% ) were categorized into group ii ( no aci after embolization ) . the neurological outcomes of the ischemic stroke patients included brain swelling , motor dysfunction , abulia , aphasia , etc . fifty - four acoa aneurysms ( 100% ) were ruptured in patients presenting with subarachnoid hemorrhage . univariate analysis of the clinical and laboratory data of the anterior circulation cerebral infarction and no anterior circulation cerebral infarction groups only two of the analyzed variables were significantly different between groups . univariate analyses of clinical and laboratory data showed that negative fluid volume balance and modified fisher grade were more prevalent in the aci group than in the no aci group [ table 1 ] . other clinical and laboratory data , including age , sex , hypertension , diabetes mellitus , hunt - hess grade , and ventricular hemorrhage showed no differences between groups . multiple logistic regression analyses adjusted for possible confounding factors . the adjusted or for modified fisher grade ( or : 4.968 , 95% ci : 1.013 - 24.360 , p = 0.048 ) was significantly higher in the aci group [ table 2 ] . results of multiple logistic regression analysis applied to the anterior circulation cerebral infarction and no anterior circulation cerebral infarction groups although the risk factors of ischemic stroke in the anterior circulation have been extensively researched , there have been fewer studies focusing on the risk factors of aci after embolization ; however , these studies do reveal risk factors comparable to those reported in the present study . previously , it was demonstrated that modified fisher grade was the most influential risk factor for anterior circulation stroke . given these findings , we evaluated the risk factors of aci after embolization and report the resulting effects on cerebral stroke here . in the present study , the prevalence of aci after embolization ( 13.0% ) was found to be high in patients with the endovascular treatment of acoa aneurysms and modified fisher grade was found to be the major determinant for anterior circulation infarction after embolization . seven of 55 patients undergoing coil embolization of acoa aneurysms developed aci after embolization , in which the patency of anterior circulation during embolization was observed . thromboembolic events during endovascular treatment of intracranial aneurysms were reported to be between 2.9 and 6% . fang et al . , showed morbidity or mortality caused by perioperative stroke occurred at a 3% rate in patients with acoa aneurysms . why then , was the stroke rate ( 13% ) so high in our study ? the higher stroke rate is likely because the patients undergoing coil embolization of cerebral anterior communication artery aneurysms in this study had all ruptured once or twice prior to embolization . the possible mechanisms for aci after embolization were previously thought to be vasospasm , thrombosis , and emboli . no pulmonary embolism , cardiogenic embolism , deep venous thrombosis , cerebral vascular arteriosclerosis , nor cerebral vascular stenosis were found in the current study . our study confirmed that vasospasm and thrombosis were the most common etiology for aci after embolization . in patients with subarachnoid hemorrhage due to acoa aneurysm , the significant risk factors determining outcomes were age , glasgow coma scale ( gcs ) , hunt - hess grade , vasospasm , ventricular hemorrhage , and hydrocephalus . however , the associations between aci after embolization of acoa aneurysm and the risk factors , such as , age , sex , negative fluid volume balance , modified fisher grade , aneurysm size , embolization time , and so on are not being determined because of the limited number of studies undertaken . in the present study , multiple logistic regression analysis revealed that modified fisher grade is independently associated with aci after embolization ; whereas , no association is found for other variables , which include negative fluid volume balance , aneurysm size , and embolization time . the previous studies reported that a closed relationship was observed between modified fisher grade and delayed ischemic neurological deficit . these findings suggest that modified fisher grade is a strong determinant for aci after embolization , and that a patient with a high modified fisher grade is at higher risk of aci after embolization . aci after embolization is a potent predictor of a poor outcome in acoa aneurysm patients . accordingly , modified fisher grade prior to stroke is required for prevention of aci after embolization . as much higher grade of modified fisher grade , we can enhance our prevention and treatment for vasospasm and thrombosis . first , this is a retrospective study , and inaccurate or insufficient assessment might have occurred . second , this is a relatively small study , so the predictor of outcome could be significant by chance . third , because the patients were recruited from a tertiary hospital , they may not represent the general stroke population after embolization . fourth , aneurysm characteristics and assessment of embolization were assessed using aneurysm size , neck width , and raymond classification ; but not using murayama classification , aneurysm volume in cubic millimeters , neck - to - volume ratio , or volumetric packing density . fifth , ct was used in this study , which is not very sensitive in the evaluation of stroke . thus , some of the stroke patients may not be ruled out . finally , all our patients were chinese , and our data may not be generalizable to other ethnic groups . as such , we await the outcome of a prospective , randomized , controlled trial when treating cerebral aneurysms . overall , the present study provides clues regarding the prevalence and causative risk factor of aci after embolization . our results showed high prevalence of aci after embolization , and a modified fisher grade was identified as the most important risk factor therefore . further studies on the mechanism , prevention , and treatment of aci after embolization are needed to provide specific guidance on its long - term management .

background : despite increasing acceptance of endovascular coiling for treating anterior communicating artery ( acoa ) aneurysms , anterior circulation cerebral infarction ( aci ) after embolization remains a limitation . with higher incidence , higher morbidity and higher mortality , it is one of the main factors influencing the acoa aneurysms prognosis . determining the risk factors leading to aci after embolization will have clinical significance . through retrospective case analysis , this study investigated the risk factors related to aci after embolization in order to provide information to serve the clinical practice.materials and methods : a retrospective review was performed of patients who had undergone coiling of acoa aneurysms from 2008 to 2012 . all patients had ruptured prior to the completion of embolization . cases with acute stroke symptoms without alternative diagnoses after embolization were diagnosed as aci . a total of 32 risk factors such as age , sex , hypertension , diabetes mellitus , modified fisher grade , hunt - hess grade , ventricular hemorrhage , etc . were analyzed using univariate and logistic regression analysis.results:univariate analysis showed that negative fluid volume balance ( p = 0.041 < 0.05 ) and modified fisher grade ( p = 0.049 < 0.05 ) reached statistical significance , suggesting that they might be risk factors for aci after embolization . multiple logistic regression analysis showed that modified fisher grade was significantly associated with aci after embolization , suggesting that it was an independent risk factor ( odds ratios ( or ) : 4.968 , 95% confidence intervals ( ci ) : 1.013 - 24.360 , p = 0.048).conclusion : modified fisher grade is an independent risk factor for aci after embolization .

neck pain is a common musculoskeletal disorder in modern society that can produce severe pain . 67% of the population suffers from it at least once in a lifetime and the prevalence is about 23%1 . the pain exacerbates and fades periodically , and many patients do not fully recover from the symptoms2 . neck pain occurs in the upper thoracic spine area including the shoulder , and it is mechanical pain caused by bad postures and habits in most cases3 . according to sharon , the upper thoracic spine is involved in the physiologic motion of the neck4 . the decreased movement of the upper cervical spine can cause excessive movement of the lower cervical spine , increase fatigue in the sternocleidomastoid , anterior scalenus , and upper trapezius , cause changes of neck postures and breathing patterns , and a decrease in the range of motion5 . patients with chronic neck pain experience functional impairments including weakening of deep bending neck muscles due to the activation of neck surface muscles6 , increased deformity of the forward head posture7 , proprioception impairment8 , and poor balance9 . in addition , decreased movement of the cervical spine restricts the range of motion of the spine and decreases breathing function10 , 11 . slightly bent positions like the forward head posture may cause mechanical neck pain , cause a greater load by affecting the mobilization order of the muscles operating when the arm is raised , and restrict the range of motion12 . these changes reduce the ability to maintain balance and increase the risk of falls and injury of the musculoskeletal system9 . physiotherapists are using methods such as electrotherapy , therapeutic exercise , and manual therapy to intervene in the neck pain13 . in a cochrane review study of neck pain , stretching exercises for the neck and upper limbs , strengthening exercises , static and dynamic stabilization exercises maitland mobilization grade 3 and 4 treatment for the cervical spine and upper spine significantly decreases neck disability index ( ndi ) , the pain index15 , and increases the range of motion16 . in addition , an the intervention combining therapeutic exercise and manual therapy was significantly more effective compared to manual therapy alone17 . recent evidence - based studies report that treatment combining therapeutic exercise and manual therapy is more effective18 , but research of combined interventions is sparse . the purpose of this study was to apply joint mobilization and therapeutic exercise to the cervical spine and upper spine , investigate the effects on functional impairments caused by the neck pain , and examine differences between groups by comparing the intervention group with the group to which only therapeutic exercises were applied . this study was implemented with non - specific neck pain patients with no medical findings who had visited the cheongju st . all participants received verbal and written information about the study and signed a consent form . the patients who had undergone or would have surgery in the spine , and those who had neurological damage , a cervical spine fracture , osteoporosis , arthritis , a malignant neoplasm , a vascular disease , or a psychiatric problem were excluded . the subjects were randomly assigned to one of two groups of nine people each . group i was the therapeutic exercise group , and group ii was the group to which joint mobilization was applied in combination with therapeutic exercise ( table 1table 1.general characteristics of the subjects ( n=18)group igroup iigender ( female)99age ( years)58.0 1.659.0 2.4height ( cm)158.0 3.9157.0 4.7weight ( kg)58.0 4.659.0 6.6values are expressed as mean sd . * significant difference between pre - post test ( p<0.05 ) . group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic exercise ) ) . the pretest included the visual analog scale ( vas ) , neck disability index ( ndi ) , active cervical range of motion ( acrom ) , static balance ability , muscle tone of the upper trapezius , and respiratory function . after the pretest , group i performed therapeutic exercise and group ii performed both therapeutic exercise and joint mobilization for 60 minutes a day , three times a week for two weeks under the guidance of a physical therapist . the participants did not receive any other interventions associated with the neck pain while this research was being conducted . the post - test was carried out with the same protocol as the pretest after two weeks . values are expressed as mean sd . * significant difference between pre - post test ( p<0.05 ) . group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic exercise ) therapeutic exercises involved enhancing mobility , stability and muscular strength of the neck , improving proprioception , and performing reeducation of movement , and the intensity of exercises was adjusted according to the physical abilities of the individuals . for the joint mobilization , after the painful sites were located by examination , the active movement test was conducted to find where joint mobilization would be applied , and the amount and quality of motion were examined . afterwards , passive physiological intervertebral movement ( ppivm ) testing and passive intervertebral accessory movement ( paivm ) test were conducted to find which joints had restricted or excessive movement , and determine where joint mobilization would be applied . the vas was employed to measure the intensity of pain19 , the ndi was used to measure neck pain disability20 , and crom instrument to assess the cervical range of motion21 . in order to check static balance , myoton pro ( myoton as , estonia ) measured the muscle tone of the upper trapezius22 , and a spirometer ( pony fx spirometer ; cosmed , rome , italy ) measured the respiratory function23 . , an ibm company , chicago , il , usa ) . for the normality test , the shapiro wilk test was conducted , confirming that the data are normally distributed . assuming homogeneity between groups , the independent samples t - test was performed , and the paired sample t - test was conducted for the within - group comparison of the measurements before and after the interventions . in addition , the independent samples t - test was conducted to compare the differences between groups . null hypotheses of no difference were rejected if p - values were less than 0.05 . vas and ndi led to significant changes in both groups , and group ii improved significantly more than group i ( tables 2table 2.visual analog scale of the subjectsgroup igroup iipre4.8 0.44.9 0.3post2.7 0.5 * 1.4 05**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) , 3table 3.neck disability index of the subjectsgroup igroup iipre15.1 2.317.2 3.1post8.9 1.5 * 8.6 1.9**significant difference between pre - post test ( p<0.05 ) . the acrom increased significantly in both groups , and group ii improved significantly more on the right lateral flexion and rightward rotation than group i ( table 4table 4.active cervical range of motion of the subjects ( )group igroup iiflexionpre49.2 8.748.9 4.7post54.2 4.5 56.1 6.8*extensionpre54.9 4.356.4 4.9post 64.3 3.2 * 67.6 3.9*rt . rotationpre60.8 3.259.7 5.1post 67.6 3.6 * 67.7 4.2**significant difference between pre - post test ( p<0.05 ) . both groups improved significantly in static balance , with no difference between groups ( tables 5table 5.center of gravity sway velocities of each group under different conditions ( /s)group igroup iifirm - eyes openpre0.04 0.00.05 0.0 post0.03 0.00.04 0.0firm - eyes closedpre0.15 0.00.15 0.0post0.14 0.00.14 0.10.37 0.1post 0.30 0.1 * 0.32 0.1**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) , 6table 6.center of gravity total sway distances of each group under different conditions ( /s)group igroup iifirm - eyes openpre294.6 64.1304.6 97.9 post270.4 66.4281.9 109.2foam - eyes closedpre618.2 82.7687.3 182.8post 529.1 88.2 * 548.4 172.5**significant difference between pre - post test ( p<0.05)significant difference between groups ( p<0.05 ) ) . muscle tone in the left and right upper trapezius muscle improved significantly in both groups , and there was no difference between groups ( table 7table 7.muscle tone of the subjectsgroup igroup iirt . upper trapeziuspre16.0 1.5 16.0 1.2post13.7 1.3 * 14.2 1.4**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . chronic neck pain patients suffer diverse functional impairments , including a deterioration of balance ability , respiratory function , range of joint motion , and proprioception11 . research related to neck pain suggests that there are more efficient interventions based on evidence through systematic reviews , and the interventions combining manual therapy and therapeutic exercise are described as more effective in recent literature18 . in this study , therapeutic exercises were conducted that are reported to be effective for patients with mechanical neck pain , namely , stretching exercise for the neck and upper limbs , static and dynamic stability exercise , strengthening exercise , and exercise reeducation14 . in addition , as manual therapy , mobilization to reduce pain and improve the movements of the soft tissue exhibiting contracture and restricted motions of joints ( maitland grade iii and iv)13 , 24 were applied to group ii . the effect of the two kinds of interventions , and group differences based on those interventions were examined . in this research , a significant reduction in the vas and ndi was observed in both groups , and the comparison between the groups showed that the intervention was more effective in group ii . this result is different from studies that found no difference between groups based on a short - term intervention17 . these differences may result from our smaller number of subjects and differences of individual characteristics . for the acrom , both groups improved significantly overall , and group ii improved more on right lateral flexion and rightward rotation . these results are consistent with studies that reported that mobilization applied to both the neck and spine at the same time improved cranial vertical angle and cranial rotation angle25 and research that showed that endurance exercise of the neck , stabilization exercise , and strength training improved the joint range of motion26 . in this study , static balance was measured under a variety of conditions , including hard or soft bearing surfaces and open or closed eyes . a significant decrease in the sway velocity of the center of gravity and in the sway distance occurred only under eyes closed , and there was no difference between groups . although it was reported that patients with neck pain and forward head posture may exhibit reduced balance ability9 , it was possible to measure the static balance ability most accurately when the soft bearing surface was used and eyes were closed to block the visual feedback . muscle tone of the upper trapezius decreased significantly in both groups , and there was no difference between the groups . this may indicate that the tension and fatigue of the upper trapezius were reduced due to pain reduction , enhancement of mobility of the neck joints , and change in the mobilization sequence of movements caused by afferent information activation of proprioception5 . deterioration of mobility of the spine and thoracic cage was reported in patients of chronic neck pain , and respiratory function was also affected27 . however , in this research , no significant change in the respiratory function was observed . this may be because the patients were not given direct training for functional improvement of breathing muscles , although the range of motion of the cervical and upper thoracic spine was improved through the study interventions . the number of subjects was small , and since the period of intervention was not long , it was difficult to produce a mechanical change of muscles . therefore , future research is required to investigate the effect of joint mobilization and therapeutic exercise on the functional impairments caused by chronic neck pain using diverse subjects and intervention periods , and research on the persistence of the effect also needs to be conducted . in conclusion , joint mobilization and therapeutic exercise for functional impairments caused by chronic neck pain significantly improved several types of functional impairment . in addition , in the group to which both joint mobilization and therapeutic exercise were applied , significantly more improvement in the pain index , neck disability levels , and acrom was seen than in the group that received only therapeutic exercise .

[ purpose ] this study evaluated joint mobilization and therapeutic exercise applied to the cervical spine and upper thoracic spine for functional impairment caused by chronic neck pain . [ subjects and methods ] eighteen study subjects were randomly assigned to two groups of nine people each . therapeutic exercise only was applied to the cervical and upper thoracic spine for group i , while both therapeutic exercise and joint mobilization were applied to group ii . the visual analog scale , neck disability index , active cervical range of motion , static balance capacity , and muscle tone were assessed with a pre - test . the intervention was carried out for 60 minutes a day , three times a week , for two weeks for each group , followed by a post - test using the same protocol as the pre - test . [ results ] the visual analog scale , neck disability index , and active cervical range of motion improved significantly in both groups . group ii improved significantly more on right lateral flexion and rightward rotation . muscle tone improved significantly in the upper trapezius in both groups . [ conclusion ] the joint mobilization and therapeutic exercise for functional impairments caused by chronic neck pain had a significant effect on several types of functional impairment .

the indications for laparoscopic pyeloplasty are flank pain , infection , stone formation , and decreased renal function . many studies have reported that laparoscopic pyeloplasty has an excellent success rate ranging from 9098% [ 15 ] . success is usually defined as improvement in hydronephrosis , as determined by ultrasound ( us ) or intravenous pyelography ( ivp ) , and the improvement of function on diuretic renography along with a decrease in washout time . although diuretic renography may be the most accurate modality with which to evaluate surgical outcome , renal us is more commonly used for long term follow up because it is less invasive and more cost effective . although there have been some published reports on the longitudinal renal sonographic changes after this procedure in children [ 68 ] , there is no similar report in adults . we evaluated the improvement of hydronephrosis longitudinally using us and ivp after retroperitoneal laparoscopic dismembered anderson between january 2006 and june 2012 , 16 patients with upjo were treated by retroperitoneal laparoscopic dismembered anderson the group included 8 females ( 50% ) and 8 males ( 50% ) with a mean age of 30 years . all 16 patients had upjo as confirmed by renal us , ivp , retrograde pyelography , and a diuretic renal scan . the patients had the disease on the right side in 4 cases and on the left in 12 cases . four patients ( 25% ) had renal calculi associated with upjo ( table 1 ) . a 2cm incision was first made at the midpoint between the 12 rib and the iliac crest in the middle axillary line . after a working space was created in the retroperitoneum by balloon dissector , a 12mm blunt a second 12mm trocar was placed 7 cm dorsal to the camera port under laparoscopic guidance . a third 5mm trocar was placed in the anterior axillary line at the level of the iliac crest , and a fourth 5mm trocar was placed in the subcostal region at the anterior axillary line . the lateroconal fascia was incised along the quadratus lumborum muscle , which allowed for exposure of the psoas muscle and the posterior lamina of gerota 's fascia . the ureter was identified and dissected in the cephalad direction toward the ureteropelvic junction ( upj ) . after dissection and clear visualization of the upjo , a stay suture was placed in the medial edge of the renal pelvis and was pulled out through the abdominal wall using endo close. the medial portion of the redundant pelvis was partially excised , and the proximal ureter was laterally spatulated with a 1.0cm longitudinal incision . the renal pelvis was then dismembered with the proximal ureter and the stenotic segment of the upj was resected . the first suture , a 40 polydioxanone suture , was placed from the most interconnected portion of the renal pelvis to the most inferior point of the ureteral spatulation . after the anterior anastomosis was completed , the same suture was applied to the posterior wall of the anastomosis . if a crossing vessel was identified during dissection of the ureter , it was preserved and the ureter was transposed anterior or posterior to the vessel . after hemostasis was confirmed , a 5mm suction drain was placed in the retroperitoneum , and all wounds were closed . after the renal pelvis was transected , stones were removed individually under direct laparoscopic vision with the use of standard laparoscopic graspers . renal us was performed at 3 , 6 , 12 , 18 , and 24 months and ivp was performed at 6 and 12 months after surgery . the degree of hydronephrosis was graded as 0 to 3 according to ellenbogen 's grading system . the procedure was considered successful when patients experienced the complete resolution of pain , improvement of hydronephrosis on us and ivp , and decreased half time clearance ( t1/2 ) as measured during a diuretic renal scan . between january 2006 and june 2012 , 16 patients with upjo were treated by retroperitoneal laparoscopic dismembered anderson the group included 8 females ( 50% ) and 8 males ( 50% ) with a mean age of 30 years . all 16 patients had upjo as confirmed by renal us , ivp , retrograde pyelography , and a diuretic renal scan . the patients had the disease on the right side in 4 cases and on the left in 12 cases . four patients ( 25% ) had renal calculi associated with upjo ( table 1 ) . a 2cm incision was first made at the midpoint between the 12 rib and the iliac crest in the middle axillary line . after a working space was created in the retroperitoneum by balloon dissector , a 12mm blunt a second 12mm trocar was placed 7 cm dorsal to the camera port under laparoscopic guidance . a third 5mm trocar was placed in the anterior axillary line at the level of the iliac crest , and a fourth 5mm trocar was placed in the subcostal region at the anterior axillary line . the lateroconal fascia was incised along the quadratus lumborum muscle , which allowed for exposure of the psoas muscle and the posterior lamina of gerota 's fascia . the ureter was identified and dissected in the cephalad direction toward the ureteropelvic junction ( upj ) . after dissection and clear visualization of the upjo , a stay suture was placed in the medial edge of the renal pelvis and was pulled out through the abdominal wall using endo close. the medial portion of the redundant pelvis was partially excised , and the proximal ureter was laterally spatulated with a 1.0cm longitudinal incision . the renal pelvis was then dismembered with the proximal ureter and the stenotic segment of the upj was resected . the first suture , a 40 polydioxanone suture , was placed from the most interconnected portion of the renal pelvis to the most inferior point of the ureteral spatulation . after the anterior anastomosis was completed , the same suture was applied to the posterior wall of the anastomosis . if a crossing vessel was identified during dissection of the ureter , it was preserved and the ureter was transposed anterior or posterior to the vessel . after hemostasis was confirmed , a 5mm suction drain was placed in the retroperitoneum , and all wounds were closed . after the renal pelvis was transected , stones were removed individually under direct laparoscopic vision with the use of standard laparoscopic graspers . renal us was performed at 3 , 6 , 12 , 18 , and 24 months and ivp was performed at 6 and 12 months after surgery . the degree of hydronephrosis was graded as 0 to 3 according to ellenbogen 's grading system . the procedure was considered successful when patients experienced the complete resolution of pain , improvement of hydronephrosis on us and ivp , and decreased half time clearance ( t1/2 ) as measured during a diuretic renal scan . the degree of hydronephrosis as determined based on us was identical to that determined based on ivp . the postoperative improvement of hydronephrosis was 63% , 100% , 100% , 100% , and 100% at 3 , 6 , 12 , 18 , and 24 months , respectively . the postoperative improvement of hydronephrosis by one grade was 56% , 73% , 67% , 50% , and 40% at 3 , 6 , 12 , 18 , and 24 months , respectively . improvement in the degree of hydronephrosis by two grades was observed in 6% , 27% , 33% , 50% , and 60% of patients at 3 , 6 , 12 , 18 , and 24 months , respectively ( table 2 ) . complete resolution of the hydronephrosis was observed in 8 of 16 patients ( 50% ) . hydronephrosis was still improving even after 12 months in 5 of 12 patients ( 42% ) ( figure 1 ) . t1/2 improved in 9 of 12 patients ( 75% ) as determined during the diuretic renal scan at 12 months ( table 3 ) . three patients experienced no changes in the results of the diuretic renal scan 1 year postoperatively . among these patients , the degree of hydronephrosis decreased to grade 1 and grade 2 at 6 months , respectively , but no further improvement of hydronephrosis was observed at 24 months . in the third patient who had grade 3 hydronephrosis preoperatively , the degree of hydronephrosis decreased to grade 2 at 3 months and continued to decrease gradually until complete resolution was obtained at 24 months . this case was regarded as successful , although improvement could not be confirmed on the diuretic renal scan . rate of improvement in hydronephrosis as determined on us and ivp after laparoscopic pyeloplasty us = ultrasound ; ivp = intravenous pyelography the change of t1/2 of the diuretic renal scan after laparoscopic pyeloplasty laparoscopic pyeloplasty was introduced in 1993 by schussller and was developed worldwide as the first minimal option , which has been shown to reduce hospital stay while offering success rates equivalent to those of the open pyeloplasty [ 1 , 11 , 12 ] . an advantage in adopting retroperitoneal laparoscopic approach compared with transperitoneal counterpart for pyeloplasty is related to a more direct path to the upj and the low risk of injury to intraperitoneal organ . conversely , the retroperitoneal approach has a limited working space that may increase the difficulty of reconstruction . whereas the magnitude of pelvic enlargement correlates statistically with the likelihood of obstruction , us can not be used to diagnose obstruction because the degree of pelvic dilatation does not specifically indicate the presence or absence of obstruction , or predict whether the hydronephrosis will improve or worsen . therefore , the success of laparoscopic pyeloplasty is usually demonstrated radiographically by an improvement in drainage and/or function as visualized by the postoperative diuretic renogram and by decreased hydronephrosis on renal us . in children amling reported that only 38% of the kidneys improved during the first 6 months of follow up , while 81% were improved 2 years postoperatively , and ultimate improvement to grade 0 or 1 was noted in only 19% of cases . kis et al . reported that 102 babies who underwent pyeloplasty were investigated by us at 6 and 12 months postoperatively . one year after surgery , the renal pelvis was smaller in 76% of cases , and renal parenchyma was normal or had increased in 92% of cases . the authors reported that the resolution of hydronephrosis after surgery was quite slow in the first postoperative year , while the increase in the renal parenchyma was relatively rapid . in the series by neste et al . , postoperative us and diuretic renograms were reviewed in patients monitored an average of 26 months after pyeloplasty . improvement as determined using us was much more gradual than the change in renographic pattern with renographic improvement observed well in advance of that observed on the us . there has been no report on the longitudinal assessment of hydronephrosis after pyeloplasty in adults . in our adult series , an improvement of hydronephrosis was observed in 10 of 16 patients ( 63% ) at 3 months , and all cases showed improvement at 6 months after surgery . all patients were without symptoms or infection after 3 months , although hydronephrosis was still present in many patients for months . in 5 of 12 patients ( 42% ) , hydronephrosis was still improving gradually even after 12 months postoperatively , and the complete resolution of hydronephrosis was observed in 8 of 16 patients ( 50% ) . the results suggest that hydronephrosis starts to improve relatively earlier in adults than in children and continues to improve gradually over the long term . in children , hydronephrosis may be found as an abdominal mass . on the contrary , in adults , flank pain or urinary tract infection since most adult upjos are symptomatic , adult patients are diagnosed relatively early when hydronephrosis has increased . for this reason , hydronephrosis may improve rapidly and remain decreased for a long time after the obstruction was treated . in most institutions , diuretic renogram is usually performed at 3 months and annually thereafter [ 1 , 2 , 11 ] . in our series , patients were followed up with us ( with or without ivp ) at 3 , 6 , 12 , 18 and 24 months to assess morphologically , and diuretic renogram was performed at 12 months to assess functionally . since the improvement in hydronephrosis and the improvement observed on the diuretic renal scan were not necessarily seen simultaneously , we recommend that patients who underwent laparoscopic dismembered pyeloplasty should undergo a diuretic renal scan at 12 months postoperatively and be followed up at least 2 years postoperatively . concerning the relation between us , ivp , and renal scan , the degree of hydronephrosis as determined based on us was identical to that determined based on ivp . there were three patients who experienced no improvement in the diuretic renal scan at 12 months . among these patients , one patient had gradual improvement of hydronephrosis even after 12 months and had complete resolution at 24 months . in this patient it could have been confirmed that t1/2 decreased if the diuretic renal scan was performed at 24 months . these results showed that serial us showing improvement of hydronephrosis may be a good alternative to ivp or diuretic renal scan in the follow up of upjo patients who underwent laparoscopic pyeloplasty . longitudinal analysis of larger numbers of patients with hydronephrosis after laparoscopic pyeloplasty will be necessary . although hydronephrosis had not changed in 37% of patients at 3 months , all cases showed improvement at 6 months , and continuous improvement was found in 40% of patients who were followed up for more than 12 months . hydronephrosis improves relatively more rapidly and for a longer period of time in adults compared to pediatric patients . however , a one grade improvement in the degree of hydronephrosis does not necessarily represent true therapeutic success . follow up by diuretic renal scan as well as us is needed if improvement can not be confirmed by diuretic renal scan at 12 months postoperatively .

objectivewe evaluated the improvement of hydronephrosis longitudinally after laparoscopic dismembered anderson hynes pyeloplasty in adult patients with ureteropelvic junction obstruction.material and methodssixteen patients underwent laparoscopic pyeloplasty at our institution between january 2006 and june 2012 . hydronephrosis was assessed by ultrasound and intravenous pyelography at 3 , 6 , 12 , 18 , and 24 months after pyeloplasty.resultsthe mean follow up time was 24 months . preoperative hydronephrosis was diagnosed as grade 2 and grade 3 in 8 patients each . postoperative improvement of the hydronephrosis by one grade was observed in 56% , 73% , 67% , 50% , and 40% of patients at 3 , 6 , 12 , 18 , and 24 months , respectively . improvement of the hydronephrosis by two grades was observed in 6% , 27% , 33% , 50% , and 60% of patients at 3 , 6 , 12 , 18 , and 24 months , respectively . in 5 of 12 patients ( 42% ) , hydronephrosis was still improving even after 12 months postoperatively.conclusionsadult patients demonstrate relatively rapid improvements in the degree of hydronephrosis after laparoscopic pyeloplasty and continue to improve for a long time .

this retrospective study was approved by the institutional review board of our hospital ( 2014 - 09 - 037 ) . between november 2011 and october 2014 , 245 patients in the icu underwent bedside pdt with a ciaglia blue rhino percutaneous tracheostomy set ( cook medical , bloomington , in , usa ) due to prolonged endotracheal tube insertion . pdt was not performed in patients aged under 18 years , those with any pulsation palpated over the tracheostomy site , patients with a history of surgery or radiotherapy in the cervical region , and those with coagulopathy ( increased prothrombin time , inr > 2 ) . the first four procedures were performed by two physicians and one nurse using the standard technique , with one physician operating the bronchoscope while the other performed pdt . the next 55 procedures were performed by one physician and one nurse , with the physician using the bronchoscope only to confirm and evaluate the proper depth for the endotracheal tube before performing pdt using the simplified method described below . the remaining 186 procedures were performed using the simplified technique after repositioning the endotracheal tube at the predetermined depth ( 17 cm for males and 15 cm for females at the incisor ) . all pdts were performed by a single physician and one nurse , both of whom fully understood the entire procedure . the nurse administered sedatives and analgesics , as well as repositioning the endotracheal tube . except for patients with limited neck extension , a small pillow was placed under the patient 's shoulders to slightly extend the neck . transverse markings were made at the cricothyroid membrane and 2.0 cm toward the sternal notch ( fig . was also made in the midline of the trachea by holding the trachea between the thumb and index finger . the middle of the vertical mark intersected with the transverse mark 2.0 cm below the cricothyroid membrane ( fig . if the cricothyroid membrane could not be palpated , ultrasonography was used to identify the anatomic structures . the cricoid cartilage and tracheal rings were identified by placing the linear probe 45 degrees from the sagittal plane , and the area between the second and third tracheal rings was marked with a skin marker ( figs . the linear probe was positioned vertical to the trachea to confirm its midline ( figs . fio2 was increased to 1.0 , and 57 cmh2o positive end - expiratory pressure was applied to all patients receiving ventilator care . in patients who were receiving oxygen supplementation via a hydro - trach t - piece , the pdt site was disinfected with povidone iodine solution and a sterile drape was used before subcutaneously injecting 2% lidocaine . a vertical skin incision ( 1.5 cm ) was made through the midline vertical marking , and the endotracheal tube was withdrawn until the tube depth was 17 cm for males and 15 cm for females ( fig . 1d ) using a 5 cm - introducer needle , included in the cook ciaglia blue rhino set , and proper needle position was confirmed by air regurgitation with a syringe containing normal saline . the remaining pdt procedure was performed using the previous standard method with a # 7 tracheostomy tube ( twist , tracoe , germany ) . a # 8 tube was used only in patients in whom bronchoscopic lavage was planned ( about 10 patients ) . however , all 3 patients were receiving either extracorporeal membrane oxygenation or continuous renal replacement therapy and had an abnormal coagulation status . bleeding in all three long - term ( > 7 days ) follow - up in 150 patients showed no late complications and no deaths related to pdt . although use of a fob during pdt has been recommended for precise positioning and to avoid complications , bronchoscopy itself can cause complications . also , an additional physician is required and additional costs may result from damage to the fob . thus , several studies have assessed the safety of pdt without a fob , including technical modifications and simplified procedures . direct visualization of the larynx with a laryngoscope confirmed adequate endotracheal tube repositioning depth prior to pdt , while direct or indirect palpation of the trachea confirmed the proper tracheostomy site . although these simplified techniques have been shown to be safe , they have not been widely adopted . this study assessed the safety of a simplified pdt technique using the ciaglia blue rhino , by repositioning the existing endotracheal tube at a pre - determined depth and determining proper pdt site by superficial palpation . early complications of pdt include loss of airway , bleeding , conversion to open tracheostomy and death , with later complications including tracheostomy occlusion , tracheal stenosis and granulation . a retrospective study comparing early and late complications of pdt , with or without fob assistance , using the ciaglia blue rhino kit in 243 patients found no between group differences in early and late complications , but one patient experienced cardiac arrest during the use of a fob due to loss of airway . in this study , only 3 patients experienced bleeding after pdt , with all 3 receiving extracorporeal membrane oxygenation or continuous renal replacement therapy and having an abnormal coagulation status . however , we were unable to assess long - term complications in patients who were transferred to other facilities . precise positioning is critical when performing pdt , thus requiring use of a fob or direct palpation of the trachea . the biggest disadvantage of our modified percutaneous method was the inability to confirm the pdt site . in five patients , where the underlying structures could not be adequately palpated another disadvantage is the lack of precise endotracheal tube repositioning , which can be achieved with a laryngoscope . although a fob was used to determine the proper tube depth in 55 patients , the needle and guidewire entered the murphy eye of the tube twice . pdt was successfully performed by retracting the tube an additional 12 cm in both cases . although none of these patients experienced endotracheal cuff failure due to needle puncture , accidental cuff puncture may occur . moreover , in contrast to subcutaneous dissection , physicians using our modified technique may be unable to identify vessels between the skin and trachea . ultrasonography , which was used when anatomical structures were not readily identifiable , also has the advantage of screening such blood vessels . since all pdts were performed at a single center by two physicians , and the proper endotracheal tube depth was evaluated in a korean population , it may be difficult to generalize the safety of our simplified technique . another limitation was the absence of patients in which pdt is believed to be difficult , such as patients with severe obesity . although studies have suggested that pdt is safe in obese patients , use of additional equipment , such as a fob or ultrasonograph , has been recommended . we have not performed pdt in morbidly obese patients who may also require extra - long tracheostomy tubes . currently , therefore , a fob should be used when performing pdt in difficult situations , such as in patients with morbid obesity , anatomic alterations or neck stiffness . in conclusion , our results show that pdt with the ciaglia blue rhino technique can be safely performed without a bronchoscope and blunt dissection . this simplified technique is a relatively safe procedure that can be especially useful in situations of limited equipment and medical personnel .

when first introduced , percutaneous dilatational tracheostomy ( pdt ) was performed using a bronchoscope . the bronchoscope itself , however , has several disadvantages , including interruption of ventilation . modifications of the standard pdt technique have suggested that pdt can be safely performed without a bronchoscope . however , the safety of these modifications is unclear and many modifications have yet to be widely adopted . this study retrospectively evaluated the safety of a simplified pdt technique using the cook ciaglia blue rhino in 186 patients . after confirming the underlying structures by palpation , pdt was performed without a bronchoscope or blunt dissection , 2.0 cm below the cricothyroid membrane . ultrasonography was used only when palpation was difficult . except for bleeding in three patients with coagulopathy , none of the patients experienced any early or late complications , and there were no deaths related to pdt . these findings confirm that pdt can be safely performed using a simplified technique , which may be useful in situations with limited equipment and medical personnel .

hypertension ( htn ) is the most common comorbidity in the world with significant public health implications [ 1 , 2 ] . the overall u.s . prevalence of hypertension among adults ages 18 years in 20052008 was 30.9% and was highest among persons ages 65 years ( 69.7% ) and non - hispanic blacks ( 44% ) . the american heart association estimates that the incurred costs of hypertension are more than $ 93.5 billion per year , and that cardiovascular disease and stroke for which htn is the predominant risk factor , account for 17% of the total annual health expenditures in the united states . hypertension and its sequelae , heart failure ( hf ) , are a progressive disease . evidence shows that less than half of patients with heart failure survive five years ( after diagnosis ) , and less than a quarter of them live ten years after their initial diagnosis . framingham heart study showed that hypertensive patients were more likely to develop heart failure ( 142 cases of hf detected during the first 16 years of followup ) than those who were normotensive . the lifetime risk for development of hf among people with blood pressure ( bp ) > 160/90 mm hg is double that of those with bp < 140/90 mm hg . heart failure in comparison to the most prevalent gender malignancies ( bowel cancer in men and breast cancer in women ) , was associated with worse long - term survival . over a million hospitalizations , contributing to more than 250,000 deaths , and escalating billions of dollars in health care expenditure is the norm . the disastrous duo , htn and hf , should be recognized , and a tailored focus on management in special populations , high - risk ethnic minority groups especially blacks who are disproportionately burdened should be developed . among racial groups , african - american adults have the highest rates ( 44% ) of hypertension in the world and are more resistant to treatment . in particular , black women have the highest prevalence and the lowest blood pressure control . the relative incidence of hf is 50% higher in african americans , 3% of whom have hf , compared with 2% of the general population [ 13 , 14 ] . the disease occurs at an earlier age , and usually at a more advanced stage along with increased hospitalization and mortality . other causes of hf include coronary artery disease , valvular heart disease , diabetes , left ventricular hypertrophy and cardiomyopathies . overt clinical hf resulting from diastolic left ventricular dysfunction may be clinically indistinguishable from that resulting from systolic dysfunction . hf with preserved left ventricular function is observed in 30% to 50% of adult cases of hf . coronary artery disease and myocardial infarction is a principal cause of systolic left ventricular dysfunction followed by hypertension . a variety of neurohormonal systems , especially the renin - angiotensin aldosterone and sympathetic nervous systems are activated in response to the left ventricular dysfunction and such activation leads to abnormal ventricular remodeling . the inexorable progression to more severe stages of left ventricular dysfunction can be significantly reduced by effective therapy with neurohormonal blockade including angiotensin converting enzyme inhibitors ( aceis ) , beta blockers ( bbs ) , and aldosterone antagonists . current understanding of the pathophysiology of hf has revolved around neurohormonal activation [ 19 , 20 ] . this endothelial dysfunction may result from insufficient nitric oxide ( no ) secondary to either reduced endothelial production of no or to increase no inactivation [ 17 , 19 , 21 ] . in hf , a pronounced increase in angiotensin ii , with modestly reduced no is seen among whites . however , african americans with hf may exhibit greater no reduction ; conversely , angiotensin ii might be less elevated among whites . important studies are investigating gene variation among the races that may influence the risk of developing hypertension and its complications , such as single nucleotide polymorphisms in the genes encoding for 1-adrenergic receptors and a2c - adrenergic receptors . african americans who are homozygous for polymorphisms in both the 1-adrenergic and a2c - adrenergic receptor genes are at a 10-fold greater risk for heart failure , which is overexpressed in african - americans . groups with overexpression of transforming growth factor -1 ( tgf-1 ) , a cytokine involved in cardiac fibrosis and remodeling , vascular and renal disease , production of the vasoconstrictor , endothelin-1 , and stimulation of renin release , have a worse prognosis . other genetic polymorphisms in african americans that may influence heart failure risk include elevated production of endothelial nitric oxide synthase , aldosterone synthase , and the g protein 825-t allele . there are two major approaches to classifying the patient 's hf stage or clinical severity . the most common is the new york heart association ( nyha ) classification , mainly based on functional capacity and subjective level of symptoms severity . this scheme is fluid and its class fluctuates in the same patient depending on their subjective assessment and clinical status . the other approach , adopted by the american college of cardiology / american heart association ( acc / aha ) guidelines , emphasizes disease progression . classified as stages a , b , c , and d , each of these stages has definable characteristics and recommended interventions . to emphasize the progressive nature of hf and the need to prevent its development or progression to end - stage disease ( stage d ) [ 18 , 23 ] , current hf guidelines define patients in stage a as those at increased risk for hf without evidence of structural heart disease or symptoms of hf . stage a represents a critically important group because many of the risk factors including hypertension can be effectively controlled and by doing so may prevent development of cardiovascular diseases including hf . patients are in stage b when they present with evidence of structural heart disease but without current or prior symptoms of hf . stage c is recognized by the presence of structural heart disease and current or prior symptoms of hf . stage d patients are those who have refractory heart failure requiring frequent hospitalization and specialized interventions . several studies have shown the efficacy of treatment of hypertension in preventing hf [ 2427 ] . treatment of hypertension reduces the incidence of hf by about 50% . in recent years , significant advances have been made in increasing awareness , treatment , and control of blood pressure . several large , randomized clinical trials have shown that specific classes of medication have mortality benefit in patients with heart failure [ 2834 ] . in the most recent guidelines for the diagnosis and management of hf in adults , the american college of cardiology foundation ( accf)/american heart association ( aha ) recognized the need for specific recommendations for special population . lifestyle modifications have been shown to reduce blood pressure , increase drug efficacy , and decrease cardiovascular risk . long - term primary prevention of hypertension includes weight reduction in overweight / obese individuals . weight loss intervention was associated with a 21% reduction in the incidence of hypertension over a 36-month period . dietary practices , reducing intake of dietary sodium to < 1.5 g nacl [ 3 , 36 ] , low potassium , magnesium , and saturated fat intake , increased calcium intake , decreased consumption of alcohol , have demonstrated greater bp reduction in blacks . these practices are most successful when reinforced with comprehensive education and counseling [ 39 , 40 ] . exercise training , regular aerobic physical activity for at least 30 minutes per day most days , has been shown to reduce recurrent cardiac events in patients with lv dysfunction from ischemic heart disease and is recommended under close medical supervision . culturally cognizant and sensitive modalities should be implemented to help guide improved compliance of therapeutic lifestyle changes ( tlc ) in special populations [ 43 , 44 ] . despite lifestyle modifications , most patients with hypertension need pharmacologic therapy for better bp control . in the united states , pharmacological treatment of hypertension before development of structural heart disease or symptoms of hf is mainly based on studies looking at prevention of end - organ damage . specific classes of medication are recommended for patients in stages b - d that have shown benefit in reducing mortality and number of hospitalizations . the reader should refer to the major guidelines for full treatment recommendations of hf in the general population [ 18 , 23 , 46 ] . . however , ethnic minorities who are at greater risk for developing hf remain underrepresented in most clinical trials . according to a recent paper by mitchell et al . treatment of hf in african americans a call to action , most of the available evidence is based on limited representation of african americans in these trials or posthoc analysis of limited evidence . these papers once again summarize the available evidence in hf management in african americans and call for more therapeutic trial focused in these high - risk population . the african american heart failure trial ( a - heft ) was the first heart failure large trial to focus specifically on the african - american population and evidence represented approximately 50% of all african americans ever studied in heart failure trials . this is randomized placebo - controlled double blind study of fixed dose isosorbide dinitrate / hydralazine ( isbd / hyd ) ( figure 2 ) . in an effort to summarize efficacy of available treatments , psaty et al the database included 192,478 patients from 42 trials randomized to 7 treatment strategies including placebo . low - dose diuretics proved the most effective first - line treatment for preventing cardiovascular disease and mortality including hf . the allhat trial showed that diuretics are as effective as calcium channel blockers or acei in preventing cardiovascular disease outcomes including hf . the allhat study also has suggested that thiazide - diuretic therapy is useful in preventing disease progression . although , allhat trials and other trials showed diuretics is the first - line of antihypertensive therapy and is still the most commonly prescribed antihypertensive medications , this is challenged by recent studies as published in february jacc and presented in the european society of htn meetings . however , the diuretics used in the allhat trials was chlorthalidone a long acting thiazide ( shown to effectively lower bp and improve survival ) , not really the same as hydrochlorothiazide ( hctz ) which is the most commonly prescribed antihypertensive thiazide at a dose of 12.525 mg . ( st luke roosevelt hospital , new york , ny ) , published the analysis of 18 trials on hctz where the antihypertensive effect of hctz at the dose that is most often used12.5 to 25 mg a day is consistently inferior to that of most other antihypertensive drug . the other limitation of diuretics as first line therapy in african americans is not only not adequate but also , has multiple side effects like dm , electrolyte abnormalities and urecemia , to mention a few . however , there is still a role of diuretics in combination with neurohormonal ( renin angiotensin aldosterone axis ) blockade and when there is compelling indication . according to the recent consensus statement of the international society on hypertension in blacks ( ishb ) , chlorthalidone furthermore , the ishb consensus statement reinforces , patient above goal bp ( sbp / dbp15/10 ) with ( goal bp < 130/80 ) end organ damage ( hf , lvh , kidney disease ) or without end organ damage ( goal bp < 135/85 ) should be on at least on combination pharmacotherapy . use of beta blockers ( bb ) as treatment for hypertension has come under scrutiny . conducted a meta - analysis of 13 trials , which included 105,951 patients showing that atenolol did not improve outcomes in terms of stroke and cardiovascular events . in stage b chf ( nyha class i ) , defined by the presence of reduced left ventricular ejection fraction ( lvef ) < 40 percent in otherwise asymptomatic individuals , aceis and bbs are recommended . iii ) manifest left ventricular dysfunction and overt symptoms ; in these individuals , aceis and bbs are also indicated . bbs are also recommended in hf because of clinical studies demonstrating decreased morbidity and mortality , and improvement in hf symptoms . aldosterone antagonists may provide additional benefit in patients with severe left ventricular dysfunction , usually late stage c ( nyha class iii low - dose spironolactone ( 12.525 mg daily ) , when added to standard therapy , decreased mortality by 34 percent . in the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) , eplerenone reduced mortality by 15% in patients following a recent mi with lvef < 40% , 90% of whom had hf symptoms . patients were excluded who had a serum creatinine of < 2.5 mg / dl and hyperkalemia . the v - heft i and ii are a large - scale trial that compared different vasodilators with placebo and among themselves . v - heft i showed , the mortality rate was significantly lower for the total cohort ( african americans and whites ) in isdn / hyd arm compared to patients treated with prazosin or placebo added to digitalis and diuretics ( figure 1 ) . in v - heft ii , though isbd / hyd was statistically inferior to enalapril in effect on mortality , patients taking isbd / hyd experienced similar mortality rate in patients taking isbd / hyd in v - heft - ii as those in v - heft - i taking isbd / hyd . posthoc analysis of v - heft i showed isbd / hyd significantly reduced mortality in african american patients but not in white patients . the reanalysis of v - heft ii , demonstrated the annual mortality rate african american ( 12.9 ) patients receiving isbd / hyd was 12.9% compared with 12.8% for african americans receiving enalapril . due to difference in outcomes in african americans in v - heft , the african american heart failure trial ( a - heft ) definitive study was designed . the a - heft was terminated earlier than anticipated completion date due to a significant ( 43% ) reduction in mortality in patients receiving combination ( isdn / hyd ) compared to the placebo arm . adding the combination of a fixed - dose of isdn / hyd ( 20 mg/37.5 mg ) to a standard medical regimen for hf , including acei and bbs , is recommended in order to improve outcomes for patients self - described as african americans , with nyha functional class iii or iv chf . furthermore , genomic evidence from the grahf ( genetic risk assessment of heart failure in african americans ) arm of the african american heart failure trial ( a - heft ) showed that blacks with the more common tt phenotype ( 61% ) of the aldosterone synthase gene ( cyp11b2 ) showed greater responsiveness to the combination of isosorbide dinitrate and hydralazine . the vast majority of blacks will have no identified cause for their htn . past studies elucidating the pathophysiological mechanisms have shown conflicting information and further investigations exploring the complex relationships and interactions are warranted . there are , however , several secondary forms of htn in blacks that are prominent and preventable . we will focus on the most common , preventable and treatable secondary form of hypertension , obstructive sleep apnea ( osa ) . the prevalence of osa in hypertensive populations is estimated to range between ( 3040% ) and in heart failure at 53% ; apnea - hypopnea index ( ahi ) 10/h ) . it is an independent risk factor for htn , nondipping of blood pressure and increased bp variability and follows a dose - dependent relationship . a prospective study showed that the odds of developing hypertension over 48 years was 2.89 times more likely for participants who had ahi 15/h compared to ahi 0 , independent of known confounders . cross - sectional data has shown 2.38-times increased likelihood of having heart failure in association with osa , independent of confounders ( n = 6424 ) [ 67 , 68 ] . osa has been shown to act through various mechanisms , independent of blood pressure , in promoting left - ventricular ( lv ) hypertrophy , diastolic , and systolic dysfunction , and overt heart failure . these mechanisms include the hemodynamic consequences of repetitive increases in left - ventricular transmutable wall pressure and over time impaired vigil heart rate modulation leading to lv hypertrophy and ventricular remodeling . additionally , osa patients without overt heart failure have more impaired diastolic relaxation than those without osa . emerging evidence illustrates patients with osa have more active atherosclerotic disease with a greater degree of vessel involvement and more vulnerable plaques than do patients without the disease , resulting in greater cardiovascular burden . the definite treatment for osa , long - term adherence to continuous positive airway pressure ( cpap ) therapy , has shown to suppress sympathetic activity , lower blood pressure , reduce right ventricular volume , and improve systolic function in patients with hf . systematic reviews , meta - analyses , and randomized controlled trials have shown statistically significant net reduction in blood pressure with cpap , especially in patients with increased ahi events [ 7883 ] . a recent 3-year study ( n = 340 ) by durn - cantolla et al . reported that participants assigned to cpap therapy for 3 months had mean 24-hour ambulatory blood pressure decreased by 2.1 mmhg ( 0.4 to 3.7 ) mmhg ( p = .01 ) for systolic and 1.3 ( 0.2 to 2.3 ) mm hg ( p = .02 ) for diastolic blood pressures . similar evidence from randomized trials showed up to 9% increase in lvef and increased quality of life ( qol ) and functional capacity among patients treated with 13 months of cpap therapy . furthermore , among patients with no overt failure , cpap therapy has been shown to reverse impaired diastolic relaxation . there is a need for further randomized clinical trials with cpap therapy among high - risk ethnic minorities especially blacks to assess whether treatment of osa has similar long - term effects and to evaluate the effect on htn and hf morbidity and mortality . taking into consideration , lifestyle and pharmaceutical interventions , our changing health care community needs to emphasize the essential nature of the patient - provider relationship and establish trust and collaboration . in the past , this has been accomplished through physician , patients , family member , and other caretaker awareness , increased education of both provider and patients , and establishment of community outreach programs . these measures have contributed to an increase in compliance and a reduction in the risk of hospitalizations [ 23 , 87 ] . we recommend an additional emphasis on the implementation of specialty care in patients with heart failure in collaboration with the primary care provider . the dramatic improvement in the management of hf and hypertension over the last 50 years has allowed us to start to target specific populations and provide more evidence - based treatment that will lead to an improvement in mortality . there are disparities in the prevalence , treatment , and control of hypertension and the incidence and morbidity and mortality of heart failure between blacks , a diverse and heterogeneous population , and whites . the management team needs to take into consideration new evidence and develop tailored strategies for effective treatment , especially for hypertension , one of the main causes of hf and many other cardiovascular complications in african africans .

hypertension ( htn ) is the most common co - morbidity in the world , and its sequelae , heart failure ( hf ) is one of most common causes of mortality and morbidity in the world . current understanding of pathophysiology and management of htn in hf is mainly based on studies , which have mainly included whites . among racial groups , african - american adults have the highest rates ( 44% ) of hypertension in the world and are more resistant to treatment . there is an emerging consensus on the significance of racial disparities in the pathophysiology and treatment options of hypertension and heart failure . however , african americans had been underrepresented in all the trials until the initiation of the a - heft trial . since the recognition of obstructive sleep apnea ( osa ) as an important medical condition , large clinical trials have shown benefits of osa treatment among patients with htn and hf . this paper focuses on the pathophysiology , causes of secondary hypertension , and treatment of hypertension among african - american patients with heart failure . there is increasing need for randomized clinical trials testing innovative treatment options for african - american patients .

strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 . cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen . frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 . ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain . cell extracts were cleared at 30,000 g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c . resins were collected at 900 g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone . bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c . eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) . strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 . cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen . frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 . ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain . cell extracts were cleared at 30,000 g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c . resins were collected at 900 g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone . bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c . eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .

substrates of the proteasome are recognized and unfolded by the regulatory particle ( rp ) , then translocated into the core particle ( cp ) to be degraded1 . a hetero - hexameric atpase ring , containing subunits rpt1-rpt6 , is situated within the base subassembly of the rp1 . the atpase ring sits atop the cp , with the rpt c - termini inserted into pockets in the cp26 . we have identified a novel function of the rpt proteins in proteasome biogenesis through deleting the c - terminal residue from each rpt . our results indicate that assembly of the hexameric atpase ring is templated on the cp . we have also identified an apparent intermediate in base assembly , bp1 , which contains rpn1 , three rpts , and hsm3 , a chaperone for base assembly . the rpt proteins with the strongest assembly phenotypes , rpt4 and rpt6 , were absent from bp1 . we propose that rpt4 and rpt6 form a nucleating complex to initiate base assembly , and that this complex is subsequently joined by bp1 to complete the rpt ring . our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process .

this was a retrospective study of 18 patients with inflammatory myofibroblastic tumors , diagnosed in the department of general pathology during the period of january 2011december 2015 . 10200 ) . the clinical details and follow - up information of each patient were noted from the clinical work station . all classical tumors fulfilled the morphologic criteria described in the who classification of tumours of soft tissue and bone . atypical cases were defined by increased cellularity , cellular atypia with large ganglion - like round to polygonal cells , multinucleated or anaplastic giant cells , atypical mitosis and necrosis . the archival tissues obtained from institutional and consultation files were fixed in 10% buffered neutral formalin and paraffin - embedded tissue blocks were prepared . ihc slides and h&e slides prepared from formalin fixed , paraffin embedded tissue blocks were reviewed . ihcs had been done on ventana benchmark xt ( ventana medical systems , tucson , az , usa ) . the stain was considered to be positive if the tumor cells showed specific cytoplasmic and/or nuclear membrane staining for the particular antibody . the immunohistochemical stains were evaluated semiquantitatively as follows : 0 , negative ; 1 + , < 10% of cells positive ; 2 + , 10%50% of cells positive ; and 3 + , > 50% of cells positive . intra - abdominal tumors with epithelioid morphology , nuclear or perinuclear accentuation of alk immunopositivity and aggressive clinical behavior were categorized as epithelioid inflammatory myofibroblastic sarcoma . ihc slides and h&e slides prepared from formalin fixed , paraffin embedded tissue blocks were reviewed . ihcs had been done on ventana benchmark xt ( ventana medical systems , tucson , az , usa ) . the stain was considered to be positive if the tumor cells showed specific cytoplasmic and/or nuclear membrane staining for the particular antibody . the immunohistochemical stains were evaluated semiquantitatively as follows : 0 , negative ; 1 + , < 10% of cells positive ; 2 + , 10%50% of cells positive ; and 3 + , > 50% of cells positive . intra - abdominal tumors with epithelioid morphology , nuclear or perinuclear accentuation of alk immunopositivity and aggressive clinical behavior were categorized as epithelioid inflammatory myofibroblastic sarcoma . a total of 18 cases of inflammatory myofibroblastic tumors were found during the study period . of the 18 cases , 17 were institutional cases and one was a consultation case ( case 2 ) from outside hospital . there were nine females and nine males with female to male ratio of 1:1 and the mean age was 23.8 years ( range , 3 to 44 years ) . the most common locations were lung ( six cases ) and intra - abdominal region ( six cases ) followed by suprarenal , mastoid antrum , dorsum of nose , mediastinum , sphenoid wing , and breast ( one case each ) . all the patients underwent surgical excision and a few patients had combination treatments : surgery plus chemotherapy or chemoradiotherapy . limited follow - up information was available , ranging from 3 to 65 months ( mean , 16.1 months ) . the tumor size ranged from 1 to 15 cm ( mean , 5.6 cm ) . 1 ) exhibiting soft to firm variegated appearance with grey white to tan fleshy and focal mucoid areas . one case showed variable hemorrhage and necrosis on the cut surface ( case 1 ) . three cases ( cases 13 , 15 , and 18 ) had multifocal appearance of tumor ( multicentric in origin ) in various anatomic locations ( table 2 ) . histologically , among the 18 inflammatory myofibroblastic tumors ( including recurrent and metastatic cases ) , 14 cases had classic histologic features ( fig . 2 ) and four cases had atypical histologic features , revealing diffuse and focal cytoplasmic ihc expression of alk-1 ( fig . 3 ) . classification of histological patterns was done for all the cases according to the dominant tumor area , which included cellular ( 77.8% ) , myxoid ( 11.1% ) , and fibrous ( 11.1% ) types . all the classical inflammatory myofibroblastic tumors displayed a bland spindle cell proliferation admixed with an inflammatory infiltrate composed of lymphocytes , plasma cells , and eosinophils . the atypical histologic features included increased cellularity , cellular atypia with large ganglion - like round to polygonal cells , multinucleated or anaplastic giant cells , atypical mitosis , and necrosis . among the atypical cases , one case ( case 1 ) showed dominant epithelioid morphology with focally ganglion like cells , hypercellularity , atypia , mitosis , and prominent nucleoli ( fig . 4 ) ; one case ( case 13 ) showed dominant spindle cell morphology and focal epithelioid morphology ( fig . 5 ) ; remaining two cases ( cases 2 and 6 ) showed atypical spindle cell morphology ( fig . 6 ) . among the atypical cases , three cases had recurrence ( cases 1 , 6 , and 13 ) and two cases had metastasis to the lung , liver ( case 1 ) , and pelvic bone ( case 15 ) . spindled tumor cells of inflammatory myofibroblastic tumors showed cytoplasmic or membrane positivity for smooth muscle actin ( 12/16 cases ) , muscle - specific actin ( 3/6 cases ) , vimentin ( 8/8 cases ) , desmin ( 5/11 cases ) , and the spindled tumor cells were negative for cytokeratin , myogenin , cd34 , cd21 , cd117 , h - caldesmon , tle-1 , cd30 , and cd15 ( table 3 ) . immunohistochemical stain for alk-1 expression was available in all the 18 cases , demonstrating cytoplasmic reactivity of spindled or polygonal tumor cells for alk-1 in 10 cases ( 55.6% ) and no reactivity in eight cases ( 44.4% ) ( tables 3 , 4 ) . diffuse ( 3 + ) cytoplasmic staining was observed in six cases and focal ( 2 + ) cytoplasmic staining in three cases . an intra - abdominal inflammatory myofibroblastic tumor with dominant epithelioid morphology ( case 1 ) revealed perinuclear accentuation of alk-1 immunoexpression , suggestive of epithelioid inflammatory myofibroblastic sarcoma ( fig . 4 ) ; the remaining atypical cases with spindle cell morphology ( including case 13 with focal epithelioid morphology ) were negative for alk-1 , suggestive of atypical inflammatory myofibroblastic tumors only ( figs . 5 , 6 ) . alk-1 positive tumors ( 55.6% ) included five females and five males and the mean age was 23.8 years ( range , 3 to 44 years ) , compared with the mean age of ( four females and four males ) 27.1 years ( range , 18 to 41 years ) for alk-1 negative cases . recurrence was noted in 3/10 alk-1 positive ( 30% ) and 3/8 alk-1 negative ( 37.5% ) cases , whereas metastasis to the lung , liver and pelvic bone was noted in the alk-1 positive group ( tables 4 , 5 ) . alk-1 immunopositivity in neither of the classical ( p = .275 ) , atypical ( p = .275 ) nor multifocal tumor ( p > .99 ) types was statistically significant at 5% level of significance with fisher exact test . there was no statistically significant correlation between alk-1 expression and either of the recurrence ( p > .99 ) or metastasis ( p = .485 ) of inflammatory myofibroblastic tumors . the tumor size ranged from 1 to 15 cm ( mean , 5.6 cm ) . 1 ) exhibiting soft to firm variegated appearance with grey white to tan fleshy and focal mucoid areas . one case showed variable hemorrhage and necrosis on the cut surface ( case 1 ) . three cases ( cases 13 , 15 , and 18 ) had multifocal appearance of tumor ( multicentric in origin ) in various anatomic locations ( table 2 ) . histologically , among the 18 inflammatory myofibroblastic tumors ( including recurrent and metastatic cases ) , 14 cases had classic histologic features ( fig . 2 ) and four cases had atypical histologic features , revealing diffuse and focal cytoplasmic ihc expression of alk-1 ( fig . 3 ) . classification of histological patterns was done for all the cases according to the dominant tumor area , which included cellular ( 77.8% ) , myxoid ( 11.1% ) , and fibrous ( 11.1% ) types . all the classical inflammatory myofibroblastic tumors displayed a bland spindle cell proliferation admixed with an inflammatory infiltrate composed of lymphocytes , plasma cells , and eosinophils . the atypical histologic features included increased cellularity , cellular atypia with large ganglion - like round to polygonal cells , multinucleated or anaplastic giant cells , atypical mitosis , and necrosis . among the atypical cases , one case ( case 1 ) showed dominant epithelioid morphology with focally ganglion like cells , hypercellularity , atypia , mitosis , and prominent nucleoli ( fig . 4 ) ; one case ( case 13 ) showed dominant spindle cell morphology and focal epithelioid morphology ( fig . 5 ) ; remaining two cases ( cases 2 and 6 ) showed atypical spindle cell morphology ( fig . 6 ) . among the atypical cases , three cases had recurrence ( cases 1 , 6 , and 13 ) and two cases had metastasis to the lung , liver ( case 1 ) , and pelvic bone ( case 15 ) . spindled tumor cells of inflammatory myofibroblastic tumors showed cytoplasmic or membrane positivity for smooth muscle actin ( 12/16 cases ) , muscle - specific actin ( 3/6 cases ) , vimentin ( 8/8 cases ) , desmin ( 5/11 cases ) , and epithelial membrane antigen ( 2/4 cases ) . the spindled tumor cells were negative for cytokeratin , myogenin , cd34 , cd21 , cd117 , h - caldesmon , tle-1 , cd30 , and cd15 ( table 3 ) . immunohistochemical stain for alk-1 expression was available in all the 18 cases , demonstrating cytoplasmic reactivity of spindled or polygonal tumor cells for alk-1 in 10 cases ( 55.6% ) and no reactivity in eight cases ( 44.4% ) ( tables 3 , 4 ) . diffuse ( 3 + ) cytoplasmic staining was observed in six cases and focal ( 2 + ) cytoplasmic staining in three cases . an intra - abdominal inflammatory myofibroblastic tumor with dominant epithelioid morphology ( case 1 ) revealed perinuclear accentuation of alk-1 immunoexpression , suggestive of epithelioid inflammatory myofibroblastic sarcoma ( fig . 4 ) ; the remaining atypical cases with spindle cell morphology ( including case 13 with focal epithelioid morphology ) were negative for alk-1 , suggestive of atypical inflammatory myofibroblastic tumors only ( figs . 5 , 6 ) . alk-1 positive tumors ( 55.6% ) included five females and five males and the mean age was 23.8 years ( range , 3 to 44 years ) , compared with the mean age of ( four females and four males ) 27.1 years ( range , 18 to 41 years ) for alk-1 negative cases . recurrence was noted in 3/10 alk-1 positive ( 30% ) and 3/8 alk-1 negative ( 37.5% ) cases , whereas metastasis to the lung , liver and pelvic bone was noted in the alk-1 positive group ( tables 4 , 5 ) . alk-1 immunopositivity in neither of the classical ( p = .275 ) , atypical ( p = .275 ) nor multifocal tumor ( p > .99 ) types was statistically significant at 5% level of significance with fisher exact test . there was no statistically significant correlation between alk-1 expression and either of the recurrence ( p > .99 ) or metastasis ( p = .485 ) of inflammatory myofibroblastic tumors . inflammatory myofibroblastic tumors formerly described as inflammatory pseudotumor , with an unpredictable behavior ranging from benign to low - grade malignant , are classified as intermediate grade tumors , with potential for recurrence and rare metastasis , in the current who classification of tumours of soft tissue and bone . these tumors are described in children and young adults in various anatomic sites with a tendency towards local recurrence in about 25% of abdominopelvic lesions and a few cases of inflammatory myofibroblastic tumors may appear morphologically benign but the biological nature may be aggressive even when the histologic appearance is bland . these tumors were considered as a neoplasm after the discovery of clonal rearrangement of the alk gene on the short arm of chromosome 2 at 2p23 in 50%75% of extrapulmonary inflammatory myofibroblastic tumors . in a study by wang et al . , the most common site of inflammatory myofibroblastic tumors was abdominopelvic region ( 73.9% ) followed by other locations . the present study included 33.3% each of intrapulmonary and intra - abdominal locations followed by other locations . there was slight male predominance in the literature ; however , there was an equal incidence in each gender in the present study . hussong et al . in 1999 stated that the combination of cellular atypia , presence of ganglionlike cells , p53 expression and dna aneuploidy are all possible predictors to identify the malignant change and aggressive behavior of inflammatory myofibroblastic tumors . in a study by coffin et al . the atypical features in his study were increased cellularity , cellular and nuclear pleomorphism , distinct fascicular and focal herringbone pattern , cellular atypia with large ganglion - like round to polygonal cells , multinucleated or anaplastic giant cells , atypical mitosis , and necrosis . alk immunopositivity was noted in 78% atypical , 54% recurrent and 60% classical inflammatory myofibroblastic tumors . metastatic inflammatory myofibroblastic tumors ( 10.1% ) were negative for alk expression . in another study , there were 60% classical and 40% atypical inflammatory myofibroblastic tumors . twenty - five percent of alk - positive and 63.6% of alk - negative inflammatory myofibroblastic tumors revealed recurrence . statistically significant correlation was found between atypia , recurrence and metastasis . there was a significant correlation between alk expression and recurrence . overall alk-1 immunopositivity was noted in 55.6% and negativity in 44.4% of inflammatory myofibroblastic tumors . there was no statistically significant correlation between alk-1 expression , atypia , recurrence and metastasis in the present study compared to the other studies , probably due to a small sample size and limited follow up data ; a larger study may be taken up in the future to evaluate the statistical significance . marino - enriquez et al . proposed a term epithelioid inflammatory myofibroblastic sarcoma for epithelioid variant of intraabdominal inflammatory myofibroblastic tumors with a nuclear membrane or perinuclear pattern of alk expression and aggressive clinical behavior . in the present study , one patient with an intra abdominal mass , which was categorized as inflammatory myofibroblastic sarcoma , with epithelioid morphology and perinuclear accentuation of alk-1 expression , developed liver and lung metastasis and expired within 8 months . alk gene rearrangement was demonstrated in all the tested cases of epithelioid variant of inflammatory myofibroblastic tumors by fluorescence in situ hybridization ( fish ) . multifocality of pulmonary and extrapulmonary pseudotumors had a high chance of recurrence compared with tumors confined to a single organ . intraabdominal pseudotumors were associated with > 75% of the recurrences when compared to other extrapulmonary pseudotumors . in the present study study by chaudhary pointed out that alk gene rearrangements were often seen in inflammatory myofibroblastic tumors of children and young adults compared to adults over 40 years of age . the search for alk gene rearrangement by fish is a useful tool for the diagnosis of inflammatory myofibroblastic tumors . reported a case of inflammatory pseudotumor of the head and neck mimicking a metastatic disease in a human immunodeficiency virus positive 49-year - old female . in immunocompromised patients , alk protein immunoexpression was detected in various other tumors including lipomatous tumors , rhabdomyosarcomas , ewing s sarcoma / primitive neuroectodermal tumors , malignant fibrous histiocytomas , and leiomyosarcomas . except inflammatory myofibroblastic tumors , almost all other tumors displayed low level of alk expression . the differential diagnosis of these tumors includes both benign and malignant spindle or epithelioid cell tumors . the spindle cell tumors include fibroblastic / myofibroblastic tumors , fibrous histiocytoma , and solitary fibrous tumor . the tumors with epithelioid or round cell morphology include anaplastic large cell lymphoma , epithelioid leiomyosarcoma , rhabdomyosarcoma , undifferentiated sarcoma , myxoid / round cell liposarcoma , myxofibrosarcoma , dedifferentiated liposarcoma , poorly differentiated carcinoma , malignant melanoma , and epithelioid gastrointestinal stromal tumor . anaplastic large cell lymphoma can be differentiated from epithelioid variant of inflammatory myofibroblastic sarcoma by immunoreactivity for cd30 , cd3 , and cd43 , diffuse membranous or cytoplasmic staining of alk-1 and absence of ranbp2-alk rearrangement . high - grade leiomyosarcoma usually reveals fascicles of spindle and epithelioid cells with increased cellularity , cellular atypia , pleomorphism , cigar - shaped nuclei and abundant brightly eosinophilic cytoplasm . alveolar rhabdomyosarcoma shows uniform round cells with scant cytoplasm and usually lacks myxoid background and inflammatory infiltrates . dedifferentiated liposarcoma can have features of inflammatory myofibroblastic tumor , but with expression of mdm2 and absence of alk-1 . epithelioid malignant peripheral nerve sheath tumor will be strongly and diffusely positive for s100 and negative for alk-1 and also smarcb1 . malignant melanoma shows polygonal cells with prominent nucleoli and the cells are positive for s100 and human melanoma black 45 . epithelioid gastrointestinal stromal tumors reveal immunoexpression of cd117 , discovered on gist-1 ( dog1 ) and negative for alk-1 [ 3 - 5 ] . nsaids , steroids , chemotherapy , and radiotherapy have been used as adjuvant therapy when feasible . in a study by jacob et al . , systemic ( liver and lumbar spine ) involvement of inflammatory myofibroblastic tumor in a 45-year - old female , expressing alk gene rearrangement , responded very well to alk inhibitor ( crizotinib ) . in a series of cases by butrynski et al . , multifocal intra - abdominal inflammatory myofibroblastic tumors which expressed alk gene rearrangement , were treated successfully by alk inhibitor ( crizotinib ) . fragoso et al . in 2011 stated that , although surgical resection is the preferred treatment for inflammatory myofibroblastic tumors , cases which are not amenable to surgical excision may be treated conservatively . in conclusion , this study demonstrates that inflammatory myofibroblastic tumors exhibit a wide age group distribution with an equal incidence in males and females . most of the tumors with histological atypia were associated with recurrence , irrespective of alk-1 expression . epithelioid inflammatory myofibroblastic sarcoma with perinuclear accentuation of alk-1 showed aggressive clinical course with metastasis and mortality . metastasis was seen in alk-1 positive tumors . however , there was no statistically significant correlation between alk-1 expression , tumor type , recurrence , and metastasis . overall , alk-1 ihc is a useful diagnostic aid in the appropriate histomorphologic context , especially in identifying epithelioid inflammatory myofibroblastic sarcomas . there were no definite histopathologic criteria to predict malignant transformation , recurrence , or metastasis . however , this study is limited by the very small number of cases , limited follow - up data and also the lack of identification of alk gene rearrangement by fish . a search for newer molecular tools by larger studies needs to be performed to identify prognostic features to guide treatment and predict the outcome .

backgroundinflammatory myofibroblastic tumor is a histopathologically distinctive neoplasm of children and young adults . according to world health organization ( who ) classification , inflammatory myofibroblastic tumor is an intermediate - grade tumor , with potential for recurrence and rare metastasis . there are no definite histopathologic , molecular , or cytogenetic features to predict malignant transformation , recurrence , or metastasis.methodsa 5-year retrospective study of histopathologically diagnosed inflammatory myofibroblastic tumors of various anatomic sites was conducted to correlate anaplastic lymphoma kinase-1 ( alk-1 ) expression with histological atypia , multicentric origin of tumor , recurrence , and metastasis . clinical details of all the cases were noted from the clinical work station . immunohistochemical stains for alk-1 and other antibodies were performed . statistical analysis was done using fisher exact test.resultsa total of 18 cases of inflammatory myofibroblastic tumors were found during the study period , of which 14 were classical . the female - male ratio was 1:1 and the mean age was 23.8 years . histologically atypical ( four cases ) and multifocal tumors ( three cases , multicentric in origin ) were noted . recurrence was noted in 30% of alk-1 positive and 37.5% of alk-1 negative cases , whereas metastasis to the lung , liver , and pelvic bone was noted in the alk-1 positive group only.conclusionsoverall , alk-1 protein was expressed in 55.6% of inflammatory myofibroblastic tumors . there was no statistically significant correlation between alk-1 expression , tumor type , recurrence and metastasis . however , alk-1 immunohistochemistry is a useful diagnostic aid in the appropriate clinical and histomorphologic context .

improving the effectiveness and efficiency of the informed consent process is a critical health policy issue . informed consent is an essential element of ethical biomedical research , with federal regulatory requirements that are intended to ensure autonomy of the research participant s decision to engage in research after a balanced discussion . however , a growing body of evidence suggests that the informed consent process does not fully satisfy the needs of clinical research participants , with a recent survey finding that nearly 19% of prospective participants considered the informed consent document difficult to understand and that 15% were not satisfied that their questions had been answered during the consent process . it has been more than 20 years since the last substantive revision of federal regulations for human subjects protection , including informed consent . in 2011 , the us department of health and human services issued an advance notice of proposed rulemaking with proposed reforms . although this led some to expect that revision of human subjects protection regulations was imminent , major reform has yet to occur . recognizing deficiencies in the current informed consent process , the clinical trials transformation initiative ( ctti ) launched the informed consent project . initial project activities revealed that although an extensive body of literature on informed consent exists , there is little published information examining the observations and recommendations of experts with long - standing experience with the informed consent process . accordingly , a ctti work group ( the expert interviews team ) conducted interviews and analyzed information from 25 highly experienced observers of the informed consent process who represented multiple sectors of the us clinical trial enterprise . the expert interviews team nominated interview candidates considered as having extensive knowledge of and experience with informed consent in the united states and representing a diverse set of stakeholders . interviews were conducted by the center for information and study on clinical research participation ( ciscrp ) between april and june 2014 . the 1-h telephone interviews were conducted individually with each participant and followed a pre - specified interview guide designed to gather opinions on the current informed consent process , barriers to modification , and recommendations for actionable change . transcribed interviews were analyzed for themes with the goal of identifying consensus opinions and recommendations . the project , including the interview guide , was approved by the duke university institutional review board ( irb ) , and each participant gave verbal informed consent prior to the interview ( see supplementary appendix for detailed methods and a description of the interviewees ) . although all interviewees agreed that informed consent is essential to the protection of research participants , most also agreed that the informed consent process in the united states has evolved into a rigid and cumbersome process that requires serious attention and reform . from the interviewee recommendations , three major themes emerged with broad consensus : reform of the informed consent document , enhancing participant understanding of the clinical research , and modification of irb review . the most frequent recommendation for a single actionable change was reform of the informed consent document that is required for federally regulated research specifically , making it simpler , shorter , and more understandable . interviewees observed that an unintended consequence of excessively long and complex informed consent documents is that research participants may not read or comprehend the information and simply sign the document . half of the interviewees believed that the informed consent document was a deterrent to participation in clinical trials . some of the proposals could be implemented within the current regulatory framework , such as using modified formatting and graphics , ensuring that documents are written at the appropriate grade and health literacy level , and explaining technical terms in clear language . however , interviewees called attention to confusion around the extent of written information that must be provided to potential research participants under federal regulations . these regulations require that informed consent documents include eight mandatory elements ( basic elements of informed consent ) and six additional elements , if applicable ( additional elements of informed consent ) . interviewees specifically raised concerns about excessively lengthy and detailed information related to the required disclosures of the procedures to be followed in the study , any benefits to the subject or to others which may reasonably be expected from the research , and any reasonably foreseeable risks or discomforts to the subjects . interviewees found the required description of any reasonably foreseeable risks to be particularly problematic . in recent draft guidance , the us food and drug administration ( fda ) recommended , all possible risks do not need to be described in detail in the informed consent form , especially if it could be overwhelming for subjects to read . information on risks that are more likely to occur and those that are serious should be included . despite this regulatory guidance , the acceptable format and depth of how any reasonably foreseeable risks are presented to research participants may require future policy debate and potential modification of the federal regulations . the importance of reforming the document to better inform research participants and increase participation in clinical research has been identified in other national policy forums , including those conducted by the institute of medicine . in the advance notice of proposed rulemaking cited above , the federal government also identified the improvement in informed consent documents , including addressing their excessive length and legalistic language , as one of eight major objectives . the second most frequent recommendation from the interviews centered on creating a more interactive and ongoing process to enhance research participant understanding . the interviewees proposed multiple interventions that would not require national policy change and could be adapted to meet the needs of the specific study population ( figure 1 ) . examples included improving research staff training on the conduct of an informed consent discussion , conducting the process with adequate time and in a setting conducive to participant privacy and comfort , and providing an opportunity for prospective research participants to speak with other participants as a resource for information . the interviewees also emphasized the need for integration of the patient s voice by including patients in the design of the informed consent process , including the informed consent document . enhancing research participant understanding of a clinical trial . there was consensus regarding a lack of evidence - based information about participant decision - making in clinical research . the interviewees also identified a lack of evidence - based tools and metrics to gauge participant comprehension specific to the informed consent process for clinical research . among many scholars in this area , brehaut et al . conducted research that supports the notion that current informed consent documents and processes do not meet validated standards for promoting a decision - making process that allows people to make explicit choices among clearly described options . several interviewees proposed that research participants should be tested to demonstrate a certain level of comprehension about the clinical investigation , but there was no consensus on this issue . notably , although federal regulations generally require that a written consent document that embodies the required elements be signed by the participant or legally authorized representative , the regulations only require that the investigator shall give either the subject or the representative adequate opportunity to read it before it is signed . thus , current federal regulations do not require evidence of comprehension , and reform in this area is likely to be controversial . a third consensus issue from the interviews was the need for modification of practices in irb review of clinical research . for example , several interviewees proposed that processes be implemented to help ensure that irbs do not require the addition of language in informed consent documents that is intended to protect institutions from liability rather than enhance research participants understanding . the most substantive recommendation was the transition to use a single irb of record for multi - site clinical trials . a related ctti project has recommended the use of a single irb of record to improve the quality and efficiency of multi - center clinical trials and is working toward addressing barriers to adoption . the national institutes of health recently developed a model of a single irb of record for multi - site translational clinical research . in the advance notice of proposed rulemaking , the department of health and human services observed that the current multiple irb review system may actually be leading to weaker protections for subjects and proposed streamlining of irb review of multi - site studies . however , a federal statutory barrier exists for the use of a single irb of record in fda - regulated multi - site medical device clinical trials , unless a local irb does not exist or its review is determined to be inadequate . the most frequent recommendation for a single actionable change was reform of the informed consent document that is required for federally regulated research specifically , making it simpler , shorter , and more understandable . interviewees observed that an unintended consequence of excessively long and complex informed consent documents is that research participants may not read or comprehend the information and simply sign the document . half of the interviewees believed that the informed consent document was a deterrent to participation in clinical trials . some of the proposals could be implemented within the current regulatory framework , such as using modified formatting and graphics , ensuring that documents are written at the appropriate grade and health literacy level , and explaining technical terms in clear language . however , interviewees called attention to confusion around the extent of written information that must be provided to potential research participants under federal regulations . these regulations require that informed consent documents include eight mandatory elements ( basic elements of informed consent ) and six additional elements , if applicable ( additional elements of informed consent ) . interviewees specifically raised concerns about excessively lengthy and detailed information related to the required disclosures of the procedures to be followed in the study , any benefits to the subject or to others which may reasonably be expected from the research , and any reasonably foreseeable risks or discomforts to the subjects . interviewees found the required description of any reasonably foreseeable risks to be particularly problematic . in recent draft guidance , the us food and drug administration ( fda ) recommended , all possible risks do not need to be described in detail in the informed consent form , especially if it could be overwhelming for subjects to read . information on risks that are more likely to occur and those that are serious should be included . despite this regulatory guidance , the acceptable format and depth of how any reasonably foreseeable risks are presented to research participants may require future policy debate and potential modification of the federal regulations . the importance of reforming the document to better inform research participants and increase participation in clinical research has been identified in other national policy forums , including those conducted by the institute of medicine . in the advance notice of proposed rulemaking cited above , the federal government also identified the improvement in informed consent documents , including addressing their excessive length and legalistic language , as one of eight major objectives . the second most frequent recommendation from the interviews centered on creating a more interactive and ongoing process to enhance research participant understanding . the interviewees proposed multiple interventions that would not require national policy change and could be adapted to meet the needs of the specific study population ( figure 1 ) . examples included improving research staff training on the conduct of an informed consent discussion , conducting the process with adequate time and in a setting conducive to participant privacy and comfort , and providing an opportunity for prospective research participants to speak with other participants as a resource for information . the interviewees also emphasized the need for integration of the patient s voice by including patients in the design of the informed consent process , including the informed consent document . enhancing research participant understanding of a clinical trial . there was consensus regarding a lack of evidence - based information about participant decision - making in clinical research . the interviewees also identified a lack of evidence - based tools and metrics to gauge participant comprehension specific to the informed consent process for clinical research . among many scholars in this area , brehaut et al . conducted research that supports the notion that current informed consent documents and processes do not meet validated standards for promoting a decision - making process that allows people to make explicit choices among clearly described options . several interviewees proposed that research participants should be tested to demonstrate a certain level of comprehension about the clinical investigation , but there was no consensus on this issue . notably , although federal regulations generally require that a written consent document that embodies the required elements be signed by the participant or legally authorized representative , the regulations only require that the investigator shall give either the subject or the representative adequate opportunity to read it before it is signed . thus , current federal regulations do not require evidence of comprehension , and reform in this area is likely to be controversial . a third consensus issue from the interviews was the need for modification of practices in irb review of clinical research . for example , several interviewees proposed that processes be implemented to help ensure that irbs do not require the addition of language in informed consent documents that is intended to protect institutions from liability rather than enhance research participants understanding . the most substantive recommendation was the transition to use a single irb of record for multi - site clinical trials . a related ctti project has recommended the use of a single irb of record to improve the quality and efficiency of multi - center clinical trials and is working toward addressing barriers to adoption . the national institutes of health recently developed a model of a single irb of record for multi - site translational clinical research . in the advance notice of proposed rulemaking , the department of health and human services observed that the current multiple irb review system may actually be leading to weaker protections for subjects and proposed streamlining of irb review of multi - site studies . however , a federal statutory barrier exists for the use of a single irb of record in fda - regulated multi - site medical device clinical trials , unless a local irb does not exist or its review is determined to be inadequate . while we recognize that modifying the informed consent process is a complex policy issue , our findings from interviews of a diverse group of experts not only solidify the recognition of major problems that require reform but also provide a framework for meaningful change at both the local and national levels . several of the recommendations proposed by interviewees with broad consensus are feasible for rapid implementation within the current regulatory framework .

backgroundinformed consent is the cornerstone for protection of human subjects in clinical trials . however , a growing body of evidence suggests that reform of the informed consent process in the united states is needed.methodsthe clinical trials transformation initiative conducted interviews with 25 experienced observers of the informed consent process to identify limitations and actionable recommendations for change.resultsthere was broad consensus that current practices often fail to meet the ethical obligation to inform potential research participants during the informed consent process . the most frequent single recommendation , which would affect all participants in federally regulated clinical research , was reform of the informed consent document . the interviews also identified the need for reform of clinical research review by institutional review boards , including transitioning to a single institutional review board for multi - site trials.conclusionthe consensus recommendations from the interviewees provide a framework for meaningful change in the informed consent process . although some proposed changes are feasible for rapid implementation , others such as substantive reform of the informed consent document may require change in federal regulations .

one of the main objectives of orthodontic treatment and/or orthognathic surgery is to improve facial harmony and beauty . although the perception of these characteristics varies according to cultures , socioeconomic status , and era , clinicians have traditionally measured the distance , angulation , and proportion of hard and soft tissues to evaluate the facial type and profile.12345678 in terms of facial profile , the preferred chin projection has been well investigated because it has a strong impact on the harmony between the upper , middle , and lower face ; the shape and contour of the lower lip ; and the mentolabial sulcus.56910 although the chin can be placed in a normal position by orthognathic surgery , some patients prefer a slightly more retrusive or protrusive chin than a normal chin . the reasons for the difference in preferred chin projection among patients might be the following : first , the final goal of chin projection is different for clinicians and patients ; and second , the effect of chin projection on frontal facial appearance has not been properly assessed . the assessment of frontal facial type ( fft ) is important because patients usually evaluate their face by using a mirror before and after orthodontic treatment and/or orthognathic surgery . the most commonly used index , which can describe the fft , is the facial index.41112131415 it represents the ratio between the height of the middle and lower face ( soft - tissue nasion soft - tissue gnathion ) and the interzygomatic width ( soft - tissue zy on the right side soft - tissue zy on the left side ) ( figure 1 ) , and is classified as euryprosopic , mesoprosopic , or leptoprosopic.411121415 although numerous two - dimensional methods , including silhouettes , photographs , and digitally modified images , have been used to investigate the preferred chin projection,216171819 these methods have some limitations in terms of evaluating the esthetic chin position , such as unrealistic impression and distortion of the facial images.52021 recently three - dimensional ( 3d ) morphing programs have been introduced to predict soft - tissue changes related to orthodontic treatment and/or orthognathic surgery.2223 these 3d morphing techniques can modify the extent of chin projection without distorting images of facial areas , thereby resulting in a more realistic impression of diverse facial types and profiles . the perception of facial harmony and beauty can also vary according to the occupation and age of the evaluators , sex and age of the subjects , as well as other conditions.41617181920212223 therefore , to avoid bias , the experimental conditions should be standardized as follows : 1 ) the occupation of the evaluators should be the same ; 2 ) the age of the evaluators should preferably match that of the patients to reflect trends in society ( e.g. , twenties and early thirties ) ; 3 ) the same subjects should be evaluated by both male and female evaluators ; and 4 ) the facial images should be realistic despite the extent of modification of chin projection . although numerous previous studies have investigated the preferred chin projection in the profile view by using samples from various ethnicities and evaluators from different professions,222324252627282930 few studies have explored the relationship between an esthetically preferred chin projection and fft . therefore , the purpose of this study was to investigate the effects of fft and sex on esthetically preferred chin projection in 3d facial images , especially in the korean population . the null hypothesis was that there would be no significant difference in esthetically preferred chin projection in terms of fft and sex . the fft was determined using the facial index , which is the ratio between the height of the middle and lower face ( soft - tissue nasion soft - tissue gnathion ) and the interzygomatic width ( soft - tissue zy on the right side soft - tissue zy on the left side).14 the facial indices in each sex are enumerated in table 1 according to the martin - saller scale.15 six 3d facial images ( three of men and three of women with class i skeletal and dental relationships ; and with the following three ffts for each sex : euryprosopic [ eury - fft ] , mesoprosopic [ meso - fft ] , and leptoprosopic [ lepto - fft ] ) were acquired using a 3d facial optical scanner ( morpheus 3d scanner ; morpheus co. , ltd . , the actual height of the middle and lower face , interzygomatic width , and facial indices of the six 3d facial images are enumerated in table 1 . using the morpheus 3d aesthetic solution software ( ver . 2.0 ; morpheus co. , ltd . ) , the normal chin projection in each 3d facial image was set to 10 of the soft - tissue facial profile angle ( glabella subnasale - pogonion ) based on the findings of previous studies on korean adult facial profiles.678 the chin projections were then morphed by gradations of 2 from the normal projection ( 10 ) ( moderately protrusive [ 6 ] , slightly protrusive [ 8 ] , slightly retrusive [ 12 ] , and moderately retrusive [ 14 ] ) . for each chin position , the survey participants were shown images in the 45 angled and 90 lateral views ( figures 2 , 3 , 4 , 5 , 6 , 7 ) . the survey participants were 75 dental students from the school of dentistry , seoul national university , seoul , korea ( 48 men and 27 women ; mean age , 28.9 3.3 years ) , who were asked to rate the five chin projections ( 6 , 8 , 10 , 12 , and 14 ) from the most to least esthetically preferred in each 3d image . this study was reviewed and approved by the institutional review board of seoul national university school of dentistry , seoul , korea ( s - d20140028 ) . the kolmogorov - smirnov test , kruskal - wallis test , and bonferroni correction were performed for statistical analyses to determine the effect of fft and sex on the preferred chin projection by using ibm spss statistics for windows ver . the fft was determined using the facial index , which is the ratio between the height of the middle and lower face ( soft - tissue nasion soft - tissue gnathion ) and the interzygomatic width ( soft - tissue zy on the right side soft - tissue zy on the left side).14 the facial indices in each sex are enumerated in table 1 according to the martin - saller scale.15 six 3d facial images ( three of men and three of women with class i skeletal and dental relationships ; and with the following three ffts for each sex : euryprosopic [ eury - fft ] , mesoprosopic [ meso - fft ] , and leptoprosopic [ lepto - fft ] ) were acquired using a 3d facial optical scanner ( morpheus 3d scanner ; morpheus co. , ltd . , the actual height of the middle and lower face , interzygomatic width , and facial indices of the six 3d facial images are enumerated in table 1 . morpheus co. , ltd . ) , the normal chin projection in each 3d facial image was set to 10 of the soft - tissue facial profile angle ( glabella subnasale - pogonion ) based on the findings of previous studies on korean adult facial profiles.678 the chin projections were then morphed by gradations of 2 from the normal projection ( 10 ) ( moderately protrusive [ 6 ] , slightly protrusive [ 8 ] , slightly retrusive [ 12 ] , and moderately retrusive [ 14 ] ) . for each chin position , the survey participants were shown images in the 45 angled and 90 lateral views ( figures 2 , 3 , 4 , 5 , 6 , 7 ) . the survey participants were 75 dental students from the school of dentistry , seoul national university , seoul , korea ( 48 men and 27 women ; mean age , 28.9 3.3 years ) , who were asked to rate the five chin projections ( 6 , 8 , 10 , 12 , and 14 ) from the most to least esthetically preferred in each 3d image . this study was reviewed and approved by the institutional review board of seoul national university school of dentistry , seoul , korea ( s - d20140028 ) . the kolmogorov - smirnov test , kruskal - wallis test , and bonferroni correction were performed for statistical analyses to determine the effect of fft and sex on the preferred chin projection by using ibm spss statistics for windows ver . 21.0 ( ibm corp . , armonk , ny , usa ) . a value of p < 0.05 was considered statistically significant no significant difference was observed between the male and female evaluators in the distribution of the preferred chin projections in each fft ( table 2 ) . the normal chin projection angle ( 10 ) was the most preferred without any sex difference ( n = 25/75 in male 3d images and n = 27/75 in female 3d images ; table 3 ) . however , in eury - fft , the slightly protrusive chin projection angle ( 8 ) was favored in male 3d images ( n = 37/75 ) , but the normal chin projection angle ( 10 ) was preferred in female 3d images ( n = 25/75 ) ( table 3 ) . in lepto - fft , the normal chin projection angle ( 10 ) was favored in male 3d images ( n = 32/75 ) , whereas the slightly retrusive chin projection angle ( 12 ) was favored in female 3d images ( n = 38/75 ) ( table 3 ) . the means of the preferred cp angles in each fft were as follows ; eury - fft : male , 8.7 and female , 9.9 ; meso - fft : male , 9.8 and female , 10.7 ; and lepto - fft : male , 10.8 and female , 11.4 ( table 3 ) . a significant difference was observed in the preferred cp angle according to fft ( eury - fft male < [ meso - fft male , eury - fft female , meso - fft female , lepto - fft male ] < [ meso - fft female , lepto - fft male , lepto - fft female ] ; p < 0.001 ; table 3 ) . no significant difference was observed between the male and female evaluators in the distribution of the preferred chin projections in each fft ( table 2 ) . in meso - fft , the normal chin projection angle ( 10 ) was the most preferred without any sex difference ( n = 25/75 in male 3d images and n = 27/75 in female 3d images ; table 3 ) . however , in eury - fft , the slightly protrusive chin projection angle ( 8 ) was favored in male 3d images ( n = 37/75 ) , but the normal chin projection angle ( 10 ) was preferred in female 3d images ( n = 25/75 ) ( table 3 ) . in lepto - fft , the normal chin projection angle ( 10 ) was favored in male 3d images ( n = 32/75 ) , whereas the slightly retrusive chin projection angle ( 12 ) was favored in female 3d images ( n = 38/75 ) ( table 3 ) . the means of the preferred cp angles in each fft were as follows ; eury - fft : male , 8.7 and female , 9.9 ; meso - fft : male , 9.8 and female , 10.7 ; and lepto - fft : male , 10.8 and female , 11.4 ( table 3 ) . a significant difference was observed in the preferred cp angle according to fft ( eury - fft male < [ meso - fft male , eury - fft female , meso - fft female , lepto - fft male ] < [ meso - fft female , lepto - fft male , lepto - fft female ] ; p < 0.001 ; table 3 ) . several studies have investigated the preferred projection of the lip and chin in various ethnicities by using facial profiles.71819222331 coleman et al.7 reported that caucasian orthodontists and laypersons preferred a retrusive chin position in the normal profile . based on their studies on chinese participants , soh et al.18 indicated that laypersons , dental students , and dental professionals preferred normal or bimaxillary retrusive facial profiles . chong et al.19 also suggested that chinese individuals preferred more retrusive chin positions than did caucasian individuals . similarly , mantzikos31 reported that japanese participants preferred normal , bimaxillary retrusion , bimaxillary protrusion , mandibular retrusion , and mandibular protrusion in the decreasing order of preference . on the basis of these studies , it can be assumed that a normal or slightly retrusive chin projection is preferred in diverse ethnicities . however , few studies have assessed the relationship between ffts and preferred chin projection . in this study , we found a significant difference in the distribution of preferred chin projection according to ffts and sex ( eury - fft male < [ meso - fft male , eury - fft female , meso - fft female , lepto - fft male ] < [ meso - fft female , lepto - fft male , lepto - fft female ] ; p < 0.001 ; table 3 ) . the age of the survey participants is an important factor in determining the preferred chin projection . park et al.32 reported that young adults ( 2039 years old ; mean age , 26 years ) favored a straight facial profile , which was in accordance with the findings of this study . a normal chin projection ( 10 ) was preferred in most facial types except for a slightly protrusive chin projection in the eury - fft male ( n = 37/75 ; mean preferred angle , 8.7 ) and a slightly retrusive chin projection in the lepto - fft female ( n = 38/75 ; mean preferred angle , 11.4 ) ( table 3 ) . these results might be attributed to the similar age of the survey participants in this study ( mean age , 28.9 3.3 years ) and those in the study by park et al.32 however , they demonstrated that middle - aged ( 4054 years ; mean age , 47 years ) as well as older individuals ( 5570 years ; mean age , 62 years ) preferred a slightly retrusive chin as an esthetically pleasing one . in addition , shimomura et al.20 suggested that as one 's age passes the thirties , one tends to prefer a slightly retrusive chin to a protrusive one . the ratio between middle and lower facial height and interzygomatic width of the eury - fft was smaller than that of the meso - fft ( less than 0.85 vs. between 0.85 and 0.89 ; table 1 ) . if the chin is retruded in the eury - fft , the interzygomatic width might be viewed as being relatively wider than that in the meso - fft . therefore , a normal or slightly protrusive chin projection can be considered more esthetically acceptable than a retrusive chin projection . in the present study , a slightly protrusive chin projection ( 8 ) n = 37/75 ; table 3 ) and the second most acceptable for female eury - fft 3d images ( n = 23/75 ; table 3 ) . the ratio between middle and lower facial height and interzygomatic width of the lepto - fft was greater than that of the meso - fft ( greater than 0.87 vs. between 0.84 and 0.87 ; table 1 ) . when the chin is protruded in the lepto - fft , the height from the soft - tissue nasion and soft - tissue gnathion can appear relatively longer because of the increased height of the lower third of the face . if the chin is retruded in the lepto - fft , the height of the lower face can appear shorter than its actual height and , as such , the retrusive chin projection may be preferred . in the present study , a slightly retrusive chin ( 12 ) was the most preferred one for female lepto - fft 3d images ( n = 38/75 ; table 3 ) and the second most preferred for male lepto - fft 3d images ( n = 20/72 ; table 3 ) . for example , in the male eury - fft 3d images , a slightly protrusive chin projection ( 8 ) was the most preferred ( eury - fft male < [ meso - fft male , lepto - fft male ] ; p < 0.001 ; table 3 ) . this finding suggested that if orthognathic surgery or adjunctive esthetic surgery were planned for male patients with class iii relationships and a eury - fft , it would be better to establish a slightly protrusive chin projection , rather than a retrusive one , for ensuring the patient 's satisfaction with the esthetic facial change . in addition , this finding may be related to a mature and responsible impression that is conveyed by a normal or slightly protrusive chin projection in males . in the female lepto - fft 3d images , a slightly retrusive chin projection ( 12 ) was considered the most esthetically pleasing ( [ eury - fft female , meso - fft female ] < [ meso - fft female , lepto - fft female ] ; p < 0.001 ; table 3 ) . for female patients with class ii relationships and a lepto - fft , a slightly retrusive chin projection , rather than a protrusive one , might be an esthetically appropriate treatment goal . this finding may be related to a feminine facial impression that is conveyed by a slightly retrusive chin projection in females . a limitation of this study is that the survey was completed only by young adults who were dental students . in addition , the number of male survey participants was almost double that of female participants , and this might have influenced the results . therefore , future studies should include survey participants with various occupational backgrounds and from different age groups with a similar proportion of sexes . since the preferred chin projection varied according to ffts and sex , the null hypothesis was rejected . these findings might serve as guidelines for setting the preferred chin projection according to fft and sex .

objectiveto investigate the effects of frontal facial type ( fft ) and sex on preferred chin projection ( cp ) in three - dimensional ( 3d ) facial images.methodssix 3d facial images were acquired using a 3d facial scanner ( euryprosopic [ eury - fft ] , mesoprosopic [ meso - fft ] , and leptoprosopic [ lepto - fft ] for each sex ) . after normal cp in each 3d facial image was set to 10 of the facial profile angle ( glabella subnasale - pogonion ) , cps were morphed by gradations of 2 from normal ( moderately protrusive [ 6 ] , slightly protrusive [ 8 ] , slightly retrusive [ 12 ] , and moderately retrusive [ 14 ] ) . seventy - five dental students ( 48 men and 27 women ) were asked to rate the cps ( 6 , 8 , 10 , 12 , and 14 ) from the most to least preferred in each 3d image . statistical analyses included the kolmogorov - smirnov test , kruskal - wallis test , and bonferroni correction.resultsno significant difference was observed in the distribution of preferred cp in the same fft between male and female evaluators . in meso - fft , the normal cp was the most preferred without any sex difference . however , in eury - fft , the slightly protrusive cp was favored in male 3d images , but the normal cp was preferred in female 3d images . in lepto - fft , the normal cp was favored in male 3d images , whereas the slightly retrusive cp was favored in female 3d images . the mean preferred cp angle differed significantly according to fft ( eury - fft : male , 8.7 , female , 9.9 ; meso - fft : male , 9.8 , female , 10.7 ; lepto - fft : male , 10.8 , female , 11.4 ; p < 0.001).conclusionsour findings might serve as guidelines for setting the preferred cp according to fft and sex .

intrathecal baclofen ( itb ) is becoming a popular modality for management of severe spasticity [ 13 ] . spasticity is defined as hypertonia in which one or both of the following signs are present ( 1 ) resistance to externally imposed movement that increases with increasing speed of stretch and varies with the direction of joint movement , and/or ( 2 ) resistance to externally imposed movement rises rapidly above a threshold speed or joint angle . it is a sensorimotor phenomenon related to the integration of the nervous system motor responses to sensory inputs . although most commonly considered as a velocity - dependent increase to tonic stretch , it is related to hypersensitivity of the reflex arc and changes that occur within the central nervous system ( cns ) , most notably , the spinal cord . injury to cns results in loss of descending inhibition , allowing for the clinical manifestation of abnormal impulses . although spasticity is part of the upper motor neuron syndrome , it is frequently tied to the other presentations of the said syndrome . contracture , hypertonia , weakness , and movement disorders can all coexist as a result of the upper motor neuron syndrome . spasticity is a common phenomenon in patients with a wide variety of neurological disorders like cerebral palsy , multiple sclerosis , cardiovascular accidents , strokes , traumatic brain , and spinal cord injury [ 6 , 7 ] . these patients not only suffer from severe contractures and deformities but also severe pain that incapacitates them . several oral and parenteral medications have been used to treat spasticity ; their mechanisms of action are different and they influence various aspects of the neuromuscular pathophysiology . benzodiazepines , for example , diazepam exert a medullary and supraspinal inhibitory effect mediated by gabaa receptors [ 813 ] . benzodiazepines can lead to habituation ; withdrawal symptoms may occur when the medication is abruptly discontinued [ 813 ] . dantrolene acts directly on the skeletal muscle , without interference with the neurological circuitry ; it inhibits release of sequestered calcium from the sarcoplasmic reticulum . a third group of antispasticity drugs are 2 adrenergic agonists , for example , clonidine and tizanidine [ 11 , 12 ] . they hyperpolarize the motor neuron cell membrane , and they have an antinociceptive effect , which may indirectly reduce muscular hypertonia as well . side effects include drowsiness , dizziness , nausea , light headedness , vertigo , ataxia , headache , tolerance , and physical dependence . the most useful oral medication in the treatment of spasticity is baclofen , a gabab receptor agonist that causes presynaptic inhibition of mono- and polysynaptic reflex pathways ; it also works at the spinal cord level after crossing the blood - brain barrier . an increasing oral dose easily causes side effects including sedation , dizziness , weakness , and dizziness [ 14 , 15 ] . itb administration has been introduced , therefore , to maximize baclofen 's effects and minimize its side effects . it has been shown to be useful in the majority of patients to treat severe or intractable spasticity and dystonia leading to a decrease in muscle spasms , pain , and an increase in functional motor activity and control [ 1618 ] . we set out to identify and describe complications of itb pump and to report avoidance and management of complications . sharing our knowledge regarding patients with itb is important because as a result of the increasing use of this device , familiarity with the workup of possible implant malfunction is important . complications of itb vary from overdose , withdrawal , mechanical implant dysfunction : tear at metal connector to the pump ( within protective silicon covering),inappropriate trimming of catheter , tear at metal connector to the pump ( within protective silicon covering ) , inappropriate trimming of catheter , retrieval of old intrathecal catheter fragment through limited hemilaminotomy with durostomy leads to csf leak with tract formation disturb wound healing , dehiscence , and infection . complications can decrease therapeutic effect , thus need to be identified and treated promptly . the patients were followed by single operative and clinical team at the department of neurosurgery at our hospital : henry ford and oakwood health systems . after the approval of the hospital internal review board , all itb pumps inserted at our facility through the period of 20022006 were identified ( excluding previously performed surgery outside our own personal experience with our patients ) . patients who had itb pump insertion and reasons for insertion were identified and reviewed . we identified those patients who had revision surgery , reasons for revision , and diagnostic workup needed to identify complications as well as type of complications , type of treatment instituted to alleviate complication , and how to identify these complications in the future . since 2002 itb pump was implanted in 44 patients : 24 children ( 16 f/8 m ) versus 20 adults ( 7 f/13 m ) , 30 primary - implant - patients ( table 1 ) 16 children ( 10 f/6 m ) & 14 adults ( 5 f/9 m ) , 14 revision - only - patients ( table 2 ) 8 children ( 6 f/2 m ) versus 6 adults ( 2 f/4 m ) . clinical charts and operative and imaging reports were reviewed to evaluate reasons for revision surgery and diagnostic workup requirements . we analyzed reasons for revision surgeries and diagnostic workup requirements in order to identify these complications early and manage them accordingly . slowly increasing baclofen - resistant spasticity was encountered in seven patients ; four of them had unsuspected pump - catheter connector defects with either : subcutaneously dislocated intrathecal catheter n = 1 , ( figure 1 ) , x - ray - documented pump - catheter connector defect n = 1 , an eight - years - old boy with posttraumatic spastic quadriplegia and right - sided myoclonic intention tremor . he underwent 18 ml itb implant on 2002 with good spasticity control then on 2005 left thalamic dbs was done with good tremor control . in 2006 , he developed withdrawal symptoms and interventional radiology with pump injection showed tear at metal connector to the pump within protective silicon covering , so revision with trimming of catheter and exchange of the pump to 40 ml pump markedly reduced his baclofen needs ( figure 2 ) . x - ray demonstrated fractured catheter with intrathecal fragment n = 2 ; the first patient was a 12-year - old boy with spastic quadriplegic cp required laminectomy . he underwent multiple revisions in 2001 , 2003 , and 2004 . in 2003 , imaging showed adhesive arachnoiditis at the catheter tip . in 2005 , he developed mild withdrawal symptoms . plain x - ray showed fractured catheter with intrathecal fragment . following retrieval of old it catheter fragment through limited hemilaminotomy with durotomy , followed by placement of a new it catheter and replacement of old 10 ml pump with new 40 ml programmable pump injection studies revealed intrathecal pericatheter arachnoiditis : 53-year - old male with spastic quadriplegic cp with secondary scoliosis . plain x - ray showed perforating tear at the metal connector to pump with connector protrusion . revision was done with catheter trimming ; exchange of itb pump to 40 ml pump resulted in marked reduction of his baclofen needs . the first one was a three - year - old boy with cerebral palsy , postpump exchange secondary to manufacturer - related mechanical pump failure ; his pump was removed , covered with appropriate antibiotics , and then reinsertion of a new pump without further complications followed . the second case was a five - year - old boy with cerebral palsy , postabdominal wound dehiscence , and catheter exposure ; antibiotics failed to save the implant , so his pump was removed followed by reinsertion of a new pump without further complications . the third case was an eight - year - old girl with cerebral palsy , postabdominal wound dehiscence , and catheter exposure ; her pump was removed ; course of appropriate antibiotics was given followed by reinsertion of a new pump without further complications . the fourth case was a twenty - eight - year - old male with cerebral palsy with p. aeruginosa in catheter left in situ during previous pump removal ( > 1 yr ) at outside institution treated with appropriate antibiotic . his new implant was not infected occult csf leakage occurred in a 37-year - old male with itb implanted in 2003 with good spasticity control . in 2004 , he developed abdominal csf collection . pump injection showed leakage , catheter tear was discovered at pump connector site , and revision surgery was done . he remained to have recurrent csf collections at abdominal site with negative pump injection studies . however , no specific csf tract was found intraoperatively n = 1 , ( see figure 3 ) ; removal of the old intrathecal catheter with extensive catheter tract obliteration and placement of a new it catheter resulted in no further csf accumulation anymore . the difference in effectiveness between oral and intrathecal administration of baclofen is due to a significantly higher drug concentration that can be achieved in the csf through itb . in addition , there is a 4 to 1 gradient in drug distribution between the caudal and rostral parts of the spinal cord following itb , thus providing for a beneficial effect at the spinal level without undesired side effects in the brain . although itb is highly gratifying , it has predominant technique - related complications during implant life . meticulous surgical technique , high clinical suspicion , appropriate workup , and timely surgical management are emphasized to reduce surgical itb complications . thorough workup is crucial to rule out implant system dysfunction if clinical evaluation is atypical . obtaining preoperative imaging , at least a plain x - ray , has been proven to be a safe and effective method of evaluation , although in our centre we have a low threshold to perform pump injection studies with contrast . in our series , itb revision surgery for both groups primary - implant - patient and revision - only - patient population ( n = 44 ) . 33 additional revision procedures performed on 22 of 44 patients itb surgery carry significant morbidity that needs extended care and hospitalization requirements ( tables 1 and 2 ) . we recommend a standardised implantation technique that can be used as a guide for a safer itb implants and may help reduce the incidence of complications . during an informed consent , we elaborated on pros and cons of continuing conservative therapy versus implantation of the itb . we also acquainted patient of the goals and benefits , possible risks , and complications . preoperatively , patient 's general medical condition was assessed by excluding infection , integrity of the contemplated operative sites , and prophylactic antibiotic within one hour of the incision time . although the procedure can be performed using local anesthesia and sedation , general anaesthesia was preferred because many of the patients were children . incision should be long enough ( i.e. 1.5 to 2 ) to expose the wound sufficiently and to minimize potential interaction of the implanted catheter with the skin borders , which are the predominant source of surgical infection . then , the lumbar puncture is performed using the 14 g tuohy needle supplied by the manufacturer in the implant kit . once the needle tip is in adequate position and csf flow is achieved , the slightly curved tuohy needle tip needs to be rotated superiorly in order to guide the catheter superiorly . the first critical point is when the catheter reached the tip of the tuohy needle . it is preferable to check fluoroscopically the correct positioning of the catheter at this point : especially important is to ensure that the catheter travels into cranial direction and that it is not looping on itself . the presence of the guide wire and an additional small metallic bead at the catheter tip makes fluoroscopic tracking of the catheter usually relatively easy . once the initial correct positioning is verified , the catheter can be advanced more , which generally goes without difficulty . the final level of the catheter is determined by the relative spasticity of the upper versus the lower extremities and to what extent spasticity control is targeted for the upper versus the lower extremities . if the catheter can not be advanced easily , it is better to leave it at that level instead of forcing it more . if the surgeon decides that the catheter needs to be withdrawn because he noted a looping or the catheter is primarily going into caudal direction , the catheter should never be withdrawn by itself , but rather the tuohy needle should be withdrawn : otherwise , there is a significant risk to cause a cut in or transection of the catheter . once the catheter is in its final position , a purse string stitch is placed very close around the needle penetration site through the fascia . then the tuohy needle is withdrawn and subsequently the guide wire . following removal of the guide wire , the purse string stitch can be tied in such a way that the intrathecal catheter can not be moved easily anymore through the fascia penetration site . patency of the catheter should be obvious by the appearance and continuous dripping of csf from the catheter end . the catheter is now also anchored to the lumbar fascia with a manufacturer supplied anchor , which is attached to the catheter and the fascia with nonresorbable sutures . take care that this does not result in acute catheter angles that may lead to catheter obliteration . the catheter end is clamped gently with a rubber shod mosquito to avoid excessive csf loss . the abdominal portion of the procedure is usually done at the time of successful lumbar puncture in order to save overall procedural time . the abdominal incision is made and a pocket is created in the subcutaneous adipose layer . the pump pocket is designed such that the pump access ports will not be exactly underneath the incision line . it is useful to have the pump placed on the patient 's abdominal fascia because this facilitates secure attachment of the pump and tends to prevent pump hypermobility that can interfere with future pump access for either refill or diagnostic purposes . the pump cavity should be large enough to accommodate the pump . once the abdominal and the lumbar sites are prepared adequately and the intrathecal catheter is in situ and anchored , a custom - bent tunneler is passed subcutaneously from the abdominal site to the lumbar site . this requires the tunneler to be substantially bent in order to follow the patient 's flank around , carefully passing the tunneler through the subcutaneous layer usually without need for counter incisions with guided assistance , tunneling direction towards the lumbar incision . it is important to make sure that enough catheter remains within the lumbar wound to make a small strain relief loop at that site . on the abdominal side , the intrathecal catheter is trimmed as needed to avoid too much looping catheter length but leaving enough material in place to be able to have a loop . at this point , patency of the catheter is double - checked when the rubber shod mosquito is removed form the catheter end . the pump - catheter connector is attached to the catheter : this requires gentle manipulation of the catheter to advance it into the connector . the pump is then placed into the pump cavity created earlier , securing the pump to the fascia using the suture loops of the pump . the excess catheter is gently placed underneath the pump prior to securing the device in situ . we feel , using the above - mentioned protocol can reduce the complications , which have been described above . the knowledge of the peculiarities of itb treatment is important , as there is an increased risk for the patients if these implants are not functioning well . the physician who may be called upon to evaluate the itb pump patient needs to have good understanding of the diagnostic challenges when faced with a possible device malfunction . this also assumes with the familiarity with diagnostic and therapeutic preimplant workup with surgical implant technique and with the postimplant maintenance management . , patients will be able to sleep better , become more independent with mobility , and their ability to do self - care helps improve urinary function . a decrease in muscle pain and fatigue that accompany spasm thus effective baclofen therapy can be delivered using itb pump , where effects of baclofen are maximised , while its side effects are minimised .

increasingly , spasticity is managed with surgically implanted intrathecal baclofen pumps . intrathecal baclofen pump revision surgery unrelated to programmable pump end - of - life is not uncommon , requiring special attention during pre- , intra- , and postoperative management . we aimed to identify and describe complications of intrathecal baclofen pump as well as to report avoidance and management of complications . methods and materials . through 20022006 , at the department of neurosurgery , henry ford and oakwood health systems , intrathecal baclofen pumps were implanted in 44 patients : 24 children versus 20 adults ; 30 primary - implant - patients ; 14 revision - only patients . we evaluated reasons for revision surgeries and diagnostic workup requirements . results . eight primary - implant - patients required 14 revisions and 7 of revision - only patients needed 13 procedures . seven patients with slowly increasing baclofen - resistant spasticity had either ( i ) unsuspected pump - catheter connector defects , ( ii ) an x - ray - documented pump - catheter connector defect , ( iii ) x - ray - demonstrated fractured catheter with intrathecal fragment . implant infections occurred in 4 cases . scintigraphy revealed occult csf leakage n=1 and intrinsic pump failure n=1 . conclusion . intrathecal baclofen pumps , although very gratifying , have a high , technique - related complication incidence during implant life . meticulous technique , high clinical suspicion , appropriate workup , and timely surgical management can reduce surgical complications of intrathecal baclofen pump implantation .

human capital and health improvement programmes are of central importance towards sustainable development and economic growth in any country . in malaysia , the health care system has changed from traditional remedies to meeting the emerging needs of the population . since the independence of malaysia in 1957 , the first reorganization started at the public primary health care services and accelerated since the alma ata declaration in 1978 . in malaysia , the ministry of health ( moh ) is the main provider of health care services to the public . the organizational structure of the moh has three levels , federal , state and district , which are decentralized to ensure efficiency . each hierarchical level determines the level of authority , information flow , accountability and supervision . this system encompasses all aspects of care such as preventive , promotive , curative and rehabilitative . the main objective is to provide a greater network of physical facilities , equity , accessibility and utilization of health care resources . at the same time , national referral centres were established to provide specialized care to enhance the basic care provided in health clinics . over the past decade there has been an explosion of tertiary level specialized care to meet the needs of the population . this is expensive , fragmented and institutionally focused and inappropriate for the majority of health consumers . in the current era this service includes a lifetime health plan that focuses on keeping the child and family well . this gives greater prominence to preventive issues and takes on healthier lifestyles by choices with risk prevention . the health care providers also need not function as controllers but act as facilitators or partners with health consumers ( figure 1 ) . figure 1transformation from industrial age medicine to information age health care ( source : amar ) . transformation from industrial age medicine to information age health care ( source : amar ) . apart from the size of the hospitals , there are differences in terms of the services provided . small district hospitals provide general medical and nursing care and their manpower consist of medical officers and other personnel . larger district hospitals and regional hospitals provide a wide range of specialist services and the public has easy access through a walk - in or referral system . moh seeks to ensure the public is informed of health issues and has access to safe water , safe food and quality medicine . the malaysian health care system focuses on primary health care ( phc ) that places social equity as important and allocates public funds for the poorest 20% of the population . in 1956 , there were only 42 phc facilities in the country . after independence , the health sector became an integral part of the national and development process and moh has been able to deliver health care to communities throughout the country . table 1 shows increasing health care facilities in secondary and tertiary care over the years . table 1health facilities of the ministry of health , malaysia in 1984 , 2001 and 2008.moh's facilities198420012008health clinics361843802rural / community clinics103919241927mobile teams35204193hospitals89 ( 21,159 beds)115 ( 29,123 beds)130 ( 33,004 beds)medical institutions8 ( 10,235 beds)6 ( 5551 beds)6 ( 5000 beds)source : adapted from merican and bin yon;planning and development division , ministry of health , malaysia . source : adapted from merican and bin yon ; planning and development division , ministry of health , malaysia . the number of hospitals , community clinics and other facilities such as special medical institutions ( national heart institute , institute of pediatrics and institute of respiratory medicine ) has increased ( table 2 ) . the total expenditure from the health department of selangor in 2006 , for instance , has increased to rm 881.3 million compared to rm 628.83 million in 2005 and rm 577.77 million in 2004 . the increase is due to new hospitals and comprehensive health services that are provided by the government . the second national health and morbidity survey in 1996 reported that 88.5% of the population stays within 5 km of a health facility and 81% lived within 3 km . findings also show that basic health care and facilities are accessible to about 70% of the population in sabah and sarawak and more than 95% of the population in peninsular malaysia . these estimates do not include other types of outreach services such as flying doctors , mobile health teams , dental clinics , travelling dispensaries and riverine services . table 2health care facilities in malaysia 2009.governmentno.beds ( official)ministry of health hospitals13033,083 special medical institutions64974 special institutions*6- national institutes of health6- dental clinics17242952 mobile dental clinics and teams5601392 health clinics808 community clinics ( klinik desa)1920 maternal & child health clinics90 mobile health clinics196non ministry of health hospitals83523privateno.beds ( official)licensed hospitals20912,216 maternity homes21102 nursing homes12273 hospice328 ambulatory care centre21108 blood bank5#- haemodialysis centre75848 community mental health centre19registered medical clinics6307- dental clinics1484-*national blood centre , national public health laboratory and 4 regional laboratories;dental chairs;#refers to 4 cord blood stem cells banks and 1 stem cell and regenerative medicine research lab and services;refers to dialysis chairs . national blood centre , national public health laboratory and 4 regional laboratories ; refers to 4 cord blood stem cells banks and 1 stem cell and regenerative medicine research lab and services ; refers to dialysis chairs . there are other government agencies that complement the role of moh to preserve the health of the people . for instance , the ministry of human resources that enforces safety and health regulations of employees , ministry of education that is responsible for the operation of the teaching hospitals and training of health personnel of the country , ministry of defence that provides health services for its population within the territory , ministry of rural development that is responsible for the health of the aborigines and ministry of housing and local government that is responsible for some of the licensing and enforcement under its purview . studies have also shown that the malaysian health standard is almost at par with those of developed countries . data from the world health report in 1999 indicated that the health indicators of malaysians were much better compared to some of the asean countries . for example , the infant mortality rate ( imr ) in malaysia is 11 per 1000 live births while in indonesia it is 48 per 1000 live births and in thailand it is 29 per 1000 live births . this figure is still high compared to the imr of singapore ( 5/1000 live births ) , united kingdom ( 7/1000 live births ) and america ( 7/1000 live births ) . equity is an assessment of fairness . despite malaysia 's effort in socio - economic development plans , there still exist issues in equity and accessibility especially for the indigenous groups , rural population and the hard - core poor . this can be seen through quality in terms of health services , manpower and equity in terms of geographical location and accessibility in terms of price and tariff . the asian economic crisis in 1998 has increased 50% of the poverty level in several countries which added difficulty for the poor and middle class in accessing health care . nevertheless , efforts are taken by the government to strengthen the rural health services in malaysia through the improvement of existing facilities and introducing new health services that range from outpatient curative care to preventive and promotive services . the rural health units consist of one health centre , four rural health units and mobile clinics . the rural health unit follows a two - tier system that provides subsidized or free health services to 15,000 to 20,000 rural population . there are 500 people per doctor in kuala lumpur and 4000 per doctor in terengganu and east malaysia . , the ratio of doctors to patients in malaysia is 1:927 compared to 1:1105 in 2008 . one of the pending concerns of the government is that there are high concentrations of private practices in the urban areas due to the demand by the affluent community . in 1993 , there are 3055 general practitioners clinics and 190 private hospitals and nursing homes in malaysia . in 2000 , 46.2% of all doctors were in the private sector and were accountable for only 20.3% of hospital beds while the rest of the 53.8% of doctors were in the public sector looking after 79.7% of the beds . it is reported that 58.8% of the specialists were in the private sector and about 41.2% were in the public sector . the findings through interviews from key personnel from moh states that the charges from private hospitals on services component range from 15% to 28% of the hospital bills and medication whereby 15% of this bill is not made known to patients . furthermore , professional fees take up almost 50% of the total bill . the difference in the public and private sectors in terms of specific services provided may have a significant effect on the equity of services and the question of efficiency and effectiveness . this leads to an imbalance of the distribution of manpower in public and private sectors in malaysia . generally , the services provided by private hospitals are curative and selective in nature , either free or subsidized and much more comprehensive which is controlled by issues of equity . access to private health services is limited to the richer society that can afford out - of - pocket payments of higher fees . immigrant health is another concern in malaysia whereby 5% of the malaysian population , which consists of about one million people , are immigrant workers . these foreign workers may harbour communicable diseases which originate from their country and this incurs health care cost when they use the health facilities in malaysia . moreover , there are many cases whereby foreign workers who have been admitted have defaulted in settling their bills and collectively with a number of other reasons unsettled hospital bills in public sectors are increasing . to address these issues , more comprehensive preventive measures and plans must be taken by designing and implementing conducive national health care financing scheme under the national health financing authority ( nhfa ) within the realm of moh . professionals in health care and the health care systems have changed at a much slower pace and are not usually suitable for the present health needs of the population . throughout the world there seems to be fundamental changes in medical care delivery systems that is in progress . asia pacific region is the most varied health region in the world because it contains the country with the largest population in the world . this includes malaysia which has about 8.3% of the population above 30 years suffering from diabetes and 29.9% from hypertension . in the less - developed countries in the region , a large percentage of the population is moving through the economic transition and about 70% of the deaths are due to chronic diseases . the united nations development program ( undp ) has published projections for changes in populations over the next 50 years . for population over 60 years , malaysia will have an increase from 5.7% in 1996 to 11% by 2020 . the world health organization ( who ) and individual countries are taking control of the progress by phc . although the definition of phc varies from country to country , it can not be denied that accessibility , quality of basic health care and equity within countries have improved . nevertheless , the populations most in need are the aborigines , the poor , the disadvantaged and the disabled . these groups have the least access to health services according to the inverse care law which explains that health care tend to operate based on active market forces . however , meeting their needs will be very challenging , because every individual has a right to health care services and it is essentially the responsibility of the government to ensure this access . health care financing is a key concern all over the world today . among others , some of the sources of funding health care are through taxation , social and private health insurance and out - of - pocket payments . the malaysian government finances the public health services through the consolidated revenue fund under the ministry of finance while the sources from the private sector are essentially from the consumers . the system of financing is inclined towards the public sector whereby only a nominal fee of rm1 for each outpatient visit is charged in accordance to the fees ( medical ) order 1976 . government employees and their family members benefit from these services even after their retirement while the social security organization ( socso ) and employees provident fund ( epf ) do not finance employees in the private sector during their retirement . comparatively , the british government initiated the 1912 national health insurance policy to compensate salaries of workers who have lost their jobs due to sickness . commercialization of health care is not financially viable for the majority of the consumers and is inappropriate because any framework of health care provision must be in line with the needs of the consumers . as a result , it has undermined the trust of individuals to the health care profession and the government . health care financing is a main challenge in many countries and should be taken into consideration in providing a safety net for the poor . the united states spends 14% of its gnp compared to asian countries that spends about 48% of their gnp on health care . with new technologies , capitalization of expensive hospital facilities and specialization has increased the cost of medical services . in 2001 , returns collected by moh malaysia in providing medical , health and dental care services amounted to 2.2% of the total operating budget . the medical price index in malaysia has increased more than the consumer price index . in some parts of the countries , where the force of the financial crisis is bigger , structural adjustments to high costs of debt servicing and reduced rates of exchange have caused cuts to the public health budget . as a result , many of the countries anxiously look for cost - containment measures and different sources of financing including cost sharing . in doing so , no one should be denied access to health care due to financial reasons and malaysia should not adopt solutions from failed regions that have failed in health care delivery . despite the high - tech medical technology in the health care sector in the united states , 45 million residents are lacking health insurance , including 10 million children who are uninsured . one possible suggestion in managing long term health problems is by looking at the chronic care model ( ccm ) that leads to improved patient care and better health care systems , which is widely practiced for ambulatory care improvement in the united states and internationally . through privatization in malaysia , the weight of the cost of care was moved to a sizeable proportion of the population that could least afford it . comparatively , the provision of medical care through the national health service in britain is committed to horizontal equity which describes equal treatment for equal need while the australian experience in health care financing is described as the classical liberal manner in which the government operates . fortunately , countries such as malaysia and thailand provided a safety net for primary care and ensured minimal essential care for the high risk groups . equity is an assessment of fairness . despite malaysia 's effort in socio - economic development plans , there still exist issues in equity and accessibility especially for the indigenous groups , rural population and the hard - core poor . this can be seen through quality in terms of health services , manpower and equity in terms of geographical location and accessibility in terms of price and tariff . the asian economic crisis in 1998 has increased 50% of the poverty level in several countries which added difficulty for the poor and middle class in accessing health care . nevertheless , efforts are taken by the government to strengthen the rural health services in malaysia through the improvement of existing facilities and introducing new health services that range from outpatient curative care to preventive and promotive services . the rural health units consist of one health centre , four rural health units and mobile clinics . the rural health unit follows a two - tier system that provides subsidized or free health services to 15,000 to 20,000 rural population . there are 500 people per doctor in kuala lumpur and 4000 per doctor in terengganu and east malaysia . , the ratio of doctors to patients in malaysia is 1:927 compared to 1:1105 in 2008 . one of the pending concerns of the government is that there are high concentrations of private practices in the urban areas due to the demand by the affluent community . in 1993 , there are 3055 general practitioners clinics and 190 private hospitals and nursing homes in malaysia . in 2000 , 46.2% of all doctors were in the private sector and were accountable for only 20.3% of hospital beds while the rest of the 53.8% of doctors were in the public sector looking after 79.7% of the beds . it is reported that 58.8% of the specialists were in the private sector and about 41.2% were in the public sector . the findings through interviews from key personnel from moh states that the charges from private hospitals on services component range from 15% to 28% of the hospital bills and medication whereby 15% of this bill is not made known to patients . furthermore , professional fees take up almost 50% of the total bill . the difference in the public and private sectors in terms of specific services provided may have a significant effect on the equity of services and the question of efficiency and effectiveness . this leads to an imbalance of the distribution of manpower in public and private sectors in malaysia . generally , the services provided by private hospitals are curative and selective in nature , either free or subsidized and much more comprehensive which is controlled by issues of equity . access to private health services is limited to the richer society that can afford out - of - pocket payments of higher fees . immigrant health is another concern in malaysia whereby 5% of the malaysian population , which consists of about one million people , are immigrant workers . these foreign workers may harbour communicable diseases which originate from their country and this incurs health care cost when they use the health facilities in malaysia . moreover , there are many cases whereby foreign workers who have been admitted have defaulted in settling their bills and collectively with a number of other reasons unsettled hospital bills in public sectors are increasing . to address these issues , more comprehensive preventive measures and plans must be taken by designing and implementing conducive national health care financing scheme under the national health financing authority ( nhfa ) within the realm of moh . professionals in health care and the health care systems have changed at a much slower pace and are not usually suitable for the present health needs of the population . throughout the world there seems to be fundamental changes in medical care delivery systems that is in progress . asia pacific region is the most varied health region in the world because it contains the country with the largest population in the world . this includes malaysia which has about 8.3% of the population above 30 years suffering from diabetes and 29.9% from hypertension . in the less - developed countries in the region , a large percentage of the population is moving through the economic transition and about 70% of the deaths are due to chronic diseases . the united nations development program ( undp ) has published projections for changes in populations over the next 50 years . for population over 60 years , malaysia will have an increase from 5.7% in 1996 to 11% by 2020 . the world health organization ( who ) and individual countries are taking control of the progress by phc . although the definition of phc varies from country to country , it can not be denied that accessibility , quality of basic health care and equity within countries have improved . nevertheless , the populations most in need are the aborigines , the poor , the disadvantaged and the disabled . these groups have the least access to health services according to the inverse care law which explains that health care tend to operate based on active market forces . however , meeting their needs will be very challenging , because every individual has a right to health care services and it is essentially the responsibility of the government to ensure this access . health care financing is a key concern all over the world today . among others , some of the sources of funding health care are through taxation , social and private health insurance and out - of - pocket payments . the malaysian government finances the public health services through the consolidated revenue fund under the ministry of finance while the sources from the private sector are essentially from the consumers . the system of financing is inclined towards the public sector whereby only a nominal fee of rm1 for each outpatient visit is charged in accordance to the fees ( medical ) order 1976 . government employees and their family members benefit from these services even after their retirement while the social security organization ( socso ) and employees provident fund ( epf ) do not finance employees in the private sector during their retirement . comparatively , the british government initiated the 1912 national health insurance policy to compensate salaries of workers who have lost their jobs due to sickness . commercialization of health care is not financially viable for the majority of the consumers and is inappropriate because any framework of health care provision must be in line with the needs of the consumers . as a result , it has undermined the trust of individuals to the health care profession and the government . health care financing is a main challenge in many countries and should be taken into consideration in providing a safety net for the poor . the united states spends 14% of its gnp compared to asian countries that spends about 48% of their gnp on health care . with new technologies , capitalization of expensive hospital facilities and specialization has increased the cost of medical services . in 2001 , returns collected by moh malaysia in providing medical , health and dental care services amounted to 2.2% of the total operating budget . the medical price index in malaysia has increased more than the consumer price index . in some parts of the countries , where the force of the financial crisis is bigger , structural adjustments to high costs of debt servicing and reduced rates of exchange have caused cuts to the public health budget . as a result , many of the countries anxiously look for cost - containment measures and different sources of financing including cost sharing . in doing so , no one should be denied access to health care due to financial reasons and malaysia should not adopt solutions from failed regions that have failed in health care delivery . despite the high - tech medical technology in the health care sector in the united states , 45 million residents are lacking health insurance , including 10 million children who are uninsured . one possible suggestion in managing long term health problems is by looking at the chronic care model ( ccm ) that leads to improved patient care and better health care systems , which is widely practiced for ambulatory care improvement in the united states and internationally . through privatization in malaysia , the weight of the cost of care was moved to a sizeable proportion of the population that could least afford it . comparatively , the provision of medical care through the national health service in britain is committed to horizontal equity which describes equal treatment for equal need while the australian experience in health care financing is described as the classical liberal manner in which the government operates . fortunately , countries such as malaysia and thailand provided a safety net for primary care and ensured minimal essential care for the high risk groups . multidisciplinary interventions are required to promote health financing , health care and disease prevention . in malaysia , the partnership between the public and private sectors should be encouraged to maximize resources and minimize duplication of health delivery in order to provide equitable health care . subsequently , the community engagement in self care , planning , organizing and management will lead to self sufficiency in health . one effective way to improve the shortage and distribution imbalance especially in rural areas which is practiced in china is to rely and train the locals as paramedical workers . three interrelated strategies to help the poor access health care are to design appropriate training for village health workers ( preventive and promotive intervention ) , design appraisals on programs implemented and introduce community participation . the proposed nhfa would be a feasible option as a health care financing mechanism in malaysia with vested authority in providing equitable and quality services both in the public and private health care services . this has also been urged by the federation of malaysian consumers associations ( fomca ) which has pointed out the benefits that will be gained in implementing the national health financing scheme ( nhfs ) , one of which will be regulating fees charged by the private hospitals and providing the public the freedom of choice to seek treatment either at public or private hospitals in malaysia . another suggestion of intervention is to establish a national health insurance fund ( nhif ) to be the main funding source for the public health care sectors which allows compulsory contribution from employers and employees . the traditional support systems in some countries were also being commented to have been taken for granted and governments need to mobilize these social networks to take care of these problems . in this respect , the development of traditional medicine is encouraged in china where traditional medicine complements western medicine and this practice is allowed in hospitals in order to give the people the choice . in australia , the demand for alternative medicine is increasing steadily and findings have shown that the consumer expenditure have doubled from $ a1 billion in 1993 to $ a2.3 billion in 2000 . a survey conducted by the malaysian moh in 2004 has concluded that 70% of malaysians have used traditional and complementary based medicine to improve their health or to treat illnesses . the fields include malay traditional medicine , chinese traditional medicine , ayurvedic medicine and natural medicine . utilization of cross - cultural traditional medicine by the various ethnic groups in malaysia is also gaining popularity . this has raised significant issues in public health policy . even though the practice of alternative medicine is recognized in statutory form under section 34(1 ) of medical act 1971 ( act 50 ) , however the safety and efficacy of these medicine must be ensured through strict regulations and public education forums . since the goal of medicine is essentially helping people to improve their health , therefore it is important for medical health professionals to work together with social workers from traditional and complementary medicine by respecting each others ' beliefs and training and working as a team . currently guidelines and the passing of the traditional and complementary medicine bill for the various fields in traditional medicine are being studied carefully by various organizations in malaysia . the malaysian government has also encouraged private hospitals to take on more social responsibility of the country and the private sectors are responding well to this . over the last couple of years , there has been an increase in efforts to improve systems and attract foreign workforce . with the tenth malaysian plan in place , it is hoped that the mechanisms set by the government will improve the situation . multidisciplinary interventions are required to promote health financing , health care and disease prevention . in malaysia , the partnership between the public and private sectors should be encouraged to maximize resources and minimize duplication of health delivery in order to provide equitable health care . subsequently , the community engagement in self care , planning , organizing and management will lead to self sufficiency in health . one effective way to improve the shortage and distribution imbalance especially in rural areas which is practiced in china is to rely and train the locals as paramedical workers . three interrelated strategies to help the poor access health care are to design appropriate training for village health workers ( preventive and promotive intervention ) , design appraisals on programs implemented and introduce community participation . the proposed nhfa would be a feasible option as a health care financing mechanism in malaysia with vested authority in providing equitable and quality services both in the public and private health care services . this has also been urged by the federation of malaysian consumers associations ( fomca ) which has pointed out the benefits that will be gained in implementing the national health financing scheme ( nhfs ) , one of which will be regulating fees charged by the private hospitals and providing the public the freedom of choice to seek treatment either at public or private hospitals in malaysia . another suggestion of intervention is to establish a national health insurance fund ( nhif ) to be the main funding source for the public health care sectors which allows compulsory contribution from employers and employees . the traditional support systems in some countries were also being commented to have been taken for granted and governments need to mobilize these social networks to take care of these problems . in this respect , the development of traditional medicine is encouraged in china where traditional medicine complements western medicine and this practice is allowed in hospitals in order to give the people the choice . in australia , the demand for alternative medicine is increasing steadily and findings have shown that the consumer expenditure have doubled from $ a1 billion in 1993 to $ a2.3 billion in 2000 . a survey conducted by the malaysian moh in 2004 has concluded that 70% of malaysians have used traditional and complementary based medicine to improve their health or to treat illnesses . the fields include malay traditional medicine , chinese traditional medicine , ayurvedic medicine and natural medicine . utilization of cross - cultural traditional medicine by the various ethnic groups in malaysia is also gaining popularity . this has raised significant issues in public health policy . even though the practice of alternative medicine is recognized in statutory form under section 34(1 ) of medical act 1971 ( act 50 ) , however the safety and efficacy of these medicine must be ensured through strict regulations and public education forums . since the goal of medicine is essentially helping people to improve their health , therefore it is important for medical health professionals to work together with social workers from traditional and complementary medicine by respecting each others ' beliefs and training and working as a team . currently guidelines and the passing of the traditional and complementary medicine bill for the various fields in traditional medicine are being studied carefully by various organizations in malaysia . the malaysian government has also encouraged private hospitals to take on more social responsibility of the country and the private sectors are responding well to this . over the last couple of years , there has been an increase in efforts to improve systems and attract foreign workforce . with the tenth malaysian plan in place , it is hoped that the mechanisms set by the government will improve the situation .

since 1957 , there has been major reorganization of health care services in malaysia . this article assesses the changes and challenges in health care delivery in malaysia and how the management in health care processes has evolved over the years including equitable health care and health care financing . the health care service in malaysia is changing towards wellness service as opposed to illness service . the malaysian ministry of health ( moh ) , being the main provider of health services , may need to manage and mobilize better health care services by providing better health care financing mechanisms . it is recommended that partnership between public and private sectors with the extension of traditional medicine complementing western medicine in medical therapy continues in the delivery of health care .

black yeasts from the family herpotrichiellaceae ( order chaetothyriales ) are fungi with a remarkable dual ecology . on the one hand , they have a unique ability to adapt to extreme environments ( exposure to toxic chemicals , high temperature , scarcity of nutrients , acidic , and/or dry conditions ) , while on the other hand , they exhibit a significant human pathogenic potential . unlike common opportunistic fungi , herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease and only occasionally in immunocompromised patients . also , more often than any other fungal group , these organisms have been reported from environments that are rich in aromatic compounds . the first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote , while their occurrence on untreated wood was comparatively low , indicating that accumulation of aromatic compounds might promote the growth of these fungi . the concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis , isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [ 26 , 27 ] . it has been hypothesized that wild berries constitute a natural niche of e. dermatitidis , which might then be ingested by birds and humans and sporadically resulting in mycoses . deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus . the related species exophiala bergeri , exophiala heteromorpha , exophiala oligosperma , and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [ 57 , 11 , 27 ] . however , the connection between route of infection and natural occurrence is less evident with these species . the tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . besides hydrocarbon exposure , environmental conditions in biofilters are characterized by a relative low water activity and acidification of the filter bed , which might lead to biomass inhibition problems . in this respect , fungal colonization in air biofilters has generally been related to an improved bioreactor performance . detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [ 3 , 20 , 29 ] , which appears to be quite an uncommon metabolic feature in the eukaryotes . molecular phylogenetic characterization of these biofilter fungi has shown a predominant affiliation to the genera exophiala and cladophialophora ( fam . herpotrichiellaceae ) , particularly to the species exophiala lecanii - corni , e. oligosperma , and the recently described e. xenobiotica , cladophialophora saturnica , and cladophialophora inmunda [ 1 , 6 ] . despite the medical and environmental relevance of herpotrichiellaceous black yeasts , little is understood on their biodiversity and natural occurrence . in order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission , the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons . different environmental samples related to the life cycle of e. dermatitidis were used as inocula . the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons . environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14 days . the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . table 1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95 n , 51045.96 ea2oak railway tie , between railsnear station , hollandsche rading521041.95 n , 51045.96 ea3oak railway tie , outside railsforest area , hilversum521220.68 n , 5119.66 ea4oak railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ea5concrete railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ec1berry , sorbus aucupariaroadside , de bilt52 717.18 n , 5 948.13 ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19 n , 5 931.42 ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec4berry , sorbus aucuparialapersveld park , hilversum521254.74 n , 51110.25 ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52 n , 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed4fresh goose feceslapersveld park , hilversum521254.74 n , 51110.25 ed5old goose feceslapersveld park , hilversum521254.74 n , 51110.25 e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . serum flasks of 100 ml were filled with approximately 25 ml of perlite granules , saturated with mineral medium . each environmental sample ( 13 g ) was washed with phosphate - buffered saline , and the suspension ( 40 ml ) was used to inoculate four different batches ( 10 ml each batch ) . as a control , these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth . the inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene . this gas phase was generated by placing 10 ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate . a solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3 months . after this time , the perlite granules in each flask were washed with 5060 ml sterile water . one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters . a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions . the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) . pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50 l volumes containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp and 2.0 u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) . amplification was performed with cycles of 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) and 72c ( 120 s ) , with a final 7-min extension step at 72c . amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) . sequence pcr was performed as follows : 95c for 1 min , followed by 30 cycles consisting of 95c for 10 s , 50c for 5 s , and 60c for 2 min . reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) . sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) . phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) . the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons . environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14 days . the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . table 1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95 n , 51045.96 ea2oak railway tie , between railsnear station , hollandsche rading521041.95 n , 51045.96 ea3oak railway tie , outside railsforest area , hilversum521220.68 n , 5119.66 ea4oak railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ea5concrete railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ec1berry , sorbus aucupariaroadside , de bilt52 717.18 n , 5 948.13 ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19 n , 5 931.42 ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec4berry , sorbus aucuparialapersveld park , hilversum521254.74 n , 51110.25 ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52 n , 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed4fresh goose feceslapersveld park , hilversum521254.74 n , 51110.25 ed5old goose feceslapersveld park , hilversum521254.74 n , 51110.25 e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . serum flasks of 100 ml were filled with approximately 25 ml of perlite granules , saturated with mineral medium . each environmental sample ( 13 g ) was washed with phosphate - buffered saline , and the suspension ( 40 ml ) was used to inoculate four different batches ( 10 ml each batch ) . as a control , these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth . the inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene . this gas phase was generated by placing 10 ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate . a solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3 months . after this time , one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters . a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions . the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) . pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50 l volumes containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp and 2.0 u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) . amplification was performed with cycles of 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) and 72c ( 120 s ) , with a final 7-min extension step at 72c . amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) . sequence pcr was performed as follows : 95c for 1 min , followed by 30 cycles consisting of 95c for 10 s , 50c for 5 s , and 60c for 2 min . reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) . sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) . phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) . a total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons ( table 2 ) . of those , a single mold ( aspergillus fumigatus ) other than black yeasts was isolated from berries of sorbus aucuparia ( sample c1 ) . the highest isolation rates were achieved with samples taken from creosoted wooden railway ties , particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate ( 39 isolates , samples a1 and a3 ) . these samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates ( 17 and 10 strains , respectively ) , but not a single fungus was isolated when naphthalene was supplied as the sole carbon source . though in lower numbers , black yeasts were also isolated from the tie section located between the rails ( 14 strains , samples a2 and a4 ) . besides creosote , these samples were likely to contain fecal and mineral oil contamination as well . table 2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments , black yeasts were isolated in two out of four berries from s. aucuparia ( 12 isolates , samples c1 and c2 ) , but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna ( samples c5 and c6 , respectively ) . soil mixed with bird feces ( guano ) also yielded melanized fungi in one out of five tested samples ( six isolates , sample d2 ) . no black yeasts were isolated when suspensions of the samples described above ( table 1 ) were directly plated onto sga plates , without the enrichment step on volatile aromatic hydrocarbons , while several common and heavily sporulating fungal species were encountered . phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups ( fig . 1 ) . sequences from reference strains deposited at the cbs collection ( utrecht , the netherlands ) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis . those encompassed the type strains of e. xenobiotica , e. bergeri , exophiala spinifera , e. oligosperma , e. heteromorpha , e. dermatitidis , e. lecanii - corni , and phialophora sessilis , as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons : cladophialophora immunda , c. saturnica , and cladophialophora psammophila sp . nov . , the latter species being in the process of description ( badali et al . , submitted ) . strains of e. xenobiotica , e. bergeri , and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . figure 1neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate . sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches were also included in the analysis ( genbank sequence codes are given between square brackets ) neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate . sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches were also included in the analysis ( genbank sequence codes are given between square brackets ) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica ( 32 strains ) . the majority was highly homologous ( 99% ) to the ex - type strain , and some were slightly deviating ( 97% homology ) . a second group of isolates ( nine strains ) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain . the cluster encompassing e. bergeri also included a major group ( 24 strains ) , the sequence of which deviated significantly from the ex - type strain ( 94% sequence homology ) , so that its affiliation to e. bergeri might be put into question . blast searches on this latter group into the genbank genomic database revealed a close match ( 98% homology ) with an unidentified exophiala strain isolated from a rock surface . a minor e. bergeri sibling group was composed of two strains for which no known sequence match was found . a third group ( three strains ) did not match any known reference type strain , but genbank searches revealed a high sequence homology ( 98% ) to the sequence of an uncultivated fungus from a municipal composting plant . except for a single isolate ( dh18150 ) obtained from a s. aucuparia berry ( sample c1 ) , the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails ( samples a1 and a3 ) . within this group , three strains were obtained under benzene enrichment , while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates . besides e. xenobiotica , sample a3 also yielded five isolates upon benzene enrichment , which were identified as e. bergeri . the related between - the - rails section from that same tie ( sample a4 ) and the guano - rich soil ( sample d2 ) yielded 1 and 3 strains of e. bergeri , respectively , using toluene as the enrichment substrate . the occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . under common laboratory culture conditions , numerous protocols for the selective isolation of black yeasts have been developed during the last decades ( an overview is presented in table 3 ) , which have yielded new data on the presence of these fungi in a wide range of artificial environments , sometimes with no obvious counterpart in nature . several methods are based on osmotolerant and oligotrophic abilities of target fungi . with these regimens , the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales , although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales . applying enrichment methods based on the inoculation of rodents , exposure to aromatic hydrocarbons , assimilation of rare sugars , and/or incubation at high temperature , most isolated black yeast - like fungi are members of the chaetothyriales . it should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi , and they are therefore classified with difficulty in any of the known ecological categories . table 3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala , fonsecaea , cladophialophorachaetothyrialesopportunists[2 , 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala , fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora , exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium , hortaeadothidealeshalophiles[31 , 32]suspendcryomyces , friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family . for example , under hydrocarbon atmospheres , the berry samples were free from aureobasidium pullulans , which is a member of the dothideales , otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries . this species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment . the presence of heavily sporulating airborne fungi , frequently found in soil and litter , was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus . interestingly , the dothidiaceous species amorphoteca resinae , known as creosote fungus for its common isolation from creosoted wood , was found neither in this study nor in previous fungal isolation surveys from creosoted wood [ 26 , 27 ] . consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples , such as wild berries , only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . in particular , the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments . with advanced analytical techniques , it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature , though at very low concentrations . toluene , for example , is produced biologically in different natural environments including maturing berries . on the other hand , as extremophilic and slow - growing microorganisms , incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts , when compared to traditional liquid enrichment cultures . regarding both the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions , wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts . creosote is a distillate derived entirely from tar , which is rich in a wide variety of polycyclic aromatic hydrocarbons , phenols , and cresols , and has widely been used as wood preservative against microbial decay . due to its carcinogenic character , the use of creosote has been banned in the european union . yet , creosoted wood utilities are still widely present in the open environment , such as telephone poles , railway cross ties , switch ties , and bridge timbers . also , creosote is an important soil contaminant in former wood creosoting plants . in a previous study , e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c , this protocol being quite selective for e. dermatitidis . it was hypothesized that the life cycle of this species involved the occurrence on wild berries , ingestion and passage through the human intestinal tract , and feces deposition on railway ties . subsequent enrichment on creosoted wood might then be related not only to the oligotrophy , thermotolerance ( particularly under tropical conditions ) , acidotolerance , and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds . the temperate wild berry sampling locations that were the subject of our study ( table 1 ) had previously been analyzed for the occurrence of black yeasts [ 18 , 26 ] . collected berries were homogenized , diluted , and used for spread plates or were subjected to the raulin s pre - incubation protocol . in these earlier studies involving several hundreds of samples , only a single strain of e. dermatitidis the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics . this hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil , using the mineral oil - flotation protocol [ 15 , 24 ] . from the present enrichment program on volatile alkylbenzenes , it appears that exophiala species , though other than the thermophilic e. dermatitidis , are regularly present on substrates that are characteristic from the latter species ( berries , bird feces , and creosoted railway ties ) . it is known to be associated with mild cutaneous infections in humans and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [ 6 , 24 , 26 ] . , it was regarded to be an extremely rare opportunist , although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . using the selective oil - flotation method , e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives . a distinct cluster was identified within the e. bergeri group , which might belong to a yet - undescribed species . strains from this group were predominantly isolated from the section between the rails of sampled wooden ties , which were partly contaminated by machine oil and human feces . the remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails , where this contaminating effect was less marked . three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere . no close sequence homology against reference strains was found for these fungi , indicating that a yet - undescribed species is concerned . the phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study . the extremophilic nature of black yeast , in combination with their capacity to metabolize aromatic pollutants , makes them ideal candidates for specific bioremediation purposes . prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard , but , as phylogenetic data on these organisms accumulates , it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings ( de hoog , unpublished ) . indeed , enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories , such as cladophialophora bantiana or e. dermatitidis . this suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . yet , more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches . the application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons .

black yeast members of the herpotrichiellaceae present a complex ecological behavior : they are often isolated from rather extreme environments polluted with aromatic hydrocarbons , while they are also regularly involved in human opportunistic infections . a selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology . samples from natural ecological niches and man - made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons . benzene , toluene , and xylene were provided separately as the sole carbon and energy source via the gas phase . the assayed isolation protocol was highly specific toward mesophilic exophiala species ( 70 strains of this genus out of 71 isolates ) . those were obtained predominantly from creosote - treated railway ties ( 53 strains ) , but isolates were also found on wild berries ( 11 strains ) and in guano - rich soil samples ( six strains ) . most of the isolates were obtained on toluene ( 43 strains ) , but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi ( 17 and 10 isolates , respectively ) . based upon morphological characterizations and dna sequences of the full internal transcriber spacers ( its ) and the 8.5s rrna genes , the majority of the obtained isolates were affiliated to the recently described species exophiala xenobiotica ( 32 strains ) and exophiala bergeri ( nine strains ) . members of two other phylogenetic groups ( 24 and two strains , respectively ) somewhat related to e. bergeri were also found , and a last group ( three strains ) corresponded to an undescribed exophiala species .