Doctor insights on:
Medicine For Bergers Disease
Clinical Trials: Although available, experimental drugs are given in clinical trials, usually in academic or private institutions with doctors involved in research, where patients that have failed to existing treatment are treat. There are a variety of immunotherapy medications like Remicade (infliximab) that are available for Crohn's you could discuss with your doctor about. Ustekinumab is a clinical trial drug for Crohn's ...Read more
Some injections: Non-surgical treatments for peyronie's include: waiting (as some penises improve without treatment), or injections of medications such as verapamil or interferon (medications that can decrease the collagen fibrous material in the plaque). An experienced urologist can evaluate to be sure peyronie's is cause of the curvature, and then he may recommend non-surgical treatments as a first step. ...Read more
Wilson's disease: Check out http://www.Mayoclinic.Com/health/wilsons-disease/ds00411 for info re wilson's disease which is autosomal recessive cause of excess copper. Both Penicillamine & trientine are used to chelate & remove excess copper. There's no best drug for everyone but rather a best drug for you. Talk to your doc about what's available to you, what's affordable, and what side effects are tolerable to you. ...Read more
Testing: Unfortunately, without knowing what part of your testing was abnormal, it is not possible to suggest a treatment. Please give some more specifics, or go see a doctor to review your test results. ...Read more
Can you tell me an example of a time when medicine was shipped elsewhere to another country in order to stop an epidemic or disease?
Yes: The Canadian Health Minister indicated that Canada will donate between 800 to 1,000 of their 1,500 doses of an experimental Ebola Vaccine - VSV-EBOV to help with the Ebola-Zaire outbreak in West Africa. ...Read more
How does nuclear medicine cure diseases? Can anyone give an example of how nuclear medicine is used to cure diseases? I don't understand how it works.
The : The best example of how nuclear medicine is used to cure diseases is treatment of thyroid cancer or graves' disease (a form of hyperthyroidism). In both cases the goal is to destroy thyroid tissue. The thyroid gland contains a large amount of iodine which it uses to produce thyroid hormone. Once iodine is ingested, a large amount is concentrated within the thyroid gland. Nuclear medicine harnesses this principal to destroy thyroid tissue by giving the patient a radioactive form of iodine (i-131). This radioactive iodine is concentrated in the thyroid gland. The isotope primarily releases beta radiation which destroys the surrounding tissue (most of which is thyroid tissue). If there is thyroid cancer in the area, the cancer is also destroyed. ...Read moreSee 3 more doctor answers
Hello, I'm 25 years,helthier, no diseases,181cm high,56kg weight,male, I'm too thin,I want a possible drug to increase my weight,my apetite is little.
Drugs to gain weight: are dangerous if no medical condition indicates their use. Better to eat higher protein such as eggs, dairy, etc. Exercise will tone up your body and help muscle mass. I'm glad your health is good. Enjoy that and focus on being a good person to others, neat and clean. Peace and good health. A smile goes a long way also. ...Read more
Which diseases are treated with nuclear medicine? I've read online about the diagnostic properties of nuclear medicine, but there is little information about the diseases which can be treated with nucleur medicine. Which diseases are treated with nuclear
Mostly thyroid.: Thyroid overactivity (hyperthyroidism which may be due to graves disease) and thyroid cancer can be treated with radioactive iodine administered orally. Nuclear medicine can also be used to treat the bone pain due metastases (cancer which has spread to the bone.). ...Read moreSee 2 more doctor answers
A variety: Of non steroidal anti inflammatory medication as long as there are no contraindications like allergies, use of blood thinners, kidney or liver disease or ulcer/reflux issues . Tylenol (acetaminophen) is an option. Other types of drugs like cymbalta, Elavil and neurontin/lyrica may also help. Ideally, not smoking, regular exercise and weight control are also helpful. Good levels of vitamin d also are important. ...Read more
No!!!!!: Many people live with celiac disease and are highly functioning professionals. Strict adherence to a gluten-free diet is crucial. That also means reading labels very carefully. You should shop at the gluten-free section, available in most groceries, be under competent medical supervision. Also see www.celiac.nih.gov/materials.aspx; www.celiac.org/ ...Read moreSee 2 more doctor answers
Antibiotics: Antibiotics depending on the disease.Get a more detailed answer ›


Invasive species often evolve rapidly in response to novel introduced environments, making them excellent models for the study of evolution.
Invasive species often evolve rapidly in response to novel introduced environments, making them excellent models for the study of evolution. However, there remains an unsolved paradox: how can introduced populations that typically have low genetic diversity respond so quickly to selection? Is this response primarily based on novel mutations or are there other mechanisms (e.g. epigenetic modifications) through which rapid evolution is occurring in these systems? In this seminar, I will describe my research on rapid evolution in Australian invasive species and discuss future directions that aim to improve our knowledge of the fundamental mechanisms driving evolution during invasion. I will describe data from a range-edge population of invasive starlings (Sturnus vulgaris) in which we have found evidence of rapid evolution of mitochondrial DNA that is linked to changes in mtDNA copy number. I will also discuss changes in gene expression identified in invasive cane toads (Rhinella marina) from original sites of introduction in Australia as compared to range-edge populations. We now are investigating to what degree these changes in gene expression are related to genetic polymorphism, whether they are driven by changes in DNA methylation and the heritability of methylation in this system. Ultimately, I aim to address the possibility that rapid evolution during invasion may be initially driven by environmentally induced epigenetic mechanisms.
Dr Lee Ann Rollins is a UNSW Scientia Fellow in the Evolution & Ecology Research Centre. Dr Rollins' research uses genetic and epigenetic data to examine population and evolutionary processes. Specifically, she employs molecular techniques to investigate mechanisms underlying rapid evolution often seen in invasive populations, using iconic Australian invaders such as the cane toad and starling as models. She is also interested in using genetic data to improve the management of invasive and conserved species.


Heterotrophic plate count (HPC) bacteria are naturally present in all aqueous environments.
These bacteria undergo multiplication cycles in drinking water, especially in closed containers (bottled water) or in tap water when chlorine levels are dissipated, such as in dead ends in water mains or household plumbing.
A study was undertaken to estimate health risk from these naturally occurring bacteria by the determination of cytotoxicity and invasiveness in a human enterocyte cell line.
HPC bacteria were isolated from bottled and tap water samples by enumerating them under physical and chemical conditions analogous to human physiology.
All HPC bacteria were examined at both log and lag phase of their growth cycles.
Bacterial broth supernatant fluids were also tested to serve as critical negative controls.
Naturally occurring HPC bacteria demonstrated low invasiveness and cytotoxicity with more than 95% of isolates showing equivalency to broth supernatant fluid.
When showing either invasiveness or cytotoxicity, only a small number of cells from the culture were positive.
Of those that were positive, log phase HPC bacteria were significantly more cytotoxic and invasive than those from stationary phase.
Bacterial broth controls demonstrated varied, but often marked, cytotoxicity.
Mots-clés Pascal : Bactérie, Eau potable, Analyse risque, Virulence, Cytotoxicité, Pathogène, Homme, Côlon, Lignée cellulaire, Lignée CACO2
Mots-clés Pascal anglais : Bacteria, Drinking water, Risk analysis, Virulence, Cytotoxicity, Pathogenic, Human, Colon, Cell line
Notice produite par :
Inist-CNRS - Institut de l'Information Scientifique et Technique
Cote : 97-0297613
Code Inist : 002A05B10. Création : 15/07/1997.


Try eating canned salmon to excess fat. Some people do not feel comfortable cooking fresh, raw seafood. If you are one of the people people, consider buying your fish in cans. Alternatively, you could find fish sold in tins, the freezer section, or even individually sealed packages. Many of these fish products require little to no cooking.
Another thing that certainly give appreciation of is insulin resistance. That is also because starvation all forms. When you introduce carbohydrates into the diet, hyperinsulinemia and blood glucose level swings may likely occur. This particular really is as a consequence of the advance in the degrees of enzymes in the body system. The enzymes that are chiefly affected are the individuals that are involved with carbohydrates or fats burning. When the human body had not been fed with carbs, stopping a ketosis diet will also imply how the ‘down regulation’ will be changed. Staying on the cyclical ketogenic diet will keep insulin needs in stability. Carbs have always created difficulties if you are with troubles.
One reason the low-carb or no-carb (also called ketogenic) diets are so attractive could be because of the large initial weight reduction. However, http://ketocontrol.org/ this weight is certainly not fat. When carbohydrates are restricted h2o has a backup store of them located from the liver and muscles in something called glycogen. The body can store approximately 400 grams of glycogen. In larger individuals this number can enhancement. In addition to this, for every gram of glycogen stored in the human body, 3 grams of water are also stored. If you figure it out, this would equate to around 1600 grams (3.5 pounds) of glycogen and stream.
Secondly, to burn the fat easily you need to build a correct personal ketosis diet plan menu for women. Knowing your metabolic type will allow you to research and exploit resources moves through your personal fat loss diet. Some sort of daily ketosis diet plan menu for womenning guide will allow you to determine just what kinds of foods it is advisable to be snacking. The easy weight loss meal guide will aid you determine ideal proportions and meal dimensions.
The faster food is converted into blood sugar, the faster your with the aid of rise. When blood sugar levels are high, method secretes insulin, its primary storage junk food diet. When insulin is present in the bloodstream, energy nutrients such as fat or carbohydrates are far able to be stored rather than burned. When you’re thinking of fat loss, this means fat is not readily mobilized from fat cells and fat burning slows or stops.
Can you utilize machines from a gym or at your house? The machine based cardio programs are sometimes a better option if you could have injuries mindful about will be less body impact stress on your metabolism. And it really doesn’t matter what piece. My only advice is in case you are going cord less mouse with machines planet gym, alternate between the various types. Maybe the step mill one day, rower the next, seated recumbent bike position, maybe a spin class, or http://ketocontrol.org/ jogging on the treadmill. Snappy to break it up so that you don’t do precisely type generally and give your body different movement patterns to adjust to while preventing repetitive demand.
The cyclical Keto Control Diet guidelines restricts carbohydrates. By restricting carbohydrates, but, maintaining caloric consumption, your body will just have one use of fuel expenditure. That is fat; which is what ketosis are. You are essentially turning on your fat burning machine. Ketones are sent out of your body and fat reduction becomes outstanding. How does this happen? The largest internal organ in your body is the key player. Your liver. The liver features the job of converting fat into ketones. These ketones are then excreted out from the body, weight/fat loss. It is a natural concept.
Ketones also appear to use a diuretic effect, which will mean a good greater lowering of normal .Moreover to normal water, if you have been working out recently to hurry along your “weight loss” (you indicate body fat decline, correct?) progress you most likely have gained some muscle doing thereby. This acquire in muscle additionally be impact the numbers you see on the scale. Muscle is also far more dense than fat.You might be wondering a person can might be going to measure your progress now how the scale doesn’t indicate as very much as it helpful to. Well, the numbers of numerous techniques to measure your bodyfat commission.


+44 1803 865913
By: Michel Peissel
253 pages, B/w photos
Describes how in 1994, the author made history twice over - by discovering the source of one of the world's mightiest rivers, the Mekong, high in the ice-strewn fields of the Tibetan Plateau; and by finding a living fossil, the Riwoche horse, a previously unknown species which may prove to be a missing link in equine evolution.
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DPP2 Rat anti-Human, Clone: 398011, Invitrogen
Rat Monoclonal Antibody
Manufacturer: Invitrogen MA524133
Reconstitute at 0.5 mg/mL in sterile PBS. Endoxin level is <0.10 EU per 1 µg of the antibody by the LAL method.The protein encoded by this gene is a post-proline cleaving aminopeptidase expressed in quiescent lymphocytes. The resting lymphocytes are maintained through suppression of apoptosis, a state which is disrupted by inhibition of this novel serine protease. The enzyme has strong sequence homology with prolylcarboxypeptidase and is active at both acidic and neutral pH.
|PBS with 5% trehalose and No Preservative|
|DPP7, Carboxytripeptidase, Dipeptidyl aminopeptidase II, Dipeptidyl peptidase 2, DPPII, Dipeptidyl arylamidase II, Dipeptidyl-peptidase 7, Dipeptidyl-peptidase II, Dipeptidylpeptidase 7, Dipeptidyl peptidase 7, Dipeptidyl peptidase II, DPP II, DPP2, QPP|
|-20° C, Avoid Freeze/Thaw Cycles|
|Mouse myeloma cell line NS0-derived recombit human DPPII/QPP/DPP7 Gly22-Leu492|
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Excellent mini or gherkin cuke which can be picked either at the midget size or small pickle stage and processed. 24" semi-vining compact plants set masses of scrumptious sweet, crisp little black-spined cukes! 10-12"H vigorous plants produce about 20-25 fruits. Best picked 2-4" long. They are delectable raw, sliced, processed and adds a dash of pizazz to any dish! Can be grown in the garden as well as in containers.
Created from a controlled method of pollination in which the pollen of two different species or varieties is crossed by human intervention. Typical F1 varieties are deliberately created to breed a desired trait. Gardeners who use hybrid plant varieties usually purchase new seed every year, however, over several years the seed could eventually be used for open pollination
Cucumbers will grow both female and male flowers. Such cucumber plants do not need another cultivar cucumber plant for pollination. They require bees, other insects or wind, however, to spread their pollen from their male flowers to their female flowers.
* This is an estimated guide. Conditions may vary considerably due to location.


- Research article
- Open Access
Hydrogen peroxide-producing pyruvate oxidase from Lactobacillus delbrueckii is catalytically activated by phosphotidylethanolamine
BMC Microbiology volume 20, Article number: 128 (2020)
Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo.
We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0–18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation.
To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors.
Pyruvate oxidase (Pox) is an enzyme in the oxidoreductase family. There are hydrogen peroxide-producing and acetate-producing Pox enzymes. In hydrogenperoxide-producing Pox enzymes, pyruvate, phosphate, and oxygen are converted to acetylphosphate, carbon dioxide, and hydrogen peroxide with the cofactors flavin adenine dinucleotide (FAD) and thiamine pyrophosphate (TPP) . Acetate-producing Pox enzymes catalyze the oxidative decarboxylation of pyruvate to form carbon dioxide and acetate with electrons being transferred directly to the electron transport chain via the membrane-embedded electron carriers [2, 3].
The most well studied acetate-producing Pox is from Escherichia coli. E. coli Pox is dependent on the same cofactors as other Pox enzymes and is also homo-tetrameric . E. coli Pox produces acetate and does not utilize oxygen as its final electron acceptor, but rather the membrane embedded ubiquinone 8 electron transport molecule . CidC is another acetate-producing Pox enzyme from Staphylococcus aureus which transfers electrons to menaquinone and has an important role in cell death pathways . Pyruvate:Quinone Oxidoreductase (PQO) from Corynebacterium glutamicum also produces acetate and transfers electrons to a quinone . One characteristic of E. coli Pox, PQO, and CidC is that they are catalytically activated by phospholipids, which is thought to facilitate efficient transfer to membrane electron shuttles by activating the enzyme at a membrane peripheral position [3, 5, 6]. Mutants in E. coli Pox that disrupt lipid activation of the enzyme are localized to the C-terminal region of the protein. Further biochemical analysis identified a lipid activation helix in the C-terminal region (amino acids 558–568) that upon lipid interaction induce the preceding alpha helix to move out of the active site, which in turn positions phenylalanine 465 into the active site enhancing electron transfer between TPP and FAD [7,8,9]. The phospholipid activation of E. coli Pox is a hybrid of Km-driven (K-type) and velocity-driven (V-type) allosteric activation where the km for pyruvate decreases along with an increase in the enzyme turnover rate . However, in CidC the activation is V-type where the enzyme turnover rate is substantially increased .
Hydrogen peroxide-generating Pox enzymes are produced by multiple different bacteria. Pox enzymes from Lactobacillus plantarum and Streptococcus pneumoniae are the most well studied. These enzymes form homo-tetrameric structures [11, 12]. The role of Pox enzymes in central metabolism is thought to be increasing ATP production in concert with acetate kinase during aerobic metabolism . In S. pneumoniae, which has no tricarboxylic acid (TCA) cycle, additional ATP production from glucose is believed to come from this pathway . L. plantarum Pox is considered to be an important component of the enhanced biomass that is observed in aerobic growth by generating acetyl phosphate as a substrate for acetate kinase . In addition, the production of hydrogen peroxide from Pox could also be important on a community level. Since hydrogen peroxide is toxic to some bacteria, Pox activity could confer a fitness advantage to the producer organism. In S. pneumoniae, pox-deficient mutants display decreased virulence in a rat model of disease likely through decreased adhesion properties, but also potentially through decreased competition with commensal bacteria . Streptococcus gordonii and Streptococcus sanguinis are known to produce enough hydrogen peroxide primarily through Pox to inhibit the oral pathogen Streptococcus mutans . Recently, Lactobacillus delbrueckii strains harboring intact pox genes were shown to produce enough hydrogen peroxide to inhibit the growth of the oral pathogen Porphyromonas gingivalis . The characterization of hydrogen peroxide-producing strains and their respective encoded hydrogen peroxide-producing enzymes could be beneficial in the development of novel treatments for diseases like periodontitis.
In the current study, we characterize a hydrogen peroxide-producing Pox enzyme from L. delbrueckii that is catalytically activated specifically by phosphotidylethanolamine, a common bacterial membrane component. This activation is largely dependent on the C-terminal region of the protein. Further, we showed that the L. delbrueckii Pox adopts a pentameric structure and potentially a dimer of pentamers, which is novel for Pox enzymes. These characteristics are unique for hydrogenperoxide-producing Pox enzymes and demonstrate substantial variability in the structure and function of Pox from different bacterial species.
Characterization of L. delbrueckii pox
The E. coli Pox has been well studied, and it is known that this enzyme is catalytically activated by phospholipids [6, 7, 17]. In addition, CidC and PQO from S. aureus and C. gluticaticum, respectively, are other pyruvate oxidases that are lipid activated [3, 5]. Another well studied Pox enzyme is from Lactobacillus plantarum [1, 13, 18]. This enzyme has not been shown to be catalytically activated by phospholipids indicating that there is variability in the catalytic activation of the Pox enzymes. Amino acid identity between the enzymes ranges from 25.9% between L. plantarum Pox and PQO to 46.4% between E. coli Pox and PQO (Table 1). For DNA sequence identity, the identity ranges from 50.7% between CidC and PQO to 56.3% for CidC and L. plantarum Pox (Table 1).
First, we wanted to determine the biochemical properties of the L. delbrueckii Pox enzyme. We previously purified L. delbrueckii Pox and used it for subsequent testing . We determined the km values for pyruvate and phosphate and the kcat value. The km for pyruvate and phosphate were 342.2 μM and 8 mM respectively, and the kcat was 0.367 s− 1 (Fig. 1 and Table 2). The optimum pH for several other pyruvate oxidase enzymes is near 5.5 and so we tested the activity of the L. delbrueckii Pox at various pHs to determine its pH activity profile. In agreement with other Pox enzymes, the L. delbrueckii Pox was most active at the pH range of 5.5 to 6 (Fig. 1c).
L. delbrueckii pox forms pentameric structure in solution
Both the L. plantarum and E. coli Pox are known to form tetrameric structures [1, 4]. To determine whether the L. delbrueckii Pox also formed a tetramer, we performed gel filtration chromatography with the purified enzyme. Interestingly, L. delbrueckii Pox eluted in two major peaks; the first with a calculated molecular weight of 694 kDa and the second with a calculated molecular weight of 345 kDa (Fig. 2a and b). The molecular weight of an STYM1 L. delbrueckii Pox monomer is 68 kDa according to the translation of the gene sequence; therefore, the first peak corresponded to a decamer and the second peak to a pentamer. The last peak corresponded to unbound FAD (Fig. 2a). Analysis of the fractions by non-reducing SDS-PAGE revealed two distinct bands that migrated distances consistent with monomer and dimer forms of the L. delbrueckii Pox at approximately 68 kDa and 136 kDa (Fig. 2c). Fractions 13 and 14, which correspond to the last peak from gel filtration, did not contain any protein and also were yellow in color confirming the presence of unbound FAD (Fig. 2c). To confirm that the 136 kDa protein band was in fact a dimer of L. delbrueckii Pox, we analyzed the L. delbrueckii Pox on non-reducing and a reducing SDS-PAGE. Under reducing conditions, L. delbrueckii Pox migrated exclusively as a monomer indicating that the 136 kDa band is a dimer, presumably linked by a disulfide bond at position 72 since it is the only cysteine in the amino acid sequence (Fig. 2d).
Phospholipid activation of L. delbrueckii pox
We hypothesized that L. delbrueckii Pox may be activated by phospholipids similar to E. coli Pox, CidC, and PQO [2, 3, 5]. We tested whether addition of either phosphotidylethanolamine (PE), phosphotidylcholine (PC), and phosphotidylglycerol (PG) could catalytically activate L. delbrueckii Pox. L. delbrueckii Pox was catalytically activated specifically by PE, but not PC or PG (Fig. 3a). The phospholipids used contained the same acyl chain structure (16:0–18:1) and only varied in the headgroup indicating that there is specificity in phospholipid activation of L. delbrueckii. To determine the full activation profile of L. delbrueckii Pox by PE, we measured the fold activity over a wider range of PE concentrations and observed activation upwards of 6-fold relative to the enzyme without PE (Fig. 3b). This level of activation exceeds that of the CidC Pox (3-fold activation) from S. aureus .
To gain mechanistic insight into how PE activates the enzyme, we tested the kinetic parameters of L. delbrueckii Pox in the presence of PE (Fig. 4). With the addition of PE, the km for pyruvate and phosphate were 519.9 μM and 1.25 mM respectively and the kcat was 6.85 s− 1 (Table 3). The approximately 18-fold increase in kcat and decrease in phosphate km relative to the enzyme in the absence of PE suggest that these parameters are the primary drivers of catalytic activation, which is consistent with the increase in kcat observed in E. coli Pox and CidC upon phospholipid activation [3, 19]. The activation observed here is a hybrid of V- and K-type activation since the kcat increased and the phosphate km decreased, which is also consistent with E. coli Pox [9, 19].
We also wanted to determine whether L. delbrueckii Pox inserts into PE micelles. We performed co-precipitation experiments with L. delbrueckii Pox in the presence and absence of PE. Nearly all of the enzyme remained in the soluble fraction and did not co-precipitate with PE, which is in contrast to E. coli Pox  (Fig. 5).
The C-terminus of L. delbrueckii pox is important for lipid activation
The C-terminus of E. coli Pox is important for lipid activation of the enzyme . To test if the C-terminus was important in the L. delbrueckii Pox, we generated a C-terminal truncation that lacked the last 34 amino acids. We purified the enzyme and tested it for its ability to be activated by PE (Fig. 6a). The enzyme had a specific activity of approximately 1.32 U/mg in the absence of PE, which is greater than the specific activity of the full-length enzyme (Fig. 1c and Fig. 6b). This is consistent with the E. coli pox truncation having greater activity partially mimicking lipid activation . Furthermore, the Δ34 enzyme was only weakly activated by PE to approximately 2-fold greater than the enzyme without PE compared to an approximately 6-fold activation with the full-length enzyme (Fig. 3b and Fig. 6c). This indicates that the C-terminal sequence is important for the lipid activation of the Pox enzyme.
Here, we demonstrated that the hydrogen peroxide-producing pyruvate oxidase enzyme from L. delbrueckii is catalytically activated specifically by PE, but not other phospholipids, and the last 34 amino acids of the enzyme are important for this activation. To our knowledge, this is the first hydrogenperoxide-producing Pox enzyme that has been shown to be activated by phospholipids. In addition, we showed that the L. delbrueckii Pox forms a pentameric structure in solution of which at least two subunits form a disulfide bond between cysteine 72. Other Pox enzymes form tetrameric structures, and there has been no evidence of disulfide bond formation indicating that the L. delbrueckii Pox has a different structure than other Pox enzymes [1, 4].
It is interesting that only PE had an impact on catalytic activity. This suggests that the size, shape, and charge of the phospholipid head group is important for catalytic activation. If the Pox enzyme is interacting with phospholipid that is already incorporated into the membrane, the head group would be exposed to the cytoplasm and be in the closest proximity to the enzyme. The interaction with the cytoplasm-facing ethanolamine likely occurs through the C-terminal 34 amino acids. Similar to the E. coli Pox, we hypothesize that this interaction induces a conformational shift in the enzyme that enhances electron transfer between FAD and TPP and decreases the phosphate km. This interaction is likely mediated by electrostatic forces. In fact, it is believed that CidC interaction with phospholipids is driven primarily by electrostatic forces suggesting there is interaction with the headgroup . Furthermore, PE is known to be an abundant component of bacterial membranes, whereas PC is more commonly a eukaryotic and mammalian membrane component [21, 22]. Thus, activation only by PE is reasonable since L. delbrueckii Pox is a bacterial enzyme. Enhanced hydrogen peroxide production by L. delbrueckii could be beneficial in a complex microbial community. In fact, hydrogen peroxide production by Pox has been shown to be important in the inhibition of dental pathogens like P. gingivalis .
The pentameric structure of L. delbrueckii Pox is different from what has been observed with E. coli Pox and L. plantarum Pox [1, 4]. Based on our gel filtration data, the L. delbrueckii also forms a decamer or a dimer of pentamers, but it is unclear which of these may be occurring. Interestingly, we also observed formation of dimers linked by a disulfide bond which are a component of the pentameric structure since they were present in fractions with an elution volume consistent with pentameric molecular weight. Therefore, the structure of the pentamer must be comprised of either three monomers and one dimer or one monomer and two dimers.
E. coli Pox lipid activation is a mixture of K- and V-type allosteric activation where the pyruvate Km decreases and the kcat increases [19, 23]. The lipid activation observed with L. delbrueckii Pox was also a mixture of K- and V-type allosteric activation, however, the specific parameters that changed were different. The pyruvate km increased slightly while the phosphate km decreased. The Kcat did increase substantially (18-fold) similar to the E. coli Pox. Since the changes in pyruvate and phosphate km could offset one another, we believe that the lipid activation of L. delbrueckii Pox is primarily driven by increasing the enzyme turnover rate. This is consistent with structural data of E. coli Pox demonstrating that lipid activation’s primary effect on the enzyme is to shift a phenylalanine closer to the interface between TPP and FAD to increase the efficiency of electron transfer . This is thought to increase the enzyme turnover rate substantially. It is also in agreement with the activation of CidC where the activation of the enzyme was exclusively driven by a 10-fold increase in kcat .
E. coli Pox, CidC, and PQO are the only known lipid-activated Pox enzymes [3, 5, 7]. These Pox enzymes likely evolved this ability so that they are most active near or at the membrane where it can readily transfer electrons to a membrane electron shuttle [3, 9]. For this reason, E. coli Pox inserts and co-precipitates with lipids in the presence of substrate . Likewise, the CidC pyruvate oxidase from S. aureus interacts with phospholipids, but the interaction is believed to be mediated by electrostatic forces indicating that there does not necessarily need to be insertion into the membrane . We did not observe any co-precipitation of L. delbrueckii Pox with PE suggesting that there is no insertion or that the interaction may be unable to withstand centrifugal forces. The fact that L. delbrueckii Pox does not strongly associate with or insert into the membrane may be a mechanism to regulate hydrogen peroxide production. If the association were too strong or there was insertion into the membrane, excessive hydrogen peroxide production may intoxicate the cell.
L. delbrueckii Pox does not need to interact with the membrane since it does not transfer electrons to a membrane electron shuttle, but rather to oxygen. Therefore, the utility of lipid activation of L. delbrueckii Pox is less clear than it is for other pyruvate oxidase enzymes that transfer electrons to membrane-bound carriers. One hypothesis is that the lipid activation of a hydrogen peroxide-producing Pox could be beneficial because it would position hydrogen peroxide production at a peripheral point of the cell. Passive diffusion of hydrogen peroxide from this point would subject the interior of the cell to less hydrogen peroxide and excrete more through the membrane than if the enzyme were positioned in the middle of the cell. Thus, the lipid activation of the Pox enzyme in this case may reduce the self-toxicity of hydrogen peroxide production by L. delbrueckii by activating the enzyme only at the edges of the cell.
In this report, we describe the first hydrogen peroxide-producing Pox enzyme that is activated by the phospholipid phosphotidylethanolamine. We also demonstrated a different oligomeric structure than other known Pox enzymes. Together, these data demonstrate that Pox enzymes from different bacterial species can vary in their structure and function. As Pox is an important enzyme in mediating bacterial interactions and microbial community structure, enhancing pathogenicity, and for certain biosensor applications, further understanding of the biochemical characteristics of Pox are warranted.
Pyruvate oxidase enzyme or pyruvate:oxygen 2-oxidoreductase (phosphorylating) (EC 18.104.22.168) was derived from Lactobacillus delbrueckii STYM1 strain . The pyruvate oxidase enzyme was purified as previously described .
Purification of L. delbrueckii pox Δ34
The L. delbrueckii pox deletion mutant was PCR amplified from STYM1 genomic DNA using the primers listed in Table 4. The PCR product was ligated into the pFLAG-CTC vector (Sigma, St. Louis, MO) using the NdeI and XhoI (New England Biolabs, Ipswich, MA) restriction sites. The product was transformed into DH5α E. coli and the construct was confirmed by PCR and sequencing. The construct was transformed into LOBSTR E. coli (Kerafast, Boston, MA), in which the L. delbrueckii Δ34 Pox was expressed as a C-terminal His6-Pox. A starter culture was diluted 1:100 into 40 mL of LB supplemented with 100 μg/mL of carbenicillin (Sigma, St. Louis, MO) and grown at 37 °C until the OD600 reached 0.4–0.8. The cells were cooled on ice for 10 min and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma, St. Louis, MO) was added and the cells incubated at 25 °C with shaking for 20 h. The cells were harvested by centrifugation at 3200 x g for 20 min at 4 °C and resuspended in 1 mL 20 mM Bis-Tris 150 mM NaCl pH 7 with EDTA-free cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). The cells were lysed by sonication on ice and the soluble cell lysate was harvested by centrifugation at 16000 x g for 15 min at 4 °C. The soluble cell lysate was filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter and applied to a 1 mL bed volume of HisPure Ni-NTA resin (Thermofisher, Waltham, MA). The column was washed twice with two column volumes of buffer containing 25 mM imidazole (Fisher, Pittsburgh, PA) and the bound protein was eluted from the column in buffer containing 250 mM imidazole. Protein purity was assessed by SDS-PAGE and Coomassie staining.
Pyruvate oxidase activity assay and kinetic parameters
Pyruvate oxidase activity was measured as previously described . Briefly, 50 mM sodium pyruvate, 15 μM FAD (Sigma, St. Louis, MO), 300 μM TPP (Sigma, St. Louis, MO), 0.03% N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (Sigma, St. Louis, MO), 0.015% 4-aminoantipyrine (Sigma, St. Louis, MO), and 33 μg/mL horseradish peroxidase (Sigma, St. Louis, MO) were combined with purified pox enzyme. After a 10-min incubation at room temperature, sodium phosphate pH 5.6 was added to 50 mM final concentration to initiate the reaction. The formation of a quinoneimine dye at 25 °C was measured by an increase in absorbance at 550 nm and the specific activity was calculated as described. 1 unit of Pox activity is defined as the production of 1 μmol of hydrogen peroxide per minute and this was converted to μM/min for kinetic analysis.
For km determination of pyruvate and phosphate and determination of kcat, dilutions of substrate were used instead of the 50 mM concentration. Enzyme concentration was 1.76 μM. V0 for each substrate concentration was plotted and the data were fitted using Prism software (Graphpad, San Diego, CA) kcat non-linear regression function. For km and kcat determination in the presence of phosphotidylethanolamine (PE), PE was used at a concentration of 7.2 mM and the enzyme concentration was 170 nM.
Phospholipid activation assay
Phosphotidylethanolamine (16:0–18:1) (Avanti Lipids, Alabaster, AL), Phosphotidylcholine (16:0–18:1) (Avanti Lipids, Alabaster, AL), and Phosphotidylglycerol (16:0–18:1) (Avanti Lipids, Alabaster, AL) dissolved in chloroform were purchased commercially at a concentration of 10 mg/mL. The desired volume of 10 mg/mL phospholipid solution was evaporated under a steady stream of argon gas (Airgas, Radnor, PA) and dried in a vacuum desiccator for 1 h to evaporate residual chloroform and yield the desired weight of dried phospholipid. The phospholipids were resuspended in the appropriate volume of 20 mM Bis-Tris 150 mM NaCl pH 6 to achieve the desired concentration and incubated at 37 °C with shaking at 225 rpm for 30 min. The suspension was vortexed for 30 s and vortexed immediately prior to each use.
Phospholipids were added to the pyruvate oxidase assay mixture at various concentrations and enzyme activity was measured as described above. Fold activity was calculated relative to enzyme activity without phospholipid.
Gel filtration analysis
Prior to analysis of the L. delbrueckii Pox, protein standards were mixed together and applied to the Superdex 200 column (GE healthcare, Chicago, IL) using a duo-flow system (Biorad, Hercules, CA). The protein standards were thyroglobulin (670 kDa), gamma-globulin (150 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa), and vitamin B12 (1.35 kDa). Protein was eluted from the column using a mobile phase of 20 mM Bis-Tris pH 6150 mM NaCl at 0.5 mL/min flow rate. A standard curve was plotted using the peak elution volume for each standard versus the Log10 of the molecular weight in daltons. Eight hundred forty μg of purified L. delbrueckii Pox was incubated in the presence of 50 mM pyruvate, 300 μM TPP, and 15 μM FAD for 10 min at room temperature prior to application to the column. The absorbance at 280 nm was recorded over the elution. One mL fractions were collected after the void volume of 10 mL until the end of the elution. The peak volume for the Pox fractions was used to calculate the molecular weight according to the standard curve.
L. delbrueckii Pox in each fraction from gel filtration was visualized after non-reducing SDS-PAGE analysis with Coomassie staining. For reducing SDS-PAGE analysis, 5% β-mercaptoethanol (Sigma, St. Louis, MO) was included in the sample buffer.
PE was prepared as described above for activation experiments. 3 μg of L. delbrueckii Pox was incubated with 50 mM pyruvate, 300 μM TPP, and 15 μM FAD with either 1 mM PE or no PE for 30 min at room temperature. The PE fraction was precipitated by centrifugation at 16,000 x g for 20 min at 4 °C. The supernatant was aspirated as the soluble fraction and the PE pellet was resuspened in an equal amount of buffer. The fractions were visualized by Coomassie staining after SDS-PAGE.
Availability of data and materials
All data generated or analyzed during this study are included in this published article. Raw data can be made available upon request from the corresponding author.
Flavin adenine dinucleotide
Sedewitz B, Schleifer KH, Götz F. Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum. J Bacteriol. 1984;160(1):273–8.
Cunningham CC, Hager LP. Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids. J Biol Chem. 1975;250(18):7139–46.
Zhang X, Bayles KW, Luca S. Staphylococcus aureus CidC is a pyruvate:Menaquinone Oxidoreductase. Biochemistry. 2017;56(36):4819–29.
Wang AY, Chang YY, Cronan JE. Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding. J Biol Chem. 1991;266(17):10959–66.
Schreiner ME, Eikmanns BJ. Pyruvate:Quinone Oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization. J Bacteriol. 2005;187(3):862–71.
Chang Y-Y, Cronan JE. An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme. Proc Natl Acad Sci. 1984;81(14):4348–52.
Grabau C, Chang YY, Cronan JE. Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region. J Biol Chem. 1989;264(21):12510–9.
Cunningham CC, Hager LP. Crystalline pyruvate oxidase from Escherichia coli III. PHOSPHOLIPID AS AN ALLOSTERIC EFFECTOR FOR THE ENZYME. J Biol Chem. 1971;246(6):1583–9.
Neumann P, Weidner A, Pech A, Stubbs MT, Tittmann K. Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli. Proc Natl Acad Sci. 2008;105(45):17390–5.
Taniai H, Iida K, Seki M, Saito M, Shiota S, Nakayama H, et al. Concerted action of lactate oxidase and pyruvate oxidase in aerobic growth of Streptococcus pneumoniae: role of lactate as an energy source. J Bacteriol. 2008;190(10):3572–9.
Muller YA, Schulz GE. Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science. 1993;259(5097):965–7.
Ramos-Montañez S, Tsui H-CT, Wayne KJ, Morris JL, Peters LE, Zhang F, et al. Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae. Mol Microbiol. 2008;67(4):729–46.
Lorquet F, Goffin P, Muscariello L, Baudry J-B, Ladero V, Sacco M, et al. Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum. J Bacteriol. 2004;186(12):3749–59.
Spellerberg B, Cundell DR, Sandros J, Pearce BJ, Idänpään-Heikkilä I, Rosenow C, et al. Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae. Mol Microbiol. 1996;19(4):803–13.
Kreth J, Zhang Y, Herzberg MC. Streptococcal antagonism in Oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol. 2008 Jul 1;190(13):4632–40.
Cornacchione LP, Klein BA, Duncan MJ, Hu LT. Interspecies inhibition of Porphyromonas gingivalis by yogurt-derived Lactobacillus delbrueckii require active pyruvate oxidase. Appl Environ Microbiol. 2019;85(18):01271–19.
Grabau C, Cronan JE. In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site. Biochemistry. 1986;25(13):3748–51.
Risse B, Stempfer G, Rudolph R, Möllering H, Jaenicke R. Stability and reconstitution of pyruvate oxidase from lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction. Protein Sci. 1992;1(12):1699–709.
Bertagnolli BL, Hager LP. Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate. J Biol Chem. 1991;266(16):10168–73.
Recny MA, Grabau C, Cronan JE, Hager LP. Characterization of the alpha-peptide released upon protease activation of pyruvate oxidase. J Biol Chem. 1985;260(26):14287–91.
Vance JE. Phospholipid synthesis and transport in mammalian cells. Traffic. 2015;16(1):1–18.
Zhang Y-M, Rock CO. Membrane lipid homeostasis in bacteria. Nat Rev Microbiol. 2008;6(3):222–33.
Mather MW, Gennis RB. Kinetic studies of the lipid-activated pyruvate oxidase flavoprotein of Escherichia coli. J Biol Chem. 1985;260(30):16148–55.
This work was funded by the NIH/NIDCR R01 DE024308 (to L.T.H) and F31 DE025523 (to L.P.C). The funding agencies had no role in the design of the study, data collection, analysis, interpretation of data, or in writing the manuscript.
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Cornacchione, L.P., Hu, L.T. Hydrogen peroxide-producing pyruvate oxidase from Lactobacillus delbrueckii is catalytically activated by phosphotidylethanolamine. BMC Microbiol 20, 128 (2020). https://doi.org/10.1186/s12866-020-01788-6
- Catalytic activation
- Hydrogen peroxide
- Lactobacillus delbrueckii
- Pyruvate oxidase


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Pharmacokinetic (PK) has improved our knowledge about the most appropriate dosing and timing of administration of FVIII/FIX concentrates in patients with Haemophilia. However, although several studies have recently addressed the relevance of PK of clotting factors, usual practice is still mostly based on empiric approaches since individual PK estimation is difficult to obtain unless the patient is formally enrolled in a study. In fact, several plasma samples collected over several hours and/or days are required to establish a half-life curve confidently and this may be a relevant problem, especially in children. Recently however population PKs has emerged as an important tool to overcome this drawback. Targeted prophylaxis could take advantage of knowing the individual response to factor concentrate administration. On the clinical ground, age and body weight (BW) are roughly used to guide dosing because usually in vivo recovery is lower and clearance is faster in children than in adults.
Keywords: Factor VIII, Factor IX, Haemophilia A, Haemophilia B, Pharmacokinetics.


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Studies concerning genetic association aim to test whether single-locus alleles or genotype frequencies (or more generally, multilocus haplotype frequencies) are different between 2 groups (usually diseased subjects and healthy controls). Genetic association studies are based on the principle that genotypes are measured "directly", i.e. by sequencing the actual genetic code.


Thanks to successful research, three forms of liver disease – hepatitis A, B and D* – can be prevented by immunization.
*although there is no specific vaccine for hepatitis D, being immunized against hepatitis B will prevent you from contracting hepatitis D.
Hepatitis A is a serious liver disease caused by a virus that attacks the liver. The virus is transmitted through contaminated food, such as raw or insufficiently cooked shellfish and contaminated water. It can be passed by someone infected with the virus who doesn’t wash his/her hands properly after a bowel movement and then touches something that you eat.
The good news is that hepatitis A can be prevented by vaccination. A variety of vaccines are available to make immunization fit your needs. The vaccine will prevent you from contracting hepatitis A and from being a potential source of infection to others.
If you are planning to travel to destinations such as Mexico, the Caribbean, Africa, Asia, Latin America or Eastern Europe, you should get vaccinated against hepatitis A. These areas can be high risk for hepatitis A due to improper food handling and preparation and inadequate sewage and water purification systems. You do not necessarily need to be vaccinated if you are travelling in Canada, the U.S., Western Europe, Australia, New Zealand or Japan. In the past several years however, there have been hepatitis A outbreaks or alerts in North America due to contaminated produce used in a restaurant or sold in a supermarket and an infected individual involved in food preparation or food serving. The hepatitis A vaccination should also be considered for those who work in day-care settings and especially those who diaper infants.
There are several different hepatitis A vaccines licensed for use in Canada. These vaccines are well-tolerated and are at least 85-90 percent effective in preventing hepatitis A. Adverse reactions are generally mild and short-lived and may include reactions at the injection site, including pain, redness and swelling.
For more information on the hepatitis A vaccination, please consult your doctor.
Hepatitis B is another form of liver disease caused by a virus that attacks the liver. The hepatitis B virus is 100 times more contagious than the virus that causes AIDS. It is spread through infected blood or body fluids or by sexual contact with an infected person. Hepatitis B is not spread by water, food or through casual contact.
Like hepatitis A, hepatitis B is also preventable by vaccine. Three injections of this vaccine within a six-month period provide long-lasting protection against hepatitis in the majority of people. People most at risk are those whose jobs may expose them to blood or body fluids ( i.e. firefighters, health care professionals, law enforcement officers, etc.), sexual partners of hepatitis B carriers and babies born to mothers who are hepatitis B carriers. Immigrants from countries where hepatitis B is common (i.e. Asia, Southern and Eastern Europe, sub-Saharan Africa and the Pacific Islands) also carry greater risk.
In Canada, there are hepatitis B vaccines licensed for use in adults and separate formulations for use in children. There is also a combined hepatitis A and B vaccine available.
The above mentioned vaccines are generally well tolerated by adults and children, with a few mild and transient adverse effects. The most common adverse effects include pain, soreness and redness at the injection site and mildly elevated temperature.
Most Canadian provinces and territories provide universal hepatitis B vaccination programs for school-aged and pre-teen children. These vaccinations are usually offered to children in grades 5 to 7 depending upon the province.
For more information on the hepatitis B immunization, please contact your doctor or public health nurse.
For those who have had recent contact (seven days or less) with infected blood or body fluids, immunization with the hepatitis B immune globulin may be effective in preventing an acute form of the illness.
Infants born to mothers infected with the hepatitis B will receive hepatitis B immune globulin at birth as well as the first of three doses of the hepatitis B vaccine. These infants will receive the second dose at one month and the third at six months of age. This treatment will give the baby a 95 percent chance of not becoming infected with the hepatitis B virus.
At this time, there is no vaccine for hepatitis C. However, all people with chronic liver disease, including hepatitis C, should be immunized against both hepatitis A and B. A second infection by either virus can compound the damage to your liver. For those whose hepatitis C is more advanced, drug treatment may be appropriate and must be administered after careful assessment by your physician.


Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.
© 2011 OSA
Skin cancer is one of the most common forms of cancer in the UK totaling around 84,500 registered cases in 2007 with 10,672 case of these being malignant melanoma (MM) [1–5]. The total cost of skin cancer in the UK in 2002 was estimated to be in excess of £190 million . In that year, 63% of the total costs of skin cancer were attributable to MM alone, amounting to 1770 cases  and the number continues to rise (1852 in 2006), resulting in a doubling of reported cases since the early 1970’s (Quinn et al., 2001).
Globally, there were more than 161,000 reports of MM alone in 2002, with Australia/New Zealand having the highest incidence followed by Northern America, Northern Europe and Western Europe (67, 27.1, 18.5 and 17.6 cases/100,000, respectively) . These numbers are predicted to continue rise over the next few decades. Although MM becomes more common with increasing age, these trends have changed more recently with an abnormally high incidence in the young; with MM becoming the most common form of cancer in people aged 15-34, a 300% increase between 1975 and 2006 . The greatest increase was found in the 60-79 year old group, particularly in males, which has seen an increase of over 600% in the same period .
Early diagnosis and treatment can prevent death for the majority of these patients. The normal route for diagnosis is a Post-Hoc method that builds-in an inherent delay in the diagnostic procedure. This involves histological processing of the tissue followed by analysis/diagnosis by an expert pathologist to assess the severity of the MM and the margins of the invading cancer cells based on Breslow thickness measurements. This may result in the patient having to undergo two or more operations to surgically remove the entire tumour, if some of the affected tissue was not removed at the first biopsy stage. For irregular shaped cancers, simply applying Breslow depth measurements to calculate the margins of the cancer to excise the lesion is inadequate and results in further excision biopsies being necessary to remove all the diseased tissue. This delay can lead to the cancer metastasizing to other tissues and significantly worsen the prognosis for the patient.
During their invasion of surrounding tissues, both Melanoma and Non-Melanoma Skin Cancer (NMSC) utilize the same biological events [8–13]. They are characterized by active invasion and destruction of surrounding connective tissue. Penetration of the cancer through the basement membrane (BM) is the initial step in the complex process of invasion and metastasis. This process when simplified consists of three distinct events: 1. attachment to the BM, via various cell surface receptors. 2. degradation of the BM and extracellular matrix by a series of matrix metalloproteinases (MMPs). 3. cancer cell locomotion into the surrounding tissue induced by chemotaxis. Therefore, a key features of all skin cancers is the degrading and remodeling of the extracellular matrix along the borders of the invading cancer, principally collagen type I, by matrix MMPs [8–13], but little is known about the differential disruption of collagen density and morphology between MM and NMSC . We propose that this degradation of collagen along the borders of the invading cancer can be used to, a) accurately define the margins of the skin cancer, and b) aide in the diagnosis of a malignant condition.
Confocal microscopy has been the subject of intensive investigation in the scientific community in recent years, not least in the field of cancer biology [14,15]. More recently, multiphoton fluorescence microscopy (MPFM) has been used for non-invasive imaging of the epidermis in a number of studies [16,17]. Since MPFM is a non-linear fluorescence phenomenon, only tissues at the focal point of the objective are excited and hence the technique is inherently confocal, i.e., the only two-photon events that can occur in the tissue will be at the focal point of the objective and therefore no confocal pinhole is required to remove out-of-focus blur. Similarly, second harmonic generation (SHG) occurs only at the focal point of the objective in tissues that have a non-centrosymmetric crystalline structure, such as collagen, to produce a strong SHG signal at exactly half the wavelength of pump laser wavelength [18–21]. However, with SHG imaging, there is substantially less absorption of energy by the tissue compared to single-photon excitation if the fundamental wavelength is off the resonance wavelength of cellular constituents, and hence, there is negligible thermal or phototoxic damage . As both SHG and MPFM utilize pulsed infra-red light this allows deep imaging into live tissues (400–1000 µm depending tissue turbidity, pigmentation and wavelength) with less scattering of light [15,22,23]. Importantly, the SHG signal is generated without the need to stain the tissue in any way, and so this technique lends itself well to be used as a non-invasive imaging tool [24,25]. Using second harmonic confocal microscopy we have demonstrated that the borders of malignant skin cancers can be easily identified in unstained human biopsy skin sections that correlate well with clinical diagnosis of MM.
We propose that by using SHG imaging the diagnostic process currently used and surgical planning can be dramatically enhanced by accurately delineating the borders of invading cancer cells at the patient’s first presentation. This could significantly reduce the time taken for diagnosis and alleviate the patient of repeated operations and possibly decrease mortality rates.
2. Materials and methods
Sections from human MM biopsies were acquired from the University of Szeged, Hungary with full ethical approval from the host institute and patient consent. Tissue devitalization protocols were standardized for all patients and no samples showed any signs of necrosis prior to excision. Histological preparation of the tissue was identical for all samples. Five samples were diagnosed as superficial spreading MM, one nodular MM and two lentigo mailgnia MM. Three samples were from female patients and five from males with an age range of 58–75 years. Breslow thickness assessments ranged from 0.304 to 1.672 mm and post-MM excision survival was 30–84 months.
Multiple adjacent fields of unstained human melanoma skin sections (4µm) (n = 8 patients) were imaged using SHG imaging to reveal fibrillar collagen distribution (principally Type I and III), followed by single-photon transmission (bright field) imaging. A detailed description of the experimental setup is described in . Image acquisition was performed on a Leica SP2 AOBS confocal multi-photon laser scanning microscope coupled to a Leica DMRE upright microscope (Leica, Milton Keynes, UK), equipped with a pulsed, mode locked femtosecond (fs) Ti:sapphire Tsunami laser synchronously pumped by a Millenia VII (Spectra-Physics, Mountain View CA), diode pumped solid state frequency doubled laser capable of delivering up to 8.5W pumping power at 532nm.
All SHG image acquisition was performed at 880nm, which has previously been found to be the optimum wavelength for collagen types I and III . The pulse width of the Tsunami was 80 fs with a pulse repetition rate of 80 Mhz. Laser power output at the microscope objective was recorded with a coherent power meter and calibrated for all samples to deliver peak power of 0.4 × 108 Wcm−2 and was consistently maintained below the damage threshold of the samples, which for collagen in sections was found to be 1.5 × 108 Wcm−2 . An IST laser spectrum analyzer coupled to a Tektronix TDS 210 oscilloscope was used to tune the laser to the desired wavelength. An electro-optical modulator (EOM) (Linos LIV20) received the laser output before delivery to the confocal microscope through a series of optical mirrors. The EOM allowed the laser intensity at the objective to be controlled and optimized. EOM was set at 90% to circularly polarize the pump laser beam, so as to ensure the incident beam laser remained consistent across all specimens. Thus the potential effects of sample polarization versus fundamental pump beam polarization were minimized.
In order to image the complete section we used a motorized stage (Mietzhouser, GmbH), controlled by the Leica software to perform automated montages from multiple fields consisting of up to 42 x 19 fields with up to 10 z-sections of 1µm thickness. Samples were imaged with a 20x, 0.7NA dry objective and 2x zoom producing an image measuring 375 by 375 µm at 512 by 512 pixel resolution. The SHG signal in the backscattered geometry was captured in the de-scanned detector with the pinhole set to the maximum of 600 µm. The Leica microscope incorporates a programmable, prismatic beam splitter, which is capable of single and multi-chromatic beam splitting. The backscattered photons are subsequently focused through the objective lens with an estimated beam diameter of 0.24 µm (λp = 880 nm) and delivered through the prismatic beam splitter, programmed to collect light at 435-445nm, before passing it on to the photomultiplier tube (PMT). This calculation is based on the Guoy phase shift and its relationship to the NA of the objective lens, the fundamental wavelength and refractive index of the sample . Thus the beam diameter = 0.32λ / √2NA. The coherent SHG signal formed in the transmission geometry was detected by the transmission detector via the microscope condenser with 445 nm df 30nm band pass filter inserted in the light path (Chroma Inc. UK). In addition, a non-confocal, bright-field image was acquired (sequentially to the SHG image) using single photon excitation at 488nm, which was collected in the transmission PMT. This image served as a reference image to overlay the H&E or Melan-A with the SHG images during the assessment stage. At the end of image acquisition the automatic montage software produced a final compilation image of the all z-projected fields.
Sister-sections were then imunohistochemically stained for the melanoma marker, Melan-A, and imaged using conventional light microscopy, then overlaid on the SHG images for comparison. The extent of invasion and limits of the borders between the two imaging techniques (H&E and or Melan-A) was assessed and marked by two independent histopathologists. The lateral boarders of cancer invasion were marked on the SHG images by an independent researcher, blinded to the histopathologists assessments, then checked for the degree of correlation between the SHG and H&E/Melan-A findings, using Mann-Whitney U-Test and Kendall-Rank Correlation Analysis. The criteria used for the independent evaluation of the MM borders of the SHG images were that one could clearly discriminate undisrupted collagen fiber bundles at the dermal-epidermal junction, adjacent to the MM lesion.
Collagen fiber density was quantified from seven regions using ImageJ image analysis software (Freeware, rsbweb.nih.gov) to calculate the percentage area of collagen fibers for that region. The images were segmented using an adaptive threshold algorithm with a fixed kernel size and offset for all samples. Data was expressed as Area %, calculated using area of collagen fibers/ area of measuring frame x 100. Generally SHG produces very high contrast images with very low or no background and since pump laser beam polarization was circular, segmentation based on threshold values proved to be a robust method. The regions quantified are highlighted in Fig. 2A and were an area of non-lesion skin under the dermal-epidermal junction (labeled A), the lateral melanoma border under dermal-epidermal junction (labeled B), an area under the middle part of the lesion and under the dermal-epidermal junction (labeled C), the areas labeled D, E, F, are all below C but one field vertically deeper to each other, and the area of dermis at the same depth as F but away from the lesion (labeled G in Fig. 2A).
Fresh pieces of whole rat and human skin were cleared of subcutaneous fat and connective tissue, and hair in the case of the rat samples, and mounted onto glass slides in PBS buffer. Human skin samples were collected from The Royal London Hospital Trust, department of surgery with full patient consent and ethical approval, and imaged within four hours. One sample was from 22 year old, breast reduction patient, and the other three samples were breast reconstructions from age rages of 30–45 years. Two samples appeared heavily pigmented whilst the other two appeared very lightly pigmented. SHG image acquisition was performed in both backscattered and forward propagated (transmission) geometries from the epithelial surface and imaged at full depth (deep into the dermis) in order to demonstrate that the technique is transferable to intact live skin. A Leica 20x 0.7 NA, long working distance objective, was used for image acquisition. A single field of 512 x 512 pixels (750 x 750 µm or 430 x 430 µm) at zoom 1.0 or 1.7 at 800 Hz with 2.5µm z-step, an imaging average of 2 frames was acquired for both rat and human ex vivo samples. Typical imaging times for ex vivo skin samples were 0.7 s per frame and up to 225 z-slices per sample; therefore total-imaging times did not exceed 3 min. 3D-images were generated with Imaris visualization software (Bitplane Scientific Software. Zurich, Switzerland).
Three images were obtained for each unstained melanoma section; an SHG image in the forward propagated (Fig. 1A ) and backscattered geometries (Fig. 1B), followed by a bright field image (Fig. 1C). Bright field and SHG images superimposed gave an image of collagen distribution within the bright field background for each section (Fig. 1D).
The SHG collagen signal showed a rapid transition at the two lateral melanoma borders, from very low or no SHG signal to that of normal collagen distribution found in non-lesion areas of skin. Medial to the melanoma borders there was a dramatic reduction in collagen SHG signal making the identification of the lateral boarders of cancer invasion unambiguous (Fig. 1D, 2A, 3C and 4B ).
Quantification of collagen fiber density from different regions around the MM lesion showed varying degrees of fiber density, expressed as Area %, ranging from 0.8% in the region labeled (C) (Fig. 2A) in the middle of the MM lesion, to 38% per unit area, in non-lesion area of skin labeled (A and G) (Figs. 2A and B). In regions labeled (D, E and F) there was a gradual increase of collagen fiber density from 9.1% to 19.88% to 33.2%, respectively (Figs. 2A and B). This increase correlated with increasing distance from the MM lesion towards a more ‘normal’ collagen fiber density. Each microscopic field (one tile in the montage) is equivalent to 375µm; therefore the collagen fiber density goes from almost zero in region C to 33.2% in region F, a distance of 1500µm (Figs. 2A-B). In the non-diseased areas of all skin sections we found a normal distribution of collagen up to the dermo-epidermal junction. Linear regression of collagen fiber density from region C (minimum SHG signal) to region G (maximum SHG signal in non-diseased area) showed an R2 value of 0.9849 suggesting that collagen fiber density is a good biomarker for malignancy (Fig. 2B).
Assessment of the lateral melanoma borders derived from SHG images versus H&E images (Fig. 3A) by the histopathologist were found, on direct observation, to co-localize in almost exactly the same positions (Fig. 3C). Cross-reference control comparison showed that melanoma borders obtained from H&E and Melan-A stained sister sections were also well co-localized (Figs. 3A and B, Figs. 4A and B).
Non-parametric statistical analysis using Mann-Whitney U-Test revealed no significant difference between the lateral melanoma borders as determined by an independent histopathologist using H&E sections and those assessed by an independent scientist using the SHG images of the corresponding unstained sister section (Fig. 5A ). p-values for the right lateral border comparison was p = 0.9491 representing a mean difference of 0.85 mm ± 0.18 mm (SE) (n = 7) and for the left border p = 0.9164 representing a mean difference of 0.55 mm ± 0.13 mm (SE) (n = 8), respectively (Fig. 5B). The total p-value for both sides was p = 0.9339. Kendall-Rank Correlation Analysis revealed a highly significant correlation between the lateral melanoma borders derived from histopathologists assessment and those from SHG images. p-value for the right lateral border comparison was p = 0.0069 (n = 7) and for the left side p = 0.0260. (n = 8) The tied p-values for the right and left sides were p = 0.0056 and p = 0.0260, respectively. The total p-value for both sides correlation analysis was p = 0.0012 (Fig. 5B).
SHG imaging of whole, ex vivo rat (Fig. 6A ) and human skin (Fig. 6 B) samples yielded detailed images of collagen morphology through the entire thickness when imaged from the epidermis, in both backscattered and transmission geometries. In the case of rat skin, SHG transmission images were detected through the whole thickness of the skin, although only the upper 150 µm is shown in (Fig. 6A). The blue channel is the two-photon auto fluorescence image of the upper epidermis, whilst the red and green channels are the transmitted and back-scattered SHG signals, respectively. In the lightly pigmented human skin samples, we were able to image through the whole thickness of epidermis and dermis to depths of approximately 300µm in the back-scattered geometry (green signal) and over 1000µm in the transmission geometry (red signal) (Fig. 6B). The heavily pigmented human samples could not be imaged from the epidermal surface, presumably because of the absorption of the 880nm wavelength light by melanin. However, if the samples were imaged from the dermal surface, clear SHG images of collagen were detected in both forward and back-scattered geometries (images not shown). Scale bars = 50 µm for each orthogonal plane.
These data support our hypothesis in that there are no significant differences between the two methods of identifying the borders of the MM lesions. Furthermore, it also emphases the fact that the extent of MM lesion can be asymmetrical and hence using Breslow thickness alone to empirically define a radius from the center of the MM lesion can in many cases be insufficient. This effect can be compounded in cases where there is very irregular shaped, MM lesions and would lead to more than one attempt to surgically remove the tumor.
We have demonstrated that SHG microscopy is a rapid and reliable method for the clinical management of MM by defining the borders of the lesions and the extent of dermal invasion (Breslow thickness) based on collagen morphology and density. Our results show that we can accurately define the lateral borders in histological sections of MM biopsies, which correlate significantly with the current gold standard assessments (H&E and Mealn A). This study provides initial evidence that collagen density in and around the suspected MM lesion may possibly be used as a biomarker for skin cancer diagnostics; further studies are in progress to confirm if collagen density and or architecture can differentiate a MM from a squamous cell or basal cell carcinoma as well as other skin tumors. Therefore, the in vivo applicability and identification of skin tumors using SHG microscopy is at present independent of lesion type so clear identification/confirmation is still required. The scant data in the literature about collagen fiber density around MM and NMSC lesions’ warrants more detailed studies but until then current diagnostic practices will still require immunophenotyping. In the diseased areas we observed a gradual decline in collagen density. This was most dramatic in the areas directly under the melanoma, which showed the weakest SHG signal; in most cases there was a complete absence of SHG signal due to the destruction of collagen by MMP’s produced by the malignant melanocytes. In the MM lesion areas the collagen morphology was greatly affected and was present in very short, thin ‘wispy’ fibers. The SHG signal then intensified with increasing depth below the lesion, until the collagen took on a more normal appearance. Furthermore, we have demonstrated that SHG imaging can detect collagen in freshly excised human and rat skin samples in both the back-scattered and transmission geometries; up to 300 µm and 1mm, respectively.
SHG imaging has been used to detect collagen non-invasively in many tissues including human skin [27,28]. It is a non-absorptive, non-linear, two-photon optical effect that can detect non-centrosymmetric crystalline structures such as collagen type I and III in unstained, live and fixed tissues [27,28]. It typically employs infrared or longer wavelength pulsed lasers, enabling good depth penetration and very little or no phototoxicity to tissues. Furthermore, the non-absorptive nature of the technique results in conservation of energy and therefore little or no heat is dissipated into non-pigmented tissue from the laser source, which would otherwise damage or even destroy live tissues.
Until social attitudes to skin cancer prevention are changed, early detection and treatment is the key to reducing mortality rates. Therefore the major challenge for skin cancer diagnostics is the early detection of non-metastasized malignant lesions and to reliably differentiate these from the less life threatening malignant cancers such as SCC, BCC and benign nevi BN, at the time the patient presents to the clinician. The most significant risk to life is MM and early detection and excision is crucial if the chances of survival are to be maximized [29,30]. SCC and BCC represent a much lower risk to life expectancy and are highly treatable if the correct diagnosis is made early on in the disease [29,30]. BN present little or no risk to patient survival; however, accurate diagnosis is required, as these can often resemble MM lesions. In addition, an apparently BN may become dysplastic and progress to a MM.
One of the key features of all skin cancers is the remodeling of extracellular matrix by MMP’s, principally collagen type I and III, which is clearly seen to be disrupted along the borders of the invading cancer cells  and missing in the diseased tissue itself. However, current diagnostic procedures do not include assessment of collagen density/architecture as a parameter, even though this is a key event in skin cancer progression and can potentially be used as a biomarker. We propose that collagen architecture, and its presence or absence can be used as, 1) a reliable marker of MM invasion through tissues, and 2) that it can be used to accurately define melanoma invasion borders.
A number of non-invasive and experimental imaging techniques are currently being used in dermatology clinics throughout the world including the Dermatascope [32,33] and single- or two-photon epifluorescence confocal microscope based systems . However, anecdotal reports and clinical interpretations of these techniques have been varied, with a tendency towards over diagnosing non-malignant conditions such as SCC and BCC putting them incorrectly into the MM category [32,33]. This leads to unnecessary surgery, pain and psychological stress for patents, believing that they have a MM, and ultimately, leads to increased costs to the Health Services. More importantly however, is the reliability and accuracy in misdiagnosis of a MM where current, non-histological methods, have a maximum of 87% specificity rate compared to CLSM of 98% .
The data from this study clearly shows that there can be very significant differences in the distance from the center of the tumor to the left and right hand borders of irregular shaped MM lesions; In Fig. 5A, sample numbers 3 and 6, show an approximately 50% differences in left- and right-hand borders, illustrating that the tumors in these samples were asymmetric. If this asymmetry were significant then clinical management of MM based on Breslow thickness measurements would not be sufficient in determining how much tissue to excise from the center of the tumor so as to encompass all the borders. The current practice can lead to multiple excision biopsies being performed, and in some cases, putting the patient at increased risk of tumor metastasis. Therefore, present-day methods for skin cancer diagnostics are complicated by the lack of a reliable non-invasive assessment technique that could eliminate the excision biopsy stage of the assessment if it is found to be non-malignant. A recent study suggests that collagen SHG signature is a promising marker for MM diagnosis  and this study supports that notion.
Any future diagnostic instrument will depend on the ability to image through the surface of the skin and thus the back-scattered signal is more important in this respect. Selecting the appropriate wavelength to ensure adequate depth penetration and to overcome absorption of the light by melanin will be crucial to the success of any such instrument. We found that at 880nm we could not detect an SHG signal from heavily pigmented human skin samples from the epidermal surface, but was able to when imaged from the dermal surface. This result emphasizes the need to fully assess the tissue wavelength absorbance characteristics so that SHG imaging can be performed in pigmented as well as non-pigmented samples. Other groups have shown, with custom built microscopes that longer infra-red wavelengths (1200 −1300 nm) can overcome the absorbance of shorter infra-red wavelength (880 nm) we have used in this study . However, in lightly pigmented skin the areas immediately outside of a suspected cancerous lesion would still be penetrable by 880nm light, although longer wavelengths would be more appropriate. Our research is aimed at developing a low resolution, fiber optic, handheld device that can give rapid and reliable assessment of collagen presence/absence outlining the MM lesion, in the first instance, and then a high resolution lens-based system for differentiating between, MM, SCC, BCC and BN based on collagen architecture and density. It is important to note that the low resolution system needs only to penetrate the skin to just below the dermal-epidermal border as this will detect the presence (presumptive normal, non-diseased tissue) or absence (presumptive MM lesion) of the collagen SHG signal The clear and unequivocal data from this study provide good evidence that SHG imaging can be a viable alternative to the existing diagnostic procedures.
In conclusion, this study provides good evidence that SHG imaging has the potential to improve on the current excision biopsy stages of MM diagnosis with a single assessment at first presentation of the patient. This can potentially shorten the diagnostic process and by demarcating the boundaries accurately ensure that the entire MM is removed first time thereby saving lives.
The authors would like to thank Mr. R Ragoowansi and Mr. G Moir Constaltant Plastic Surgeons of Department of Plastic Surgery, St Barts and the London NHS Trust (London, UK) for the their help and supply of the normal human skin samples. Thanks also goes to Dr Sian Hughes, Consultant Histopathologist and Dr. Lorette Folkes Special Registrar, Department of Histopathology, University College Hospital, London, UK for their help in independently confirming MM diagnosis and indicating the borders of the MM lesions on the normal H&E and Melan-A samples. Thanks to Mr. Kieran Power MRCP, for the independent assessment of MM boarders using the SHG images. This work was supported by Proof of Concept Funding Grant from UCL Business (UCL, London, UK). All authors have no conflicts of interest to declare.
References and links
1. Cancer Research UK, “Statistical Information Team” (2010), http://info.cancerresearchuk.org/cancerstats/types/skin/incidence/.
2. ISD-Online, “Cancer incidence and mortality data” (2010), www.isdscotland.org/isd/183.html.
3. Northern Ireland Cancer Registry, “Cancer incidence and mortality” (2008), www.qub.ac.uk/research-centres/nicr/Data/OnlineStatistics/MalignantMelanoma/.
4. Office for National Statistics, “Registrations of cancer diagnosed in 2005, England” (2008), www.statistics.gov.uk/statbase/Product.asp?vlnk=8843&More=N.
5. Welsh Cancer Intelligence and Surveillance Unit, “Cancer incidence in Wales, 2003-2007” (2010), www.wales.nhs.uk/sites3/page.cfm?orgid=242&pid=35385.
8. P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003). [CrossRef] [PubMed]
9. N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005). [CrossRef] [PubMed]
10. C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003). [CrossRef] [PubMed]
11. J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000). [CrossRef] [PubMed]
12. F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996). [CrossRef] [PubMed]
13. E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002). [CrossRef] [PubMed]
16. J. Paoli, M. Smedh, and M. B. Ericson, “Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers,” Semin. Cutan. Med. Surg. 28(3), 190–195 (2009). [CrossRef] [PubMed]
17. M. H. Lien and V. K. Sondak, “Diagnostic techniques for primary cutaneous melanoma,” G. Ital. Dermatol. Venereol. 144(2), 187–194 (2009). [PubMed]
19. T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006). [CrossRef] [PubMed]
20. C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977). [CrossRef]
21. S. Roth and I. Freund, “Second harmonic-generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979). [CrossRef]
22. P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009). [CrossRef] [PubMed]
23. A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005). [CrossRef] [PubMed]
24. G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003). [CrossRef] [PubMed]
27. E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003). [CrossRef] [PubMed]
32. V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006). [CrossRef] [PubMed]
35. T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008). [CrossRef] [PubMed]


Leucorrhoea or (leucorrhoea British English) is a thick, whitish or yellowish vaginal discharge. There are numerous reasons for leucorrhoea, the standard one being estrogen imbalance.
[Citation needed] The measure of release may increment because of vaginal disease or STDs, and it might vanish and return every now and then. This release can continue happening for quite a long time, in which case it turns out to be more yellow and putrid.
It is normally a non-neurotic indication auxiliary to provocative states of vagina or cervix.
It's anything but a noteworthy issue yet is to be settled as quickly as time permits.
It tends to be a characteristic resistance instrument that the vagina uses to keep up its synthetic parity, and in addition to save the adaptability of the vaginal tissue.
The expression "physiologic leucorrhoea” is utilized to allude to leucorrhoea because of estrogen stimulation.
Leucorrhoea may happen regularly amid pregnancy.
This is caused by expanded blood flow to the vagina because of expanded estrogen. Female newborn children may have leucorrhoea for a brief timeframe after birth due to their in-uterine introduction to estrogen.
Leucorrhoea can likewise be caused by sexual stimulation.
It might likewise result from irritation or clog of the vaginal mucosa. In situations where it is yellowish or emits a smell, a specialist ought to be counseled since it could be an indication of a few ailment forms, including a natural bacterial disease (oxygen consuming vaginitis) or STD.
There are various reasons why this stream may end up over the top. These include:
Contaminations: The vagina is exceptionally helpless to contagious, bacterial and parasitic diseases. These diseases can be advanced by poor cleanliness, the utilization of engineered clothing and unprotected sex. An expansion in vaginal release is normally one of the primary indications of a vaginal disease.
Damage: Giving birth to a kid, having a fetus removal or inordinate sex may bother the covering of the vagina and cause damage. This thusly can trigger leucorrhea.
Illnesses: Diseases, for example, diabetes or paleness may debilitate the resistant framework and trigger vaginal contaminations causing an exorbitant release. An UTI (Urinary Tract Infection) or worms in the stomach may likewise spread disease to the vagina.
Contraceptives: Contraceptive showers and jams utilized amid intercourse can chafe the vagina and cause over the top releasing. Now and again, aggravation of an intra-uterine prophylactic gadget can likewise cause release and agony.
Awful cleanliness: Not changing your clothing frequently enough, neglecting to change tampons or sharing open toilets can likewise increment vaginal release.
Leucorrhea can be forestalled by keeping the genital region spotless and dry, wearing cotton clothing, maintaining a strategic distance from the utilization of beauty care products on the genital zone and having safe intercourse.
Drinking a lot of water will likewise help flush poisons from the body and control the stream of vaginal release.
Whenever left untreated, this condition can spread to the uterus, fallopian cylinders and cervix and can trigger pelvic incendiary illness.
On account of post-menopausal ladies over the top vaginal release can likewise be viewed as a side effect of cervical malignancy and henceforth should be broke down as quickly as time permits.
In any case, this condition can be effectively treated at home.
Abstain from eating sugar, hot and zesty nourishment and liquor to treat release.
Eating curd can likewise help regard vaginal release as it contains lactic corrosive that cools the body. Likewise, dodge anxiety as this can build the release.